EFFECTS OF HEMA TODINIUM INFECTION ON THE NORWAY LOBSTER, NEPHROPS NOR VEGICUS (L. ) Grant D. Stentiford Presented in candidature for the degree of Doctor of Philosophy, to the Institute of Biomedical and Life Sciences, University of Glasgow. September, 2000 Ncphrops traw1crs at Tarbert. Loch Fvne (G. D Stentiford) Candidate's Declaration I declare that the work recorded in this thesis is entirely my own, unless otherwise stated and that it is of my own composition. No part of this work has been submitted for any other degree. Grant D. Stentiford September 2000. i Acknowledgments Financially: To the Ministry of Agriculture Fisheries & Food for funding the current project, to the National Shellfish Association of the USA for providing me with a travel scholarship to attend their 91' Annual meeting in Nova Scotia, Canada in April 1999, to the Kristinebcrg Marine Research Station (KMRS) in Sweden for providing a grant (via the EC) to visit their'large scale facility in September 1999 and to the Physiological Society for providing me with a Dale & Rushton travel scholarship to visit the Bodega Marine Laboratory (BML), California in May 2000. Academically: To my supervisors Professor Graham Coombs (Division of Infection & Immunity (DII), University of Glasgow), Dr Jim Atkinson (University Marine Biological Station Millport - UMBSM), Mr Nick Bailey (Fisheries Research Services, Marine Laboratory Aberdeen - MLA) and especially to Dr Douglas Neil (Division of Environmental & Evolutionary Biology (DEEB), University of Glasgow) for their support, enthusiasm, advice and ideas. Also to Dr Alan Taylor and Professor Keith Vickerman of DEEB for continual support and sound advice throughout this project. Also at the University of Glasgow, I thank Drs Charlie McSharry, Rob Strang, Hugh Elder, Laurence Tetley and Steve Arnott for giving up their time to assist my research. Outside of the University, I thank Drs Richard Smullen (University of Stirling) and Simon Webster (University of Bangor) for advice on crustacean hormones and the hyperglycaemic response, Professor Ernie Chang (BML, California) for his kind hospitality and advice during my visit to BML, to Dr Jeff Shields (Virginia Institute of Marine Science) for sharing with me his extensive knowledge of crustacean microparasites, to Dr S Baden (KMRS Sweden) for her help and advice on the responses of Nephrops to hypoxia, to Mr Mats Ulmestrand (Institute of Marine Research, Sweden) for his discussions concerning Hematodinium infection in Swedish Nephrops populations, to Professor Robert Adlard (Queensland Museum, Australia) for providing ii nucleotide sequences and advice on PCR of Hematodinium DNA, to Dr Louis Evans (Curtin University of Technology, USA) for her advice on idiopathic muscle necrosis, to Mr Gary Cross (St Davids Sea Life Centre) for passing on his enthusiasm and knowledge about fish and fisheries to me and for allowing me to experience living in Pembrokeshire, and finally to Dr David Holdich (University of Nottingham) for introducing me to the of delights of crustacean disease during my undergraduate days at Nottingham! Technically: I extend my sincere thanks to all of the technical support staff who have advised me on how not to break laboratory machinery and how to appreciate that it really is them who keep a university running; in DEEB, to Mr Graham Adam, Ms Kate Orr, Ms Aileen Adam, Ms Pat McLaughlin, Mr John 'Don't throw that seawater away' Laurie and especially to Ms June '7 man-hours to collect Crangon' Freel. To Mr David Laughland (DII) for showing me exactly what could go wrong with an HPLC system, to Ms Margaret Mullin (DII) for assistance with electron microscopy, To Mr David Bova and Adrian Weetman (MLA) for excellent assistance during a cold, wet week chasing Nephrops around a tank with a stick in Aberdeen, Mr Steve Parker for arranging boat trips (UMBSM) and finally and perhaps most importantly, to Mr Mick Parker and the crew of the RV 'Aora' for catching all of the animals that will be talked about in this study. Socially: Where to begin? - Suffice to say, I greatly appreciate all of the friendships that I have formed while working in Glasgow and on the Isle of Cumbrae and can probably only mention (or remember) a small proportion of the key players; in particular to Stephen 'potatoes' Murnaghan (honorary DEEB member) for long runs, late night drinking and for being a great mate, to Chris Noble for agreeing with me that fish really are better than birds (he also did some of that late night drinking too! ), to Stuart Fleming for providing constant entertainment (especially in the 'early days'... I'm planning my novel about you already! ), to Mike 'plants' Kennedy for working on things without personality, to 'Hong Kong' Kenny Leung for being the most cheerful person I iii have met and to Dom 'Ibiza' Lewis and Aaron 'string theory' Johnson, who share the social room/office where I work. In addition, I need to mention some more excellent acquaintances; Andrea Fidgett, Steve 'Stato' Amott, Beth Mouat (fellow Nephrops fan), Jason Hall-Spencer (we'll continue this down south! ), Kath Sloman, Kevin Butterworth (cheers for Sweden), Gordon Goodall and the Infection and Immunity lab rats (thanks for the eclipse experience in Cornwall! ) and finally to my very good friend Melanie Bergman for her continuous hospitality (Squat lobster Thai curry) and friendship on the Isle of Cumbrae over the last three years ... we'll write that paper one day! Finally: Of course, a great debt of appreciation goes out to my mum, dad and brother who have given me continual support and encouragement during my studies in Glasgow and Nottingham. The rest of my family also disserve a big thank-you - especially to Jan, Ray and Samantha, Gran and to my nan (Mo) and grandfather (Ted) who are no longer around to see this chapter of my life completed ... I dedicate this to you. Lastly and with the greatest amount of appreciation, I thank Del for her constant love and encouragement - you've provided the balance. iv Summazy Summary 1. The Norway lobster (Nephrops norvegicus) is one of the most valuable shellfish resources in the north east Atlantic ocean and considerable knowledge exists with regard to its biology and life history. During the late 1980s, populations of N. norvegicus were shown to harbour an infection by a parasite of the genus Hematodinium (Dinoflagellata: Syndinidae). Although studies on the pathology, progression and prevalence of infection have since been carried out, considerable gaps exist in our knowledge of the effects of parasitism on host life history and cycling of the parasite in the field. This study aims to develop techniques for monitoring Hematodinium infection in natural populations of N. norvegicus and to study the interactions between parasite-induced pathological changes and the life history of host animals. 2. The two major field methods (body colour and pleopod index) for the diagnosis of infection in N. norvegicus by Hematodinium were compared. The pleopod index was then used to collect infection prevalence data in the Clyde Sea area over a period of 31 months from December 1998 to August 2000. Peak infection prevalence occurred during the spring and was highest in small lobsters and in females. Mean infection severity increased during the main infection season (Jan - May), highlighting the progressive nature of patent infection. Infection prevalence was synchronous between the sexes in some years but not in others. 'Me proportion of recently moulted, infected males in the late season extended the duration of peak infection prevalence in certain years. Sites with a smaller mean animal size showed higher infection prevalence. The methods available for monitoring Hematodinjum infection in N. norvegicus are evaluated. The relationships between fishing pressure, population structure of N. norvegicus and seasonal Hematodinium infections are also discussed. 3. While useful for studying advanced (patent) infections of the haemolymph, the pleopod diagnostic method failed to identify low-level haemolymph (sub-patent) and tissue- based (latent) infections. The development and application of an immunoassay for the detection of antigens from Hematodinium in N. norvegicus is described. Infected tissue and haemolymph samples were detected as multiple-band reactions to a polyclonal antibody (anti-Hematodinium). The sensitivity limit of the method was 204 parasites. mm3 - which is approximately ten times more sensitive than the pleopod diagnosis method. Use of the immunoassay on tissue samples taken from catches V Summary showed that the pleopod method considerably under-diagnosed infection prevalence in the early part of the season, though this under-diagnosis decreased as infected lobsters in the field progressed from latent and sub-patent to patent infections. The immunoassay failed to detect any infected lobsters during the summer months, suggesting that infection may not be carried over from one season to the next. Data are discussed in relation to the epidemiology of latent and sub-patent Hematodinium infections, to the transmission of parasites to other hosts and to the use of this immunoassay for the routine assessment of Hematodinium infection prevalence in N. norvegicus populations. 4. The haemolymph free amino acid (FAA) composition of X norvegicus at different stages of infection by Hematodinium sp. was determined by reverse phase high performance liquid chromatography (HPLQ. Uninfected animals had a total haemolymph FAA concentration of 3.79 pmol. ml-1. The concentrations of several FAAs, notably serine, were reduced in early infections (Stage 1), while at later Stages (24) several FAAs were increased in concentration. The most significant change was in taurine, which increased by 13-fold (from 0.22 to 2.56 gmol ml-1) in Stage 4, when its relative contribution to the total FAA was 41.6%. Possible causes of these changes in the lobster haemolymph FAAs are the breakdown of host tissues, haemocyte lysis, a host stress response and the release of FAAs from parasite cells. These results have implications for the biology of X norvegicus, and indicate that the taurine: serine ratio in the haemoIymph provides a sensitive diagnostic measure of patent Hematodinium infections. 5. Changes in the biochemistry and ultrastructure of the deep abdominal flexor (DAF) muscles were studied in X norvegicus at different stages of infection by Hematodinium. Muscles from infected lobsters showed slight, but significant increases in total water content, greatly depleted glycogen reserves and an altered free amino acid profile. However, protein concentration and composition remained unchanged. Parasitic infection also caused an alteration in sarcolemmal structure and localized disruption of myofibrillar bundles around the fibre periphery. The implications of these changes for normal escape swimming are evaluated. The altered carbohydrate titre could reflect the parasites acting as a carbohydrate sink in the haemolymph, a disruption of normal tissue glycogenesis, or some alteration in the host's hormonal regulation. The changes described could also adversely affect the taste, texture and marketability of infected meat. vi Summary 6. The effects of Hematodinium on carbohydrate metabolism were examined in N. norvegicus. Five stages of infection were observed, including uninfected, sub-patently infected, lightly infected (Stage 1), and moderately and heavily infected (Stages 2 and 3-4, respectively). Lobsters that were in Stages 1-4 of infection had significantly lower levels of hemolymph glucose than uninfected or sub-patently infected animals. These results were accompanied by significantly lower levels of hepatopancreatic glycogen in Stages 2-4 compared to Stages 0-1. Due to the disruption of the normal feedback loops that control the release of crustacean hyperglycaernic hormone (CHH) from the sinus gland, plasma concentrations increase with infection severity. The increase in CHH concentrations occurred concomitantly with reduced concentrations of plasma glucose and tissue glycogen. Data are discussed from the perspective that the parasite places a heavy metabolic load on the host lobster. 7. A post-capture muscle necrosis of rapid onset has been identified in N. norvegicus. Economic losses, due to mortality of these animals in transport, were encountered by Scottish wholesalers during the summer and autumn of 1999. The pathology causes a loss of the normal function of the abdomen, thus preventing the normal 'tail flip' swimming. Electron microscopy of affected tissue failed to reveal any obvious causative agent but showed a disruption of sarcomeric organization, a loss of Z-line material, a condensation of myofibrils; and an infiltration of necrotic regions by granulocytes. SDS-PAGE of affected muscle tissue revealed a great reduction of most of the major contractile proteins. The condition most closely resembles idiopathic muscle necrosis, a pathology previously reported from both wild and cultured crustaceans. Damage to the integument in conjunction with exposure to various stressors during and immediately following capture is the most likely cause of the pathology. The rapid onset of the pathology has implications for the post-capture handling procedure for N. norvegicus and their subsequent vivier transport to market, and may also be partially responsible for the high mortality rate of undersized N. norvegicus returned to the sea after capture and aerial emcrsion. 8. Various components of swimming performance were measured in uninfected N. norvegicus and compared to animals at different stages of infection by Hematodinium. Animals showed a progressive decline in overall swimming performance as infection severity increased, with reductions in the number of tail-flips performed, the number of swimming bouts and the total distance travelled by swimming. The velocity of the first (giant-fibre mediated) tail flip and average velocity over the swimming bout were also Summary significantly reduced in infected lobsters. Possible reasons for this decreased swimming performance are suggested, and the implications of this for predator avoidance of infected lobsters in the benthic habitat, and for capture of Nephrops by trawl rigs, are discussed. 9. The pattern of tail flipping in uninfected and Hematodinium-infected N. norvegicus over a complete swimming sequence, and the subsequent performance following 4h of recovery are described. Swimming in uninfected X norvegicus was divided into two phases, the first comprising 80 to 100 high power tail flips, and the second comprising a variable number of less powerful flips which precede cessation of swimming. Lightly infected X norvegicus showed a similar pattern, though the number of flips comprising the second phase was much less. Heavily infected N. norvegicus produced a smaller number of weak flips before cessation of swimming. Following recovery, uninfected N. norvegicus produced 84.3 % of the flips produced in the initial trial, this reduction being due to a smaller number of Phase 2 tail flips. Infected lobsters showed a lower recovery rate than uninfected animals, this being proportional to the severity of infection. The metabolic basis of this swimming pattern and the reasons for less Phase 2 tail flips being produced during Hematodinium infection are discussed in relation to the catchability of infected X norvegicus by trawlers and predators. 10. Time-lapse video recording was used to study the burrow-related behaviour of uninfected and Hematodinium-infected X norvegicus in aquarium conditions. Uninfected lobsters were more active during the hours of darkness, while infected lobsters were equally as active in the light and dark periods. The absolute number of departures performed by infected lobsters (70 day) was more than double that of uninfected lobsters (30.1 day-'), while the duration of burrow departures performed by infected lobsters (258.5 s. departure") was more than six times greater than in uninfected lobsters (38.7 s. departure"). This led to a more than ten times increase (from 1.7 to 19.4 0/6) in the percentage of the day spent out of the burrow by infected lobsters. The altered burrow-related behaviour could be due to the increased nutritional or oxygen requirements of infected lobsters, both of which may cause the host to emerge from the burrow onto the sediment surface. Increased time spent out of the burrow has implications for the catchability of infected lobsters by trawlers and by predators, and for the estimation of infection prevalence from trawl-caught samples. 11. In Conclusion, this study has greatly increased our understanding of the complex relationship between Hematodinium parasites and their hosts. By linking biochemical Summary and physiological data to effects observed in the field, it has been shown that Hematodinium infection may provide an excellent model system for studying stress responses in aquatic invertebrate hosts. The study has described in much greater detail than before the effect of parasitism on host lobsters, and has related these effects to the moult cycle and to host condition. Furthermore, it has shown how changes in host behaviour and locomotion during infection may be directly or indirectly linked to biochemical and physiological changes brought about by infection. The implications of these changes in the life history of N. norvegicus for the availability of infected lobsters to be captured by trawlers and predators is discussed. ix Contents Candidate's declaration Acknowledgments Summary Contents CHAPTER ONE General Introduction CHAPTERTWO The relationship of Hematodinium infection prevalence in a Scottish Nephrops norvegicus population to seasonality, moulting and sex. i ii V x I 25 CHAPTER THREE Development and application of an immunoassay diagnostic technique for studying Hematodinium infections in Nephrops norvegicus populations. 50 CHAPTER FOUR Changes in the plasma free amino acid profile of the Norway Lobster, Nephrops norvegicus at different stages of infection by a parasitic dinoflagellate (genus Hematodinium). CHAPTER FIVE Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Norway lobster, Nephrops norvegicus (L. ) during infection by a parasitic dinoflagellate of the genus Hematodinium. CHAPTER SIX Carbohydrate Dynamics and the Crustacean Hyperglycaemic Hormone (CHH): Effects of Parasitic Infection in Lobsters. 72 90 III x CHAPTERSEVEN A rapid onset, post-capture muscle necrosis in the Norway lobster, Nephrops norvegicus (L. ) from the west coast of Scotland, United Kingdom. 132 CHAPTER EIGHT An analysis of swimming performance in the Norway lobster, Nephrops norvegicus (L. ) infected by a parasitic dinoflagellate of the genus Hematodinium. 154 CHAPTERNINE Cessation of swimming and subsequent recovery in the Norway lobster, Nephrops norvegicus (L. ), and changes associated with infection by a parasitic dinoflagellate (genus Hematodinium). 173 CHAPTERTEN Alteration of burrow-related behaviour of the Norway lobster, Nephrops norvegicus (L. ) during infection by the parasitic dinoflagellate Hematodinium. 197 CHAPTER ELEVEN Conclusions and Prospects. 222 xi Chapter One - General Introduction Chapter 1 General Introduction. The Norway lobster (Nephrops norvýgicus). The Norway lobster, Nephrops norvegicus (L. ) is a burrowing decapod crustacean which is an important member of the marine benthic community on soft sediments, and over the past 50 years it has been the subject of an important fishery in the northeast Atlantic. Due to its availability in large numbers throughout the year, its adaptability to aquarium conditions and its convenience for laboratory studies on whole animals and isolated tissues, it has proved to be a model species for research on various aspects of crustacean biology and ecology. Laboratory studies and field observations, addressing aspects of its general biology; including feeding ecology (Loo et al., 1993; Cristo, 1998), reproduction (Fanner, 1974a) and moult cycle (Gonzilez-Gurriar6n, 1998) and of its behaviour (Rice & Chapman, 1971; Farmer, 1974b, c; Ar6chiga & Atkinson, 1975; Atkinson & Naylor, 1976; Newland & Chapman, 1989) have led to considerable advances in our understanding of the life history of N. norvegicus in the field. Although other studies have been concerned with N. norvegicus as a fisheries target (Tuck et al., 1997a, b; Merella et al., 1998; SardA, 1998), there are still considerable gaps in our knowledge of the population dynamics and fisheries biology of this important species (Briggs, 1997). Up until the 1950s, these lobsters were usually discarded by fishermen due to their troublesome blockage of trawl nets, thereby impeding fin-fish capture (Briggs, 1997). However, more recently they have become one of the most important shellfish species captured in the northwest Atlantic, with annual landings of around 60,000 t (Tuck et al., 1997a, b). Large fisheries for X norvegicus exist around the south and west coasts of I Chapter One - General Introduction Iceland, the North sea, the west coast of France, the Irish sea, the Kattegat, the Skagerrak, the Bay of Biscay, the Adriatic and the Tyrrhenian sea (Dow, 1980). It also supports a major fishery in the United Kingdom, with the Scottish fishery contributing over 76 % of this (22,000 tonnes, L57 million in 1999). The bulk of Scottish landings (c. 80 %) are from trawler capture, with lobsters usually being 'tailed' at sea and landed as 'scampi'. Creeling on the other hand is generally used for the capture of larger animals in sheltered waters or where trawling is not feasible. Creel-caught animals are usually landed live, after which they are transported via specially designed 'vivier' vehicles, often to supply the lucrative continental European markets (Anon, 1999). The natural history of X norvegicus impinges in many ways upon its availability to the fishery. As lobsters can only be captured when present on the surface of the sediment, both trawl and creel capture depend upon lobsters leaving their burrows (Fig. 1), and out-of- burrow activity provides the most likely explanation for the short term variations seen in catches (Fanner, 1974c). Burrow excursions are principally for the acquisition of food, and periods of maximal activity may coincide with the presence of principal prey items (Atkinson & Naylor, 1976). Female lobsters, which spend much of the winter within their burrows incubating eggs, are largely unavailable to the fishery during these times, causing a strong predominance of males in the catches (Farmer, 1974b). The feeding ecology of the female lobster during incubation is not well understood, though suspension feeding may play a significant role in nutrient supplementation (Loo et al., 1993). Following spawning, females emerge from the burrow to feed, moult and be mated by hard-shelled male lobsters. During this period, the sex ratio within the catch can return to approximately 1: 1; the different sex ratios at different times of the year represent the significant seasonal changes in the burrow-oriented behaviour of female lobsters (Farmer, 1974a). Once on the surface of the sediment, capture by trawlers is further affected by the ability of lobsters to perform escape swimming. Following disturbance by the ground rope of the 2 Chapter One - General introduction trawl (Fig. 2); lobsters undergo a series of rapid abdominal flexions and extensions (tail flips), which propel the animal backwards and away from the source of threat (Newland et al., 1992). Both the speed and endurance of tail flip swimming have implications for capture by trawl nets. In a study of the reaction of N. norvegicus to trawl nets, two capture zones have been observed: the 'catching zone' immediately in front of the net mouth, and the 'peripheral herding zone', where tail flipping lobsters are apparently herded (into the catching zone) by the otter boards of the trawl. Lobsters which are in the catching zone or herded in to this zone by the otter boards are usually overtaken and captured, while those tail flipping away from the catching zone evade capture (Newland & Chapman, 1989). Once within the net, the retention of captive lobsters is dependent upon the size of the lobster, the mesh size and the crowding of the net with other species. In addition to the possibility of physical damage from the trawling process itself (Symonds & Simpson, 1971), after landing on the deck of a fishing vessel, N. norvegicus are exposed to a significant period of air-emersion during sorting of the catch. This period of emersion may be expected to lead to considerable physiological stress caused by extremes of temperature, light, desiccation and hypoxia (Santos & Keller, 1993; Jussila et al., 1997; Paterson & Spanoghe, 1997; Chapman et al., 2000). Stress responses occur when regulated physiological systems are pushed beyond their normal limits of operation by external stressors acting upon them (Paterson & Spanoghe, 1997). Failure of all or part of these regulatory responses will lead to increasing physiological disturbance and ultimately death (Morris & Airriess, 1998) and such stresses have been blamed for considerable post- harvest losses in commercially important crustacean fisheries (Cawthorn, 1997; Paterson & Spanoghe, 1997), The cumulative effect of these stresses has also been implicated in the low survivorship of undersized discarded N. norvegicus (Uhnestrand et al., 1998). Creel- capture is thought to impart considerably less stress upon N. norvegicus than trawl-capture (Wileman et al., 1999). However, in addition to the aggressive conflicts and starvation 3 Chapter One - General Introduction which may occur within creels, lobsters will be exposed to an array of stressors during emersion, handling, pounding and transportation to the market place (Whyman et al., 1985; Spicer et al., 1990; Paterson & Spanoghe, 1997; Schmitt & Uglow, 1997). As such, any lobster in sub-optimal condition at the point of capture (e. g. recently moulted, diseased, damaged etc. ) may be less likely to survive post-capture handling, storage and transportation (see Zhou & Shirley, 1995). Additionally, loss of physiological condition in the post-harvest period has been implicated in the deterioration in the flavour and texture of crustacean meat (Boyd & Sumner, 1973). Disease as an additional stressor. Infectious and non-infectious diseases affect both wild and cultured crustacean species. An understanding of crustacean pathogens and the natural reactions of host animals to disease is necessary for the efficient husbandry of cultured species and for the management of wild stocks. Non-infectious pathologies, such as those brought about by heat, cold, detergents, pesticides and heavy metals, may be important factors in the regulation of host populations (Pauley, 1975a). They may also cause significant economic losses in aquaculture situations, with disease events normally coinciding with overcrowding, handling, hypo- or hyperthermia, hyperactivity, changes in salinity or hypoxia (see Akiyama et al., 1982; Nash et al., 1987). However, the majority of infections in crustaceans are caused by pathogenic microorganisms. The most important pathogens include representatives of the viruses, bacteria, fungi and protozoa. Over 30 species of virus have been described infecting crustaceans (Brock & Lightner, 1990), some of which, such as the one causing Taura syndrome in the shrimp Penaeus vannamei, cause significant economic losses under aquaculture conditions (Tu et al., 1999). Bacteria have been reported as residual inhabitants of the normal crustacean haemolymph (Colwell et al., 1975). The Family Vibrionaceae 4 Chapter One - General Introduction (especially Vibrio spp., Aeromonas spp. ) most commonly cause bacterial infections (Lightner, 1977), and these are often associated with host stress reactions to sub-optimal environmental conditions (Lightner & Lewis, 1975; Johnson, 1976; Stewart, 1980). External pathologies of the carapace, such as 'rust disease' and 'black spot' have been attributed to chitinivorous species of Vibrio and Pseudomonas (Baross et al., 1978). Bacterial colonization may also occur as a secondary effect of infection by other parasites. In these cases, impairment of the normal immune response of the host caused by the primary pathogen may allow infestation by secondary opportunists (Meyers et al., 1987; Field et al., 1992). Diseases caused by fungi may also have significant impact on crustacean populations. Shell diseases, commonly in conjunction with bacterially-induced lesions, have been attributed to various fungal species. One such disease in European species of crayfish is Burned Spot disease. In this, fungal hyphae growing within the cuticle cause dark lesions and breaches to appear, which allow secondary invasions by bacteria to occur. A similar disease, Black Mat syndrome (caused by the ascomycete Trichomaris invadens) has been described in Tanner crabs (Chionoecetes bairdi and C opilio) from Alaska. In this infection, the connective tissue surrounding the organs may be largely replaced by fungal hyphae, with the highest prevalence of infection being found in mature female crabs (Brock & Lightner, 1990). Ile most notable example of a fungal disease of wild crustacean populations is 'Crayfish Plague' caused by the PhycomyCete Aphanonýyces astaci. First reported in Italy in the mid- I 800s, the disease spread rapidly through mainland Europe, destroying many native populations of Astacus astacus, A. leptodactylus and Austropotamobiuspallipes by the early 1900's (Johnson, 1983). North American species of crayfish, which show resistance to the parasite, can however act as carriers. It has thus been suggested that Crayfish Plague may have been introduced to Europe with imported animals and subsequently formed cpizootic infections in native populations (Johnson, 1983). 5 Chapter One - General Introduction Infections caused by yeasts have also been described in Daphnia, Artemia, amphipods and freshwater crayfish (Brock & Lightner, 1990). It is likely that all decapod crustaceans harbour protistan parasites, although relatively few species are known due to the small number of decapod species that have been examined (for review see Sprague & Couch, 1971). The most important protozoan parasites of crustaceans are the microsporidians, with over 140 species described (Brock & Lightner, 1990). The microsporidians are intracellular parasites with great reproductive capacity. Transmission is normally by ingestion of spores by the host organism. Spores hatch and sporoplasms: gain entry to the primary site of infection (usually the gut wall) via the spores' polar filaments. From here, the infection can spread to target tissues and organs. Once inside the target cell, a period of rapid multiplication (binary fission of schizogony) is followed by sporogony, whereby sporonts divide into sporoblasts and then to spores. I'liese spores can then infect other host cells, often completely replacing the cell contents with parasite material (Canning, 1977). Microsporidian parasites are commonly found infecting the skeletal muscles (Findley el al., 198 1; Olson & Laman, 1984; Dennis & Munday, 1994) the hepatopancreas (Anderson et al., 1989, Kabre, 1992), the gut wall (Kelly, 1979) and the reproductive tissues (Baticados & Enriquez, 1982) of crustaceans. In addition to the economic threat of microsporidiosis to the survival of cultured species, the presence of infections in wild crustacean populations may impact upon reproductive output and stock recruitment (by causing parasitic castration - Breed & Olson, 1977) and may also affect the marketability of infected animals (Olson & Laman, 1984). Protozoan parasites of the subphylum Sarcomastogophora are generally thought to play a minor role in crustacean diseases (Sprague & Couch, 1971). However, one species of the family Paramoebidae, Paramoeba perniciosa, the causative agent of Grey Crab disease in the blue crab (Callinectes sapidus), causes seasonal infections along the eastern coast of the USA (Johnson, 1977). Moribund specimens have a translucent, grey appearance, with 6 Cliapter One - General Introduction internal symptoms that include lysis of muscle and blood cells and a reduction in carbohydrate and protein reserves (Pauley et al., 1975). The parasite is responsible for commercially significant mortalities in wild populations and epizootics in holding tanks (Sawyer et al., 1970; Johnson, 1988). Ciliate diseases are also relatively uncommon in crustacean hosts. The early descriptions of ciliate infections of crabs were attributed to the genus Anophrys, but more recently these forms have been transferred to the genus Paranophrys (Sparks et al., 1982). They are generally described as being problematic under hatchery and holding conditions (Bang et al., 1972) but have also been reported in wild populations (Poisson, 1930; Armstrong et al., 1981). Recently, studies on the infection by the scuticociliate Anophryoides haemophila have suggested this to be one of the three most important diseases in the American lobster (Homarus americanus), contributing significantly to post-capture economic losses (Cawthorn, 1997). Though initially described as being 'blood parasites', ciliate infections are now known to be systemic pathogens, capable of invading all major tissues (Sparks et al., 1982). Death of the host is probably due to a major disruption of normal haernolymph and tissue function caused by the massive proliferation of parasites which accompanies late infection (Armstrong et al., 198 1). In addition to their importance in the phytoplankton, the dinoflagellates are found as symbionts in a phyletically wide range of marine invertebrates (Trench, 1987). The first well-documented parasitic life style amongst the dinoflagellates was by Pouchet in 1885. He described Gymnodinium pulvisculus (later named Oodinium pauchetti) as an ectoparasite on Oik-opleura (Pouchet, 1885 in Coats, 1999). However, knowledge of parasitic dinoflagellates was poor until the contributions of Chatton were collected in a volume of parasitic dinoflagellates in the 1920s and reviewed in the 1930's (Chatton & Poisson, 1931 in Taylor, 1987). The work of Chatton was considerably expanded by Jean and Monique Cachon in the mid 1900s, who described the cytology and life history of 7 Chapter One - General Introduction numerous parasitic dinoflagellate forms (for review see Cachon & Cachon, 1987). It is now known that approximately 35 genera, representing four orders of dinoflagellates (the Phytodiniales, Gymonodiniales, Blastodiniales, and Syndiniales) contain species which are known to be parasitic. The majority of these are parasites of marine and estuarine organisms (Coats, 1999). In addition to the crustaceans, parasitic dinoflagellates are known to infect algae, protozoa, annelids, molluscs, salps, tunicates, rotifers and fishes (with over 140 species out of the 2000 described being parasitic) (Shields, 1994). Concerns over the importance of dinoflagellates as agents of disease in marine fisheries have led to a large expansion in the body of literature on this subject. In particular, much interest is now focussed on aspects of parasite-host ecology and on the molecular identification of parasitized hosts and the different parasite taxa responsible (Coats, 1999). A number of parasitic dinoflagellate 'tribes' have been distinguished: the Blastodinida, Duboscquodinida, Syndinida and the Dinococcidia (which differ in morphology of the vegetative stage, in nuclear development and in their structural and metabolic relationship with the host) (Cachon & Cachon, 1987). The Blastodinida, Duboscquodinida and the Syndinida have life cycles consisting of two phases: the vegetative phase (trophont) and the reproductive phase (sporont). The reproductive phase leads to the formation of two dissimilar forms of bi-flagellate dinospore ('swarmers') which arise from different parent individuals and ensure dispersal and new infection (Cachon & Cachon, 1987). The Syndinidae are generally considered as parasites of the haemocoel of metazoan hosts. They occur mainly as plasmodial forms which continue to grow and multiply until production of the motile spore stage. There are no chloroplasts and osmotrophy is the rule during the trophic phase (flagella not present), where lipid and polysaccharide inclusions suggest active feeding at the expense of the host. Sporogenesis is simple, with multiplication of the nuclei, plasmodia and cytoplasmic divisions up until the last generation of sporocysts, following which the bi-flagellate spores are produced and 8 Chapter One - General Introduction liberated (Cachon & Cachon, 1987). Four genera of syndinids are known to be parasitic of crustaceans; Actinodinium, Trypanodinium, Syndinium and Hematodinium. Of these, Actinodinium (in copepods) and Trypanodinium (on copepod eggs) have not been well documented, while Syndinium and especially Hematodinium have been studied by a number of workers (Shields, 1994). Infection by Syndinium turbo and S. gammarai have been described in copepods and inoculation presumably occurs by ingestion of dinospores by the host. Pathology includes degeneration of the host musculature, castration and eventually death. It is assumed that such infections may play a significant role in the regulation of host populations (Shields, 1994). Infections caused by Hemato&nium spp. Members of the genus Hematodinium are primarily parasites of decapods. Questions relating to host specificity are complicated by limited species descriptions and the lack of recognized morphological traits for sorting taxa of some parasitic dinoflagellates (Coats, 1999). Until recently, there was only one described species in the genus Hematodinium. This type species, Hematodinium perezi. was first described from the portunid crabs Carcinus maenas and Docarcinus depurator in European waters (Chatton & Poisson, 193 1). More recently, description of a second species, H. australis (Hudson & Shields. 1994) and molecular studies showing differences in Hematodinium isolates from other crustacean hosts (Hudson & Adlard, 1996), indicate a higher species diversity and host specificity than first thought (Coats, 1999). Hematodinium infection has now been reported from a number of crab hosts from around the world, including Callinectes sapidus (Newman & Johnson, 1975; Messick, 1994), Cancer pagurus (Latrouite et al., 1988), Cancer irroratus, Cancer borealis (Maclean, 1978); Chionoecetes bairdi (Meyers et aL, 1987; Eaton et al., 1991; Love et al., 1993), Chionoecetes opilio (Taylor & Khan, 1995), Necora (= Liocarcinus) Puber (Wilhelm & 9 Chapter One - General Introduction Boulo, 1988; Wilhelm & Mialhe, 1996), Ovalipes ocellatus (Maclean, 1978) Portunus pelagicus (Hudson & Shields, 1994), Scylla serrata (Hudson & Lester, 1994), Trapezia areolata and T coerulea (Hudson et al., 1993). Hematodinium-like dinoflagellates have also been described from several species of pandalid shrimp (Bower et al., 1993, Meyers et al., 1994) and in benthic amphipods (Johnson, 1986). During the early 1980s, a low incidence of N. norvegicus in a moribund state, with dull orange colouration and a milky-white haemolymph, were captured by fishermen in the Firth of Clyde, western Scotland. Due to its coincidence each spring with the main moulting period, the condition was designated 'post-moult trauma'. By the late 1980s, the increasing incidence of lobsters in this poor condition began to evoke comment from fishermen and processors and led to a regular sampling program to define its seasonality and geographic incidence (Field, 1992). Further studies on these affected lobsters led to the discovery that the condition was in fact caused by a parasitic dinoflagellate, similar to the Hematodinium species described from other species of decapod. This was the first description of Hematodinium infection in a lobster (Field et al., 1992). The N. norvegicus isolate of Hematodinium has now been serially cultured at 80C for a number of years and a putative life cycle for the parasite has been described. The parasite appears to undergo a series of developmental changes which eventually lead to the production of the motile dinospore stages. However, successful transmission of parasites to uninfected hosts has not been demonstrated to date, suggesting that the life-cycle described from in vitro cultures may not fully explain events in the field (Appleton & Vickerman, 1998). The presence of Hematodinium infection in the hermit crab Pagurus bernhardus in the Clyde fishery (Appleton & Vickerman, 1998) and the description of Hematodinium infection in several species of benthic amphipod (Johnson, 1986) means that the involvement of an intermediate/alternative host in Hematodinium transmission to N. norvegicus cannot be ruled out. The Hematodinium isolate from C sapidus has also been 10 Chapter One - General Introduction serially cultured (Shields & Messick, 1997), though here, transmission to uninfected hosts has been demonstrated by serial passage of infected haemolymph (Shields & Squyars, 2000). To date, the major field diagnostic method for the detection of Hematodinium infection in N. norvegicus has been the pleopod method of Field & Appleton (1995), in which a pleopod is assessed for the presence of agglutinated parasite and hemocyte material under low-power microscopy. In addition to its rapidity and adaptability to field conditions, a further advantage of this method is that it allows infection severity to be assessed on a four point scale, which allows the progression of infection to be charted. Ibis has been used to show that patent Hematodinium infection (present in the haemolymph) progresses from low haemolymph - burdens during light infections to the production of swarming spore masses at the termination of infection, after which the host lobster dies (Field & Appleton, 1995; Appleton & Vickerman, 1998). However, the pleopod method fails to diagnose low- level (sub-patent) haemolymph infections and tissue-based (latent) Hematodinium infections (Field & Appleton, 1996). For this reason, a polyclonal antibody, was raised against the cultured Hematodinium sp. originally isolated from N. norvegicus and used in an indirect fluorescent antibody technique (IFAT) study to show that apparently uninfected lobsters (using the pleopod method) can harbour low level infections outside of the main season (Field & Appleton, 1996). Observations made in this IFAT study suggested that the epidemiology of latent and sub-patent infections can only be investigated by using diagnostic methods which are considerably more sensitive than the pleopod method. However, the pleopod method has been used to show that epizootics of Hematodinium infection, similar to those already described in other commercially important crustacean hosts from southeast Alaska, France and the eastern United States (Newman & Johnson, 1975; Latrouite et al., 1988; Wilhelm & Boulo, 1988; Meyers et al., 1987,1990; Wilhelm & Mialhe, 1996) occur in N. norvegicus populations at various sites on the west coast of 11 Cbapter One - General Introduction Scotland (Field et al., 1992,1998). For this reason, the pleopod method remains a powerfiil field tool for the diagnosis of patent infections. Rationalefor the current study. The large fishery for N. norvegicus necessitates careful stock assessment and effort control (Tuck et al., 1997b). While an important feature of such assessment is the estimation of natural (" and fishing (F) mortalities, little information is available on the former (Chapman, 1980). However, a major contributor to natural mortality in any population is disease, with infectious organisms also affecting growth, reproduction and egg survival in their hosts. As such, assessment of natural mortality in fisheries models should make use of data on disease prevalence where available (Kuris & Lafferty, 1992). The prevalence of Hematodinium infection in N. norvegicus, especially during epizootic seasons is now being considered as an important natural mortality factor in N. norvegicus stock assessment models (Anon, 1997) and as such, estimation of the proportion of natural mortality (M attributable to Hematodinium infection requires accurate assessment of prevalence in natural populations. I'lie diagnostic sensitivity and reliability of the pleopod method therefore requires investigation, while fiirther development of the MAT method of Field & Appleton (1996) into a rapid, sensitive and non-subjective immunoassay will allow for infection prevalence data from different fisheries, at different European locations, to be compared with greater confidence. Any feature which alters the burrow emergence pattern or the escape swimming ability of N. norvegicus may be expected to impact upon their capture by trawlers. Due to pathological (Field & Appleton, 1995) and physiological (Taylor et al., 1996) deterioration which occurs in N. norvegicus with patent Hematodinium infections, it has been suggested infected lobsters may spend longer periods out of the burrow and may be less able to escape from predators and trawlers than their uninfected counterparts (Field et al., 1992, 12 Chapter One - General Introduction Field & Appleton, 1995). As such, estimations of Hematodinium infection prevalence in lobsters from trawl-caught samples may give a false estimation of true levels of infection prevalence in the natural populations. In order to assess the accuracy of infection prevalence estimates in trawler-caught samples, an investigation into the burrow-oriented behaviour and the escape swimming ability of Hematodinium-infected N. norvegicus is required. Studies on Hematodinium infections of other commercially-important crustacean hosts species have described major pathological disruptions to the muscle and haemolymph (Meyers et al., 1987; Taylor & Khan, 1995; Wilhelm & Mialhe, 1996). Sin-dlar gross observations on pathological progression of infection (such as milky-white haemolymph, infiltration of tissue and organs by parasite cells and general morbidity) have also been made for Hematodinium-infected N. norvegicus (Field et al., 1992; Field & Appleton, 1995), though to date, only one study has provided data on the physiological changes which occur in haemolymph and tissues during infection (Taylor et aL, 1996). Changes in the biochemical and physiological characteristics of Hematodinium-infected N. norvegicus tissues may have implications at several different levels. At market level, biochemical alterations to the meat of heavily-infected animals may render it unmarketable, a problem already encountered in Hematodinium-infected tanner crabs (C bairdi and C opilio) (Meyers et al., 1987; Taylor & Khan, 1995). At the fisheries level, ultrastructural and biochemical disruptions to the abdominal muscles may impinge upon the escape-swimming capability of infected lobsters, while the utilization of host resources by the parasites may lead to physiological starvation, an effect which may manifest itself as an increase in out-of-burrow foraging - both of which may lead to increased catchability of infected lobsters relative to their uninfected counterparts. Alternately, parasite-mediated biochemical and physiological alterations in the host could be used to map the pathological 13 Chapter One - General Introduction progression of infection, thereby leading to a richer understanding of the ecological relationship between parasite and host. The aims of this thesis are based on the above considerations, and are: 1. To use existing field diagnostic methods for studying the epidemiology of patent Hematodinium infection prevalence in natural populations of N. norvegicus and to investigate prevalence in relation to the size, sex, moult status and population structure of host animals (Chapter 2). 2. To develop the sensitive MAT method into a multi-sample, reproducible diagnostic method for the detection of sub-patent and latent Hematodinium infections in field-caught samples of N. norvegicus (Chapter 3). 3. To investigate the effects of Hematodinium infection on the biochemistry and physiology of the haemolymph and tissues of N. norvegicus (Chapters 4,5 and 6). 4. To investigate whether Hematodinium infection renders N. norvegicus more available to the fishery and to predators by causing changes in the locomotory ability (Chapters 8 and 9) and burrow-related behaviour (Chapter 10) of infected lobsters. 5. To integrate laboratory-based and field data to provide a detailed picture of the overall ecological effect of Hematodinium infection on N. norvegicus (Chapter II- Conclusions and Prospects). While not being an initial aim of this thesis, chapter 7 provides the first description of a seasonal, post-capture necrotic muscle pathology in N. norvegicus from the west coast fishery. This chapter reports on a disease which is distinct in both its seasonality and its pathology from Hematodinium infection, and thus provides a useful reference for the prevention of misdiagnosis in the field. 14 Chapter One - General Introduction Each of the data chapters in this thesis has been written as independent pieces of research and Chapters 2-10 inclusive have been submitted separately as scientific papers. 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Escape swimming in the Norway Lobster. Journal of Crustacean Biology 12,342-353. 20 Chapter One - General Introduction Newman, M. W. & Johnson, C. A. (1975). A disease of blue crabs (Callinectes sapidus) caused by a parasitic dinoflagellate, Hematodinium sp. Journal of Parasitology 63,554- 557. Olson, R. E. & Laman, C. N. (1984). Prevalence of microsporidian infection in commercially caught pink shrimp, Pandalusjordani. Journal of Invertebrate Pathology 43, 407-413. Paterson, B. D. & Spanoghe, P. T. (1997). Stress indicators in marine decapod crustaceans, with particular reference to the grading of western rock lobsters (Panuhrus cygnus) during commercial handling. Marine & Freshwater Research 48,829-834. Pauley, G. B. (1975). Introductory remarks on diseases of crustaceans. Marine Fisheries Review 37,2-3. Pauley, G. B., Newman, M. W. & Gould, E. (1975). Serum changes in the blue crab, Callinectes sapidus, associated with Paramoeba perniciosa, the causative agent of Gray crab disease. Marine Fisheries Review 37,34-38. Poisson, R. (1930). Observations sur Anophrys sarcophaga (= A. maggii Cattaneo) Infusiore holotriche marin et sur son parasitisme possible chez certains crustacds. Bull. Biol. Fr. -Belg. 64,288-331. Pouchet, G. (1885). Novelle contribution A I'histoire des P6ridiniens marins. Journal of Anatomy & Physiology 21,28-88. Rice, A. L. & Chapman, C. J. (197 1). Observations on the burrows and burrowing behaviour of two mud-dwelling Decapod crustaceans, Nephrops norvegicus and Goneplax rhomboides. Marine Biology 10,330-342. Santos, E. A. & Keller, R. (1993). Effects of exposure to atmospheric air on blood glucose and lactate concentration in two crustacean species: a role of the crustacean hyperglycaernic hormone (CHH). Comparative Biochemistry & Physiology 106A, 343- 347, SarcU, F. (1998). Symptoms of overexploitation in the stock of the Nonvay lobster (Nephrops norvegicus) on the "Serola bank" (western Mediterranean sea off Barcelona). Scientia Marina 62,295-299. Sawyer, T. K., Cox, R. & Higginbottom, M. (1970). Hemocyte values in healthy blue crabs Callinectes sapidus and crabs infected with the amoeba Paramoeba perniciosa. Journal of Invertebrate Pathology 15,440-446. Schmitt, A. S. C. & Uglow, R. F. (1997). Haemolymph constituent levels and ammonia efflux rates of Nephrops norvegicus during emersion. Marine Biology 127,403-4 10. Shields, J. D. & Messick, G. A. (1997). Aspects of the life cycle of Hematodinium perem . in the blue crab, Callinectes sapidus. Journal of Shellfish Research 16,357. 21 Chapter One - General Introduction Shields, J. D. & Squyars, C. M. (2000). Mortality and hematology of blue crabs, Callinectes sapidus, experimentally infected with the parasitic dinoflagellate Hematodinium perezi. Fishery Bulletin 98,139-152. Shields, J. D. (1994). The parasitic dinoflagellates of marine crustaceans. Annual Review of Fish Diseases 4,241-271. Sparks, A. K., Hibbits, J. & Fegley, J. C. (1982). Observations on the histopathology of a systemic ciliate (Paranophrys sp.? ) disease in the Dungeness crab, Cancer magister. Journal of Invertebrate Pathology 39,219-228. Spicer, J. 1, Hill, A. D., Taylor, A. C. & Strang, R-H. C. (1990). Effect of aerial exposure on concentrations of selected metabolites in the blood of the Norwegian lobster Nephrops norvegicus (Crustacea: Nephropidae). Marine Biology 105,129-135. Sprague, V. & Couch, J. (1971). An annotated list of protozoan parasites, hyperparasites and commensals of decapod crustaceans. Journal of Protozoology 18,526-537. Stewart, J. E. (1980). Diseases. In; The biology and management of lobsters (eds. Cobb, J. S. & Phillips, B. F. ). Academic Press, New York. pp. 301-342. Symonds, D. J. & Simpson, A. C. (1971). The survival of small Nephrops returned to the sea during commercial fishing. Journal Cons. Int. Explor. Mer. 34,89-98. Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The effects of Hematodinium sp. -infection on aspects of the respiratory physiology of the Norway lobster, Nephrops norvegicus (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228. Taylor, D. M. & Khan, R. A. (1995). Observations on the occurrence of Hematodinium sp. (Dinoflagellata: Syndinidae); the causative agent of Bitter crab disease in Newfoundland snow crab (Chionoecetes opilio). Journal of Invertebrate Pathology 65,283-288. Taylor, F. J. P, (1987). General group characteristics; special features of interest; short history of dinoflagellate study. In: The biology of dinoflagellates (ed. Taylor, F. J. R. ). Blackwell Scientific Publishers, Oxford, UK. pp. 1-23. Trench, R. K. (1987). Dinoflagellates in non-parasitic symbioses. In: The biology of dinoflagellates (ed. Taylor, F. I. R. ). Blackwell Scientific Publishers, Oxford. UK. pp. 530- 570. Tu, C., Huang, H-T., Chuang, S-H., Hsu, J-P., Kuo, S-T., Li, N-J., Hsu, T-L. & Lin, S-Y. (1999). Taura syndrome in Pacific white shrimp Penaeus vannamei cultured in Taiwan. Diseases of Aquatic Organisms 38,159-161. Tuck, I. D., Chapman, C. J. & Atkinson, RIA. (1997a). Population biology of the Norway lobster, Nephrops norvegicus (L. ) in the Firth of Clyde, Scotland - I: Growth and density. Journal of Marine Science 54,125-135. Tuck, I. D., Chapman, C. J. & Atkinson, RIA., Bailey, N. & Smith, R. S. M. (1997b). A comparison of methods for stock assessment of the Nonvay lobster, Nephrops norvegicits, in the Firth of Clyde. Fisheries Research 32,89-100. 22 Chapter One - General Introduction Ulmestrand M., Valentinsson D., Sangster G. I., Bova D., Kynoch R. J., Breen M., Graham G. N., Soldal AN, Cruikshank 0., Moth-Poulson T. & Lowry N. (1998). Nephrops survival after escape from commercial fishing gear or discarded from deck. ICES Nephrops life history study group meeting. La Corufia, Spain. 1998. Wileman, D. A., Sangster, G. I., Breen, M., Ulmestrand, M., Soldal, AN. & Harris, R. R. (1999). Roundfish and Nephrops survival after escape from commercial fishing gear. FAIR-CT95-0753, EC, 140 pp. Whyman, S., Uglow, R. & MacMullen, P. (1985) A study of mortality rates of the velvet crab during holding and transport. 259, Sea Fish Industry Authority. Industrial Development Unit, UK. Wilhelm, G. & Boulo, V. (1988). Infection de 1'etrille Docarcinus puber (L. ) par un dinoflagelle parasite de type Hematodinium. ICES Committee Meeting CM. 1988/K: 32. Wilhelm, G. & Mialhe, E. (1996). Dinoflagellate infection associated with the decline of Necora puber crab populations in France. Diseases of Aquatic Organisms 26,213-219. Zhou, S. & Shirley, T. C. (1995). Effects of handling on feeding, activity and survival of red king crabs (Paralithoides camtschaticus) (Tilesius, 1815). Journal of Shellfish Research 14,173-177. 23 Chapter One - General Introduction Fig. 1. The Norway lobster, Nephrops norvegicus in typical 'guarding' position at the entrance to its burrow (from Erwin & Picton, 1995). Fig. 2. The Norway lobster, Nephrops norvegicus undergoing active escape-swimming following disturbance by the ground rope of a trawl net (from Newland, 1985). 24 Chapter Two - Infection prevalence Chapter 2 The relationship of Hematodinium infection prevalence in a Scottish Nephrops norpegicus population to seasonality, moulting and sex. Abstract. The two Major field methods (body colour and pleopod index) for the diagnosis of infection in the Norway lobster, Nephrops norvegicus (L. ) by the dinoflagellate parasite Hematodinium have been compared. Using the more reliable method, the pleopod index, which scores infection severity on a four point scale, infection prevalence data have been collected from a single fishing ground in the Clyde Sea area, western Scotland over a continuous period of 31 months. Peak infection prevalence occurs during the spring and is highest in small N. norvegicus (mean carapace length 28.1 mm ± 0.67 in females and 30.9 mm ± 0.50 in males) and in females. Mean infection severity increased from 1.05 ± 0.03 during the low season (July - December) to 2.59 ± 0.19 by the end of the main infection season (May), highlighting the progressive nature of patent infection over time. Data collected separately for male and female lobsters show that infection prevalence is synchronous between the sexes in some seasons but not in others. Additionally, the proportion of recently moulted, infected males in the late season extends the duration of peak infection prevalence in certain years. Data from two adjacent fishing grounds in the Clyde Sea area has also shown that infection prevalence depends upon the population structure at a given site - the site with smaller animals showing the highest prevalence. The methods for monitoring Hematodinium infection for the assessment of natural mortality attributable to infection are evaluated. The relationships between fishing pressure, population structure of N. norvegicus and seasonal Hematodinium infections are also discussed. Submitted as: Stentiford, G. D.. Neil, D. M. & Atkinson, RTA. (2001). The relationship of Hematodinium infection prevalence in a Scottish Nephrops norvegicus population to seasonality, moulting and sex. ICES Journal of Marine Science. 25 Chapter Two - Infection prevalence Introduction. Populations of the Norway lobster (Nephrops norvegicus) provide one of the most valuable shellfish resources in the north east Atlantic, with annual landings in excess of 60,000 tonnes (Tuck et al., 1997). A major fishery for N. norvegicus exists in waters surrounding the United Kingdom, with the Scottish fishery contributing over 76% of this (07 million in 1999 - FRS, 2000, unpublished). The majority of the landings are from trawler capture, with lobsters being 'tailed' for sale as 'scampi'. Larger animals, captured by baited creels are often exported live to continental Europe by specially-designed 'vivier' vehicles. The large fishery for N. norvegicus necessitates careful stock assessment and effort control (Tuck et al., 1997) and an important feature of such assessment is the estimation of natural (" and fishing (F) mortalities. While fishing mortality is directly related to effort (E), little information is available on the rate of natural mortality in X norvegicus populations (Chapman, 1980). A major contributor to natural mortality in any population is disease, and infectious organisms may also affect growth, reproduction and egg survival in their hosts. As such, assessments of natural mortality in fisheries models should make use of data on disease prevalence where available (Kuris & Lafferty, 1992). Infections by parasitic dinoflagellates of the genus Hematodinium have been reported in a number of commercially important crustacean hosts (Newman & Johnson, 1975; Maclean, 1978; Messick, 1994; Meyers et al., 1987; Latrouite et al., 1988; Wilhelm & Boulo, 1988; Hudson & Shields, 1994; Hudson & Lester, 1994; Taylor & Khan, 1995. Wilhelm & Mialhe, 1996). Stocks of X norvegicus on the west coast of Scotland have been also been shown to harbour infections by Hematodinizim (Field et al., 1992). Since the initial descriptions of disease etiology, diagnosis and pathology (Field et al., 1992; Field & Appleton, 1995,1996), further studies have described the in vitro life-cycle of the parasite (Appleton & Vickerman, 1998) and have revealed that significant alterations in host physiology (Taylor et al., 1996), biochemistry (Stentiford et al., 1999 - Chapter 4, 26 Chapter Two - Infection prevalence Stentiford et al., 2000a - Chapter 5), locomotory performance (Stentiford et al., 2000b - Chapter 8) and behaviour (Stentiford et al. 2001a; in manuscript - Chapter 10) are associated with parasitism. A number of methods are available for the detection of Hematodinium infection in N. norvegicus. The simplest involves external assessment of the altered carapace coloration and opacity that accompanies patent infection. This assessment has the advantage that it can be performed rapidly in the field, but its value as a diagnostic tool has not been established, since no systematic study has been made of the relationship between parasite burden and colour change. The method that has been used most routinely is the pleopod infection staging technique of Field & Appleton (1995), in which a pleopod is assessed for the presence of parasite and hernocyte material under low-power microscopy. This method assigns infection severity on a four point scale, which allows the progression of infection to be charted. The current study has compared the sensitivity and reliability of the body colour and pleopod methods for the detection of patent infections. Observations on the prevalence of Hematodinium infection in crabs have suggested that there is a highly seasonal epidemiology, with peak infection occurring over a relatively narrow time period, followed by a longer period of undetectable or low level infection prevalence (see Shields, 1994). Studies on Hematodinium infection in Scottish N. norvegicus populations have shown similar features of seasonal epidemiology, along with evidence that prevalence is higher in small lobsters than in large lobsters, and in female lobsters than in male lobsters (Field et al., 1992,199 8; Field & Appleton, 1995). Meyers et al. (1990) have reported that Hematodinium infection prevalence is highest in post-moult C bairdi. Moulting has also been suggested as the major predisposing factor for Hematodinium infection of N. norvegicus, though the relationship remains unclear (Field et aL. 1992,1998). From a general survey of Hematodinium infection in N. norvegicus captured from the west coast of Scotland, it has been shown that the Clyde sea area generally has the highest 27 Chapter Two - Infection prevalence prevalence levels (Field et al., 1998). Here we report the results of a continuous long-term study of infection prevalence, whereby the pleopod diagnostic technique was applied to a major N. norvegicus fishing ground in the Clyde Sea area. Data are discussed in relation to the importance of long-term Hematodinium infection monitoring in commercially important crustacean populations, and the requirement for accurate infection prevalence data for use in stock assessment models for the X norvegicus fishery. Methods. Capture qj*lobsters. Norway lobsters (Nephrops norvegicus) were caught from a depth of approximately 80 in, using 90 minute tows of a standard otter-trawl (70 mm mesh size). The start of the towing period was always between 0900 h and 1000 h to minimise any effect of differential burrow emergence behaviour on catch composition (Atkinson & Naylor, 1976) and the effect of capture time on infection prevalence (Field et al., 1998). The main capture site was at a location immediately south of Little Cumbrae (LQ in the Clyde sea area (55.4 I*N, 4.56*W) or in one instance, from the adjacent Bute-Cumbrae channel (BC) (55.46*N, 4.591VV) in the Clyde Sea area, Scotland, UK. Both sites are fished commercially. Trawls were made each month over the period from February 1998 to August 2000 inclusive (n 31). Following capture. all non-target species were separated from the catch and a random 5 kg sub-sample of N. norvegicus was removed for assessment of sex, carapace length, approximate moult stage and infection stage. Carapace length was measured from the rim of the eye socket to the posterior mid-point of the carapace. Moult stage was assigned as either intermoult (IM - no setal withdrawal in the pleopod and a rigid carapace) or recently moulted (RM - no setal withdrawal in the pleopod and a flexible 'paper' campace) (see Aiken, 1980). Hematodinium infection diagnosis was carried out either in the laboratories 28 Chapter Two - Infection prevaIence of the University Marine Biological Station Millport (UMBSM) or at the Division of Environmental & Evolutionary Biology, University of Glasgow, Scotland, UK. All sub- samples were stored in a cool, damp environment following capture and were generally alive for Hematodinium infection diagnosis. Pleopods were viewed under low-power light microscopy (x 40 magnification). The accumulation of agglutinated parasite and haemocyte material was used to assign the appropriate stage, whereby Stage 0 is apparently uninfected and Stages I to 4 are patently infected. The accuracy of the body colour and pleopod diagnosis methods was assessed by comparing the results from two different scorers (one naive and one experienced). Both scorers assessed each lobster in a standard sub-sample for Hematodinium infection using the body colour method (loss of shell translucency, vivid coloration) and the pleopod method (as above). Results from the two scorers were compared at the end of the trial to assess the inter-operator sensitivity and reliability of the two methods. Analysis of data. Comparisons of the mean size of infected male and female lobsters and the mean size of uninfected and infected lobsters from the LC and BC sites were performed either by one- way analysis of variance (ANOVA) for normally distributed data, or by a Mann-Whitney test for non-normal distributions. Tests were considered significant if p<0.05. Results. Body colour vs pleopod diagnosis. By comparing the scores obtained by the experienced and nal-e operators, it was possible to assess the sensitivity and reliability of the two major field diagnosis methods. In relation to the pleopod method, the body colour assessment underestimated infection prevalence by approximately 50 %, even when used by the experienced operator (Fig. la). Of those 29 Chapter Two - Infection prevalence infected lobsters thus misdiagnosed by the body colour method, the majority were of Stage I infection for both the naive and experienced scorer. However, no heavily infected lobsters were missed by the colour diagnosis method by either operator (Fig. lb). The pleopod scores show that the naive operator estimated infection prevalence to be approximately 9% higher than the experienced operator (Fig. la). Of these over-estimates, again the majority were lightly-infected (Stage 1) animals. Hematodiniumprevalence at the LC site. Infection prevalence data obtained using the pleopod method at the LC site over three consecutive fishing seasons are shown in Figure 2a. During the summer and autumn (July to November), the prevalence of patent infection was minimal in both male and female lobsters (< 5%), with the initial increase occurring during December in each of the seasons studied. Prevalence of patent infection showed a marked increase during the period between January and March, and peaked in April or May. Overall infection prevalence reached a maximum of 20 - 25% in each of the three seasons studied, though prevalence in female lobsters was as high as 35% during the 1998 and 2000 seasons. However, the elevated level of infection prevalence in female lobsters had a lesser effect on the overall infection prevalence, due to the reduced proportion of female lobsters in the catch during the late winter and early spring periods (Fig. 2b). Following the peak (May to July), a sharp reduction in the prevalence of patently infected lobsters occurred. Severity qfpatent Hematodinium infection. As well as allowing Hematodinium infection prevalence to be assessed at the population level, the pleopod staging method also allows the severity of the infection in lobsters (on a scale of Stage I to Stage 4) to be assessed The mean infection severity measured in lobsters displaying patent infection, captured at the LC site over the three consecutive seasons is 30 Chapter Two - Infection prevalence shown in Fig. 2c. Over this period, all Hematodinium infected lobsters captured during the 'Low Season', i. e. outwith the main infection season of July - December, had very low parasite burdens, with a mean infection stage of 1.05 ± 0.03 (n=12). At the beginning of the main infection periods (February), the mean severity of infection in infected lobsters increased significantly to 2.04 ± 0.02 (n=3) (p < 0.05), and then to 2.59 ± 0.19 (n=3) (p = 0.05) (heavy Hematodinium parasite burden) by the end of the main infection season (May). These data indicate a progressive nature of patent infection over the main infection period. Size of infected lobsters. The monthly data for mean carapace size of lobsters captured at the LC site are given in Fig. 3. In addition to a reduction in mean carapace lengths which occurred during the autumn (September to October) of each season studied, the mean carapace length of male lobsters was significantly smaller in the 1999 season (29.2 ± 0.4 mm) than in thel998 season (32.3 ± 1.15 mm) (p < 0.05). However, no significant reduction in carapace length was seen in females over the same period (30.7 ± 1.4 mm in 1998; 27.8 ± 0.7 min in 1999) (p = 0.08 1). In the 2000 season, the mean sizes of male and female lobsters (30.7 ± 0.5 and 28.2 ± 0.6 mm respectively) were not significantly different to those in the 1998 season (p > 0.05). The mean carapace length of Hematodinium-infected female lobsters (28.1 mm ± 0.67) was significantly smaller than that of infected male lobsters (30.9 nun ± 0.50) captured from the LC site (p < 0.001). In order to test whether the mean carapace length of the X norvegicus population at a particular site affects the prevalence of Hematodinium infection in that population, two geographically-close sites (LC and BQ were sampled on the same day in February 1999. Overall infection prevalence at the BC site was 15.5%, while at the 31 Chapter Two - Infection prevalence LC site, prevalence -was 21.6%. The mean carapace length of uninfected lobsters captured at the BC site (37.5 ± 0.47 mm) was significantly greater than for lobsters at the LC site (28.6 ± 0.26 mm) (p < 0.001). Similarly, the mean carapace length of Hematodinium- infected lobsters captured at the BC site (36.2 ± 0.52 mm) was significantly greater than those from the LC site (28.1 ± 0.42mm) (p < 0.001). When the mean carapace lengths of uninfected and infected lobsters from the LC site were compared, it was found that the difference was not significant (p = 0.239). At the BC site, the mean carapace length of infected lobsters was also not significantly different from that of uninfected lobsters (p = 0.066). This suggests that the size distribution of infected lobsters capt ured at each site was not significantly different from the size distribution of uninfected lobsters captured at the same site (Fig. 4 ab). Hematodinium infection and the moulting period. 'nie relative proportion of recently moulted (RM) and intermoult (IM) X norvegicus in the catch gives an indication of the main moulting period and allows an assessment to be made of the synchrony of moulting amongst individuals in the population. Moult data from the LC site are shown in Figure 5a. Large numbers of RM lobsters appeared in the catch during the spring and summer of each year, but the relative proportion of RM lobsters appeared to vary between seasons (with a particularly high proportion of RM females during the summer of 1998). The reduction in the proportion of RM lobsters in the catch during late summer seems to be due to the progression of moulted lobsters into the IM state as the season advances. The fact that there is a higher proportion of RM female lobsters than RM male lobsters each spring and summer is suggestive of more synchronous moulting period in females than males, even though RM lobsters of both sexes are found in the catch throughout the year. 32 Chapter Two - Infection prevalence By combining data for moult status and infection prevalence and comparing the prevalence level in RM and IM lobsters, it is possible to investigate the relationship between moulting the onset of disease patency in each sex. Figure 5b shows infection prevalence data for RM and IM female lobsters at the LC site. In the 1998 season, prevalence was initially high in IM female lobsters, and increased in RM female lobsters as the season progressed. In the 1999 season, IM prevalence also rose initially, though the prevalence level in RM females did not increase to the level seen in the 1998 season. Finally, in the 2000 season, the prevalence level in IM and RM female lobsters was similar for the whole season. In contrast male lobsters displayed a similar pattern of IM and RM infection prevalence for each of the seasons studied (Fig. 5c), with RM infection prevalence being consistently higher than IM infection in the late season (April-May). In some seasons, data for overall infection prevalence at the LC site (Fig. 2a) show an apparent 'plateau' of peak infection prevalence between February and April-May (e. g. the 1999 season). When data from the 1999 season for RM and IM female lobsters are compared to those of RM and IM male lobsters, it can be seen that infection prevalence peaks later in males than in females and that the plateau of infection prevalence described above is caused by an increase in infection prevalence in RM males (Fig. 2a and 5c). During other seasons (e. g. 2000), when male and female infection prevalence peaks occur at the same time, a sharper peak of infection prevalence occurs (see dashed lines between Fig. 5b and 5c). Discussion. The body colour vs pleopod diagnostic methods. Tle body colour diagnostic method uses external features of infection (vivid colouration, opaque carapace) to estimate prevalence. It is rapid and can be carried out aboard research and fishing vessels. However, the current study has shown that it may underestimate 33 Chapter Two - Infection prevalence infection prevalence relative to that determined by the pleopod method, by approximately 50 %. No improvement in diagnostic accuracy occurred with experience, with both the naive and experienced operators failing to diagnose large numbers of early-stage (Stage 1) infections. '11iis suggests that while this method may be useful for the selection of heavily infected lobsters from the catch for pathological studies, the colour changes in lobsters with light infections are too minor to be used for accurate diagnosis. As such, the body colour method probably has little use in generating accurate data for modelling the proportion of natural mortality (" attributable to Hematodinium infection (Field et al., 1992). 'Me pleopod diagnosis method detected considerably more infected lobsters than the body colour method, but the higher prevalence estimate made by the nalve operator suggests that some subjectivity exists in this method, especially for the diagnosis of light infections. As the pleopod method determines the presence of Hematodinium parasites in the haemolymph, any other changes to the haemolymph which occur following capture arc likely to affect diagnostic accuracy. In the hours following capture, colonization of the haemolymph by bacteria was often observed, making visual assessment of light infections more difficult. Sample freshness is therefore important, especially for the diagnosis of light infections. However, overall, the pleopod diagnostic method provides a reliable, rapid and relatively transferable tool for infection assessment and is therefore useful for preliminary studies in N. norvegicus fisheries where the presence of Hematodinium infection is unconfirmed. Hematodinium infection epidemioloU. By use of the pleopod diagnostic method, the current study has highlighted the seasonal, epidemic nature of Hematodinium infection in a population of N. norvegicus contributing to the important commercial fishery in the Clyde Sea area and reinforces earlier reports of high Hematodinium infection prevalence at other sites on the west coast of Scotland (Field 34 Chapter Two - Infection prevalence et al., 1992,1998). During the study period, three discrete seasonal episodes of Hematodinium infection occurred (in each case, during the spring), in which overall prevalence reached 20-25 %. Previous data from the Clyde Sea area have shown that peak infection prevalence can be as high as 70 % in certain years (Field et al., 1992), highlighting the potential for considerable variation in the absolute level of infection prevalence between years. In the present study, infection prevalence was highest in female lobsters (especially during the 1998 and 2000 infection seasons), and this may be understood in terms of the life history of the female. In N. norvegicus, the egg-rearing period may last for up to 8 months of the year, during which time the female lobster remains for the most part, within the burrow (Farmer, 1974a). Following spawning, the female lobster emerges from the burrow (reflected by the increase in the proportion of female lobsters in the catch during late spring in this study) to feed, moult and be mated by a hard-shelled male lobster (Farmer, 1974b). It has been proposed that mechanical disruption to the soft cuticle of the female lobster during copulation may lead to infection by motile spores of the Hematodinium parasite entering at the trauma site (Field et al., 1992). However, although feasible, this would not explain transmission to male lobsters and such a route of transmission has not been demonstrated experimentally. During periods of food scarcity (e. g. in the extended time period spent within the burrow during incubation), it has been suggested that lobsters may resort to suspension feeding in order to supplement their normal nutritional requirements (Loo el al., 1993). In this way, it is possible that lobsters may ingest motile or encysted (see John & Reid, 1983) forms of the Hematodinium parasite which may exist in the sediment or in the burrow water. Similarly, the hermit crab (Pagurus bernhardus), which also harbours infections of Hematodinium on the Scottish N. norvegicus fishing grounds is known to resort to filter feeding when other food sources are scarce (Gerlach et al., 1976). Ingested parasite forms may penetrate the 35 Chapter Two - Infection prevalence gut wall and lie dormant in tissues such as the hepatopancreas until development of patent infection at some point in the future (Appleton & Vickerman, 1998). It seems likely that ingestion of spores via suspension or filter feeding, or of vegetative forms and/or spores by predation (of hermit crabs) or cannibalism are the most likely routes of transmission to and from N. norvegicus. Predation on amphipods which are also known to harbour Hematodinium infections (Johnson, 1986) is also a possible route of transmission to N. norvegicus and P. bernhardus, though studies of Hematodinium infection in Scottish amphipod species have not been made. Ile pleopod diagnosis method allows some assessment to be made of the absolute Hematodinium parasite burden in infected N. norvegicus. Field et al. (1992) showed that the majority of infected lobsters captured by trawling were of Stage I or 2 infection. However, the current study has shown that while mean infection severity in infected N. norvegicus is low during the summer months (mean approximately Stage 1), this increases as the infection season progresses (up to a mean of Stage 3). Progression of infection through the main season is suggestive of a slow incubation of the parasite in the haemolymph, and reinforces previous data which showed that the Hematodinium parasite burden increased by 100-fold over a period of 100 days in the haemolymph of aquarium- held infected lobsters (Appleton et al., 1997). The slow incubation of patent Hematodinium infection in the field may increase the chance of parasite transmission via cannibalism or predation, and occurs concomitantly with the progressive utilization of host tissue and haemolymph storage products (Stentiford et al., 1999 - Chapter 4; 2000a - Chapter 5). The final sporolation phase of the parasite, whereby parasite forms become motile and emerge from the host lobster (Appleton & Vickerman, 1998) probably occurs when all available host resources have been depleted (Stentiford el al. 2001b. in manuscript - Chapter 6) and may explain why sporolation of the parasite can occur in lobsters displaying only Stage I or 2 infection (Appleton & Vickerman, 1998). 36 Cbapter Two - Infection prevalence Hematodinium infection and the moult cycle. An association between patent Hematodinium infection and moulting has been described by a number of workers, though due to the inherent complexities of the moulting process, the details of this relationship remain unclear (Meyers et al., 1987; Field et al., 1992,1998; Messick, 1994). By assessment of shell condition, the current study has shown that in some years an extended infection season is caused by a high infection prevalence in RM male lobsters during the late season (April-May). In other years, where infection prevalence rises and falls more abruptly, prevalence in RM and IM males and RM and IM females coincides, creating a sharper infection peak. It is probable that in seasons of very high peak infection prevalence (> 70%), the infection prevalences in male and female lobsters peak at the same time. Ilerefore, in terms of absolute natural mortality (" attributable to Hematodinium infection, the length of the infection season may be as important as the absolute peak prevalence level Parasitism in relation to host condition. The general condition of the host may help to establish the degree to which parasitic infections are successful, with hosts in the best physiological condition harbouring the largest parasite burdens (Schmidt and Platzer, 1980). Female crustaceans have relatively larger amounts of hepatopancreatic tissue than mates as a means of supplying some of the nutritional requirements for egg rearing and spawning (Farmer, 1974b). The association of moulting with patent Hematodinium infection in N. norvegicus may thus be directly related to the nutritional status of the host and the nutritional requirements of the parasite. In decapods, the highest concentration of hepatopancreatic storage products (e. g. lipids and glycogen) are found in the pre-moult period (Johnson, 1980). During the final stages of the pre-moult and in the post-moult periods, storage material is mobilized (from tissue to haemolymph) to sustain the animal over the non-feeding stages of the life-cycle (Icely & 37 Chapter Two - Infection prevalence Nott, 1992). Recently, it has been shown that the plasma concentration of the crustacean hyperglycaemic hormone (CHH), a hormone involved in carbohydrate homeostasis in the tissues and haemolymph of crustaceans (Santos & Keller, 1993), is increased by up to 100- fold in the hours leading up to moulting, with levels returning to normal following ecdysis (Chung et al., 1999). In relation to this, a study of the carbohydrate and CHH dynamics of Hematodinium-infected lobsters has shown that the concentration of CHH is elevated significantly during sub-patent infection and that the increased concentration of CHH in the plasma at this time may create haemolymph conditions that are suitable for rapid growth of the parasite population (Stentiford et al. 2001b, in manuscript - Chapter 6). This may explain the coincidence of peak infection prevalence with the main moulting period for N norvegicus. Additionally, due to larger volumes of these storage tissues, the higher incidence of Hematodinium infection in female lobsters may reflect some relative advantage of the female host to the parasite. This benefit may be enhanced in smaller lobsters, which contain relatively larger amounts of hepatopancreatic material per unit size, store larger amounts of reserve material for moulting (Heath & Barnes, 1970), and in the case of males, moult more frequently than larger lobsters (Sarda, 1995, GonzAlez-Gurriardn et al., 1998). All of these features are likely to make them a better resource for Hematodinium parasite growth and transmission. The consistently higher peak in RM infection prevalence in male lobsters also suggests that, in males at least, infection may be carried through the moult. As such, lobsters with latent and low-level haemolymph infections may survive the moult, with severe infections developing after the moult, while those entering the moult with severe infections may perish during ecdysis. Further research on this complex issue is required to determine the link between Hematodinium infection and the moult. 38 Chapter Two - Infection prevalence Hematodinium infection and thefisheryfor N. nom , ýcus. A reduction in the mean carapace length of the N. norvegicus population on a particular fishing ground is one symptom of stock over-exploitation (Sarda, 1998). The data presented in this study suggest that the mean carapace length of lobsters has reduced at the LC site in the Clyde Sea area over the past 3 years. This reduction may be due to high recruitment at the same site over the same period (Marrs et al., 2000). The significant difference in mean carapace length between the BC and LC sites also suggests that the population structure of N. norvegicus may differ between geographically-adjacent sites. While Field et al., (1998) showed that the mean carapace lengths of Hematodinium infected N. norvegicus may differ between sites, they did not compare the mean size of the whole catch to the mean size of infected animals. The current study has shown that for two grounds with infected animals of different mean sizes, the mean size of infected lobsters was not significantly different to that of the whole sub-sample at either the LC or BC sites. However, the overall prevalence at the two sites was different, suggesting that population structure may play a part in the overall Hematodinium infection prevalence. Large populations of small, size-matched, rapidly growing individuals may lead to strong moulting synchrony amongst individual lobsters. As described above, it may be the populations with such characteristics that experience the highest Hematodinium infection prevalence each season. Further investigations on the effect of carapace size distributions on infection prevalence should be carried out to determine the importance of fishing pressure in the generation of population structures suitable for Hematodinium epidemics to occur. Due to the severe pathological effects associated with advanced Hematodinizim infections, the survival of infected lobsters under aquarium conditions (Field et al., 1992, Field & Appleton, 1995) and the sporolation response which leads to the death of the host lobster (Appleton & Vickerman, 1998), it is unlikely that recovery from patent infections is 39 Chapter Two - Infection prevalence possible. As such, seasons of high infection prevalence have been associated with reductions in landing per unit effort (LPUE) and burrow density (Field et al., 1998). Accurate prevalence estimates should allow a Hematodinium infection mortality factor (MH) to be incorporated into natural mortality (M, with this factor being greater during seasons in which infection prevalence is highest. Previous attempts to incorporate the high observed prevalence of Hematodinium infection into analytical stock assessments of the Clyde Sea area modelled the effect as an additional loading on the natural mortality rate M (since animals showing symptoms of patent infection usually die) (Anon, 1997). However, the exercise was not entirely successful, leading, as it did, to unrealistic estimates of recruits entering the fishery. Following this, the accuracy of infection prevalence estimates from trawl caught N. norvegicus samples has been questioned, due to the severely reduced escape swimming capacity (Stentiford et al., 2000b - Chapter 8) and the increased out-of-burrow activity (Stentiford et al. 2001a, in manuscript - Chapter 10) of infected lobsters. Increased catchability of infected lobsters relative to their uninfected counterparts may lead to considerable overestimation of true prevalence on a particular fishing ground. Furthermore, if infected animals are likely to be more susceptible to predation through a reduced ability to escape, then the infection may not necessarily add to overall natural mortality, but rather replace a proportion of it. Taken together, the altered catchability of patently infected lobsters and the subjectivity in detecting light infections raise doubts about the accuracy of the pleopod method for estimating patent Hematodinium infection prevalence in natural populations, and about the usefulness of these data for estimating natural mortality due to Hematodinizim infection. Development of sensitive molecular methods for the non- subjective detection of latent and sub-patently infected lobsters, which are less likely to show behavioural changes due to parasitism, should allow these issues to be resolved. 40 Chapter Two - Infection prevalence References. Aiken, D. E. (1980). Moulting and growth. In: The Biology and Management of Lobsters. (Eds. J. S. Cobb & B. F. Phillips). Academic Press, New York, pp. 91-163. Anon (1997). Report of the working group on Nephrops stocks. ICES CM 1997/Assess: 9 Appleton, P. L. & Vickerman, K. V. (1998). In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sP. ) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116,115-13 0. Appleton, P. L., Field, R. H., Vickerman, K., Atkinson, R. J. A., Taylor, A. C., Rogerson, A. & Neil, D. M. (1997). Mortality of Nephrops norvegicus on the west coast of Scotland. Report to MAFF CSG, March 1997. Atkinson, R. J. A. & Naylor, E. (1976). An endogenous rhythm and rhythmicity of catches of Nephrops norvegicus. Journal of Experimental Marine Biology & Ecology 25,95-108. Chapman, CT (1980). Ecology of juvenile and adult Nephrops. In: The Biology and Management of Lobsters. (Eds. J. S. Cobb & B. F. Phillips). Academic Press, New York, pp. 143-178. Chung, J. S., Dircksen, H. & Webster, S. G. (1999). A remarkable, precisely timed release of hyperglycernic hormone from endocrine cells in the gut is associated with ecdYsis in the crab Carcinus maenas. Proceedings of the National Academy of Science USA 96,13103- 13107. Farmer, A. S. D. (1974a). Burrowing behaviour of the Norway lobster, Nephrops norvegicus (L. ) (Decapoda: Nephropidae). Estuarine & Coastal Marine Science 2,49-5 8. Fanner, A. S. D. (1974b). Reproduction in Nephrops norvegicus (Decapoda: Nephropidae). Journal of Zoology 174,161-183. Field, R. H. & Appleton, P. L. (1995). A Hematodinium-like dinoflagellate infection of the Norway lobster Nephrops norvegicus: observations on pathology and progression of infection. Diseases of Aquatic Organisms 22,115-128. Field, R. H. & Appleton, P. L. (1996). An indirect fluorescent antibody technique for the diagnosis of Hematodinium sp. infection of the Norway lobster, Nephrops norvegicus. Diseases of Aquatic Organisms 24,199-204. Field, R. H., Chapman, C. J., Taylor, A. C., Neil, D. M. & Vickerman, K. (1992). Infection of the Norway lobster Nephrops norvegicus by a Hematodinium-like species of dinoflagellate on the west coast of Scotland. Diseases of Aquatic Organisms 13,1-15. Field, R. H., Hills, J. M., Atkinson, R. J. A., Magill, S. & Shanks, A. M. (1998). Distribution and seasonal prevalence of Hematodinium sp. infection of the Norway lobster (Nephrops norvegicus) around the west coast of Scotland. ICES Journal of Marine Science 55,846-858. 41 Chapter Two - Infection prevalence FRS (2000). Nephrops stocks around Scotland. Fisheries Research Service (FRS) fish stocks infonnation leaflet, Aberdeen, UK (unpublished). Gerlach, S. A., Ekstrom, D. K. & Eckardt, P. B. (1976). Filter feeding in the hermit crab Pagurus bernhardus. Occologia 24,25 7-264. Gonzdlez-Gurriardn, E., Friere, J., Farina, A. C. & Ferndndez, A. (1998). Growth at moult and intermoult period in the Norway lobster Nephrops norvegicus from Galician waters. ICES Journal of Marine Science 55,924-40. Heath, J. R. & Barnes, H. (1970). Some changes in biochemical composition with season and during the moulting cycle of the common shore crab, Carcinus maenas. Journal of Experimental Marine Biology & Ecology 5,199-233. Hudson, D. A. & Lester, RIG. (1994). A parasitological survey of the mud crab Scylla serrata (Forskal) from southern Moreton bay, Queensland, Australia. Aquaculture 120, 183-199. Hudson, D. A. & Shields, J. D. (1994). Hematodinium australis n. sp., a parasitic dinoflagellate of the sand crab Portunus pelagicus from Moreton bay, Australia. Diseases of Aquatic Organisms 19,109-119. Icely, J. D. & Nott, J. A. (1992). Digestion and absorption: digestive system and associated organs. In: Microscopic anatomy of Invertebrates Vol. 10. (Eds. F. W. Harrison, and A. G. Humes). Wiley-Liss, New York, pp. 147-20 1. John, A. W. G. & Reid, P. C. (1983). Possible resting cysts of Dissodinium pseudolunuld Swift ex elbrdchter et drebes; in the northeast Atlantic and the North sea. British Phycological Journal 18,61-67. Johnson, P. T. (1980). The gut. In: Histology of the blue crab, Callinectes sapidus. A model for the Decapoda. Praeger Scientific, New York. pp. 149-247. Johnson, P. T. (1986). Parasites of benthic amphipods: dinoflagellates (Duboscquodinida: Syndinidae). Fishery Bulletin 84,605-614. Kuris, A. M. & Lafferty, K. D. (1992). Modelling crustacean fisheries: effects of parasites on management strategies. Canadian Journal of Fisheries & Aquatic Science 49,327-336. Latrouite, D., Morizur, Y., Nodl, P., Chagot, D. Wilhelm, G. (1988). Mortalite du tourteau Cancer pagurus provoquee par le dinoflagelle parasite: Hematodinium sp. Conseil International pour I'Exploration de la Mer. CM. 1988/K: 32 Loo. L-O.. Pihl Baden. S. & Ulmestrand, M. (1993). Suspension feeding in adult Nephrops norvegicus (L. ) and Homarus gammanis (L. ) (Decapoda). Netherlands Journal of Sea Research 31,291-297. Maclean, S. A. (1978). Three new crustacean hosts for the parasitic dinoflagellate Hematodiniumperezi (Dinoflagellata: Syndinidae). Journal of Parasitology 64,158-160, 42 Chapter Two - Infection prevalence Marrs, S. J., Tuck, I. D., Ameri, E., Atkinson, R. J. A., Santojanni, A. & Stevenson, T. D. I. (2000). Improvement of Nephrops stock assessment by use of micro-scale mapping of effort and landings. Final Report to the European Commission. Reference 97/100, Study Project in support of the Common Fisheries Policy. 202pp +6 appendices. Messick, G. A. (1994). Hematodinium perezi infections in adult and juvenile blue crabs Callinectes sapidus from coastal bays of Maryland and Virginia, USA. Diseases of Aquatic Organisms 19,77-82. Meyers, T. R.. Botelho, C., Eaton, W., Koeneman, T. M., Short, S. & Imamura, K. (1990). Distribution of bitter crab dinoflagellate syndrome in southeast Alaskan tanner crabs, Chionoecetes bairdi. Diseases of Aquatic Organisms 9,37-43. Meyers, T. R., Koeneman, T. M., Botelho, C. & Short, S. (1987). Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan tanner crabs, Chionoecetes bairdi. Diseases of Aquatic Organisms 3,195-216. Newman, M. W. & Johnson, C. A. (1975). A disease of blue crabs (Callinectes sapidid) caused by a parasitic dinoflagellate, Hematodinium sp. Journal of Parasitology 63,554- 557. Santos, E. A. & Keller, R. (1993). Regulation of circulating levels of crustacean hyperglycaemic hormone: evidence for a dual feedback control system. Journal of Comparative Physiology 163B, 374-379. SarA F. (1995). A review (1967-1990) of some aspects of the life history of Nephrops norvegicus. ICES Marine Science Symposia 199,78-88. SardA, F. (1998). Symptoms of overexploitation in the stock of the Norway lobster (Nephrops norvegicus) on the "Serola bank" (western Mediterranean sea off Barcelona). Scientia Marine 62,295-299. Schmidt, S. P. & Platzer, E. G. (1980). Changes in body tissues and hemolymph composition of Culex pipiens in response to infection by Romanomerml .S cull . cl . vorax. Journal of Invertebrate Pathology 36,240-254. Shields, J. D. (1994). The parasitic dinoflagellates of marine crustaceans. Annual Review of Fish Diseases 4,241-271. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (1999). Changes in the plasma free amino acid profile of the Norway lobster, Nephrops norvegicus at different stages of infection by a parasitic dinoflagellates (genus Hematodinium). Diseases of Aquatic Organisms 3 8,15 1- 157. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (2000a). Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Nonvay lobster, Nephrops norvegicus L. during infection by a parasitic dinoflagellate of the genus Hematodinium. Diseases of Aquatic Organisms 42,133-14 1. Stentiford, G. D., Neil, D. M., Atkinson, R. J. A. & Bailey, N. (2000b). An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infected by a 43 Chapter Two - Infection prevalence parasitic dinoflagellates of the genus Hematodinium. Journal of Experimental Marine Biology & Ecology 274,169-18 1. Stentiford, G. D., Chang, E. S., Chang, S. A. & Neil, D. M. (2001b). Carbohydrate Dynamics and the Crustacean Hyperglycaemic Hormone (CHH): Effects of Parasitic Infection in Lobsters. General & Comparative Endocrinology (in manuscript). Stentiford, G. D., Neil, D. M. & Atkinson, R. J. A. (2001a). Alteration of burrow-oriented behaviour of the Norway lobster, Nephrops norvegicus (L) during infection by the parasitic dinoflagellate Hematodinium. Marine & Freshwater Behaviour & Physiology (in manuscript). Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The effects of Hematodinium sp. -infection on aspects of the respiratory physiology of the Norway lobster, Nephrops norvegicus (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228. Taylor, D. M. & Khan, R. A. (1995). Observations on the occurrence of Hematodinium sp. (Dinoflagellata: Syndinidae); the causative agent of Bitter crab disease in Newfoundland snow crab (Chionoecetes opilio). Journal of Invertebrate Pathology 65,283-288. Tuck, I. D., Chapman, CI, Atkinson, RIA., Bailey, N. & Smith, R. S. M. (1997). A comparison of methods for stock assessment of the Norway lobster, Nephrops norvegicus, in the Firth of Clyde. Fisheries Research 32,89-100. Wilhelm, G. & Boulo, V. (1988). Infection de 1'etrille Docarcinus puber (L. ) par un dinoflagelle parasite de type Hematodinium. ICES Committee Meeting CM. 1988/K: 32. Wilhelm, G. & Mialhe, E. (1996). Dinoflagellate infection associated with the decline of Necora puber crab populations in France. Diseases of Aquatic Organisms 26,213-219. 44 Chapter Two - Infection prevalence Fig. IA and B. (A) Percentage Hematodinium infection prevalence detected using the body colour and pleopod diagnostic methods by the nalve and experienced operators. (B) Number of lobsters diagnosed as infected by the pleopod method but not by the body colour method as a function of pleopod infection stage. C]cobur El pleopod F so ý 40 ý 30 20 ý 10 ý A Experienced 45 40 35 ý 30 .0 0 Z: 25 20 .0 r. ri 15 Z 10 5 o-I B I Scorer 2 Iifxtion Stap Narve Ei F-, penerred EI Naht 3&4 45 Chapter Two - Infection prevalence Fig. 2A, B and C. (A) Pleopod-derived Hematodinium infection prevalence, (B) proportion of males and females and (C) average pleopod-derived infection stage, in monthly samples of N. norvegicus captured from the Little Cumbrae (LQ site between February 1998 and September 2000. -o-Maie prevalence -. c)-- Female prevalence ,& Overall prevalence B im C 0 00 ww-3, Eý -w-ý ýý e g', ýý 2ý, e 2R gý & gý ýýýý?? 1 2e m jý, 1 -3 0' "' l' gA. ik%, _A i' eýäggý 'Z ee1- uý ýe--ý k) 3Z-4. >2 Capoze date Capare date 46 Chapter Two - Infection prevalence Fig. 3. Mean carapace length of monthly samples of N. norvegicus captured from the Little Cumbrae (LQ site between February 1998 and September 2000. --(ýFcmalc --c3- Male III 00 00 00 00 00 00 00 00 00 00 cy, CYN ce e eu 92. ý, = ýi 00 u. 'M, = ýo e, Lu ><ý'- <ý , e> 2A, 2, -- cn Capture date III a, 0, g? U< :5-, 3ý 47 Chapter Two - Infection prevalence Fig. 4A and B. Carapace length frequency distributions of X norvegicus from the Little Cumbrae (A) and Bute-Cumbrae channel (B) sites. Uninfected lobsters are shown as clear bars and infected lobsters as black bars. Bute/Cumbrac channel 16 , A (: I Uninfected M Infected 14 ý 12 10 8 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 South of Littlc Cumbrac 40 , B [3 Uninfected M Infected 35 ý 30 ý 25 20 is ý 20 22 24 26 29 30 32 34 36 39 40 42 44 46 48 50 52 Carapace length (mm) 48 Chapter Two - Infection prevalence Fig. 5A, B and C. (A) Proportions of recently moulted (RM) and intermoult (IM) male and female N. norvegicus. (B) Infection prevalence in RM and IM female and (C) male lobsters at the Little Cumbrae site between March 1998 and September 2000. Dashed lines on figures B and C compare RM and IM infection prevalence seasons in female and male lobsters (see main text). so 43 -43- RM nab , *, IM ntak ****** -. 49 Chapter Tliree - Immunoassay Diagnosis Chapter 3 Development and application of an immunoassay diagnostic technique for studying Hematodinium infections in Nephrops norvegicus populations. Abstract Patent Hematodinium infections of the Norway lobster, Nephrops norvegicus can be detected with a morphological method (the pleopod diagnosis), but this fails to identify low-level haemolymph (sub-patent) and tissue-based (latent) infections. The current study describes the development and application of an immunoassay for the detection of antigens of the parasite Hematodinium in the Norway lobster, Nephrops norvegicus. Infected tissue and haernolymph samples were detected as multiple-band reactions to a polyclonal antibody (anti- Hematodinium). By comparing haernolymph and tissues samples from the same lobsters, it was possible to distinguish latent from sub-patent infections. The sensitivity limit of the method was 204 parasites. mm3 - approximately ten times more sensitive than the pleopod diagnosis method. Use of the immunoassay on tissue samples taken from catches taken in the Clyde Sea area, Scotland, U. K. showed that the pleopod method considerably under-diagnosed infection prevalence in the early part of the season, though this under-diagnosis decreased as infected lobsters in the field progressed from latent and sub-patent to patent infections. However, the immunoassay failed to detect any infected lobsters during the summer months, suggesting that infection may not be carried over from one season to the next. Data are discussed in relation to the epidemiology of latent and sub-patent Hematodinium infections, to the transmission of parasites to other hosts and to the use of this immunoassay for the routine assessment of Hematodinium infection prevalence in N. norvegicus populations. Submitted as: Stentiford, G. D., Neil, D. M. & Coombs, G. H. (2001). Development and application of an immunoassay diagnostic technique for studying Hematodinium infections in Nephrops norvegicus populations. Diseases of Aquatic Organisms (submitted). 50 Chapter Three - Immunoassay Diagnosis Introduction The Norway lobster (Nephrops norvegicus) is the subject of an important fishery in the northeast Atlantic, with annual landings in excess of 60 000 t per annum making it one of the most valuable lobster resources in the world (Tuck et al., 1997). Populations of N. norvegicus range from Iceland and northern Norway to the Mediterranean sea and the Moroccan coast (Figueiredo & Thomas, 1967). An infection of N. norvegicus by the parasitic dinoflagellate Hematodinium was first described in Scottish populations by Field et al. (1992), and since this initial description Hematodinium infection has also been reported from populations in the Irish sea (McAliskey & Briggs, 1997), the German bight (Dr F. Redant, personal communication) and the Swedish Skaggerak and Kattegat (Mr M. Ulmestrand, personal communication). However, it remains to be determined whether this infection extends to N. norvegicus populations at more southerly locations along the coastline of mainland Europe and in the Mediterranean sea. External manifestations of Hematodinium infection of N. norvegicus include an opaque, vividly coloured carapace, general morbidity (Field & Appleton, 1995), a reduction in swimming performance (Stentiford et al. 2000a - Chapter 8) and altered burrow-related behaviour patterns (Stentiford et al. 2001a, in preparation - Chapter 10), while internal pathological effects include marked changes in host physiology (Taylor et al., 1996) and biochemistry (Stentiford et al., 1999 - Chapter 4,2000b - Chapter 5). These pathological effects are similar to those caused by Hematodinium infections of other crustacean host species (Meyers et al, 1987; Hudson & Shields, 1994; Messick, 1994; Hudson, 1995; Love et al., 1996; Shields & Squyars, 2000). A tentative life-cycle of the N. norvegicus isolate of Hematodinium has been described from in vitro cultivation and observations on aquarium- held lobsters (Appleton & Vickerman, 1998), although transmission to uninfected hosts has not been demonstrated to date. In addition, the seasonal nature of Hematodinium infection in N. norvegicus has been described in a number of studies (Field et al., 1992,1998) and 51 Chapter Ilree - Immunoassay Diagnosis has recently been examined in detail for a single fishing ground in the Clyde sea area, Scotland, UK (Stentiford et al. 200 1 b, in preparation - Chapter 2). A number of methods are available for the detection of Hematodinium infection in N. norvegicus. The simplest involves external assessment of the altered carapace colouration and opacity, which can be performed rapidly in the field. However, the insensitivity of this method, especially for detecting early infections, has recently been demonstrated (Stentiford et al., 2001b, in preparation - Chapter 2). The method which has been used most routinely is the pleopod staging technique of Field & Appleton (1995), whereby a pleopod is removed and assessed for the presence of parasites and hemocytes using low- power microscopy. This method assigns an infection severity score based on a four point scale, which allows the progression of infection to be followed over its seasonal cycle (Field et al., 1992,1998; Stentiford et al., 2001b, in preparation - Chapter 2) and has greatly facilitated laboratory studies on the pathological progression of infection (Stentiford et al., 1999 - Chapter 4,2000b - Chapter 5). It seemed probable, however, that the pleopod diagnosis method, which scores only patent infection, would fail to identify latent (tissue-based) and sub-patent (low level haemolymph parasitaemia) infections and so underestimate prevalence. Modem diagnostic methods based on molecular technology have increased the sensitivity of detection for many infective agents. Immunoassays, which detect parasite antigens, utilize specific antibodies produced against parasite antigens. These can be applied in assays based on Western blotting or the immunofluorescence antibody technique (IFAT) and in enzý, me-linked-immunosorbant-assays (ELISA). Such methods have facilitated the sensitive diagnosis of important pathogens of aquatic hosts. These include the detection of Taura syndrome virus (TSV) in penaeid shrimps (Poulos et al., 1999), Penaeus monodon- type baculovirus (MBV) in Penaeus monodon (Hsu et al., 2000), Renibacterium salmoninanim in Atlantic salmon (Salmo salar) (Griffiths et al., 1996), Sphaerospora 52 Chapter Three - Immunoassay Diagnosis dicentrarchl in the European sea bass (Dicentrarchus labrax) (Mufloz et al., 2000) and Cryptocaryon irritans in barrimundi (Lates calcarifier) (Bryant et al., 1999). An IFAT, developed using antibodies raised against in vitro cultured Hematodinium parasites originally isolated from X norvegicus, has revealed that some apparently uninfectcd N. norvegicus in fact harbour latent or sub-patent infections outside of the main infection season (Field & Appleton, 1996). However, the epidemiology of these low-level infections, and their progression towards patent infections (detectable using the pleopod method), were not undertaken. I'lie current study aimed to develop the IFAT technique of Field & Appleton (1996) into an immunoblotting assay, and apply the assay developed to analyze infection in a major Scottish N. norvegicus fishery over a 12 month period. Hepatopancreatic tissue was chosen as the study tissue as this has been suggested as a likely location of latent Hematodinium infection in N. norvegicus (Field & Appleton, 1996) and is well perfused with haemolymph, and so allows detection of sub-patent as well as latent infections. I'lie data obtained are discussed in relation to Hematodinium infection epidemiology in natural populations, the non-subjective monitoring of Hematodinium infection in other European populations, and the provision of accurate data on infection- associated mortality for use in stock assessment models. Methods. Capture of lobsters. Norway lobsters (Nephrops norvegicus) were caught using 90 min tows of a standard otter-trawl (70 mm mesh size) from a depth of approximately 80 m, at a location immediately south of Little Cumbrae (LQ in the Clyde sea area (55.41'N, 4.56M. T'he start of the towing period was always between 0900 h and 1000 h to minimise any effect of differential burrow emergence behaviour on catch composition (Atkinson & Naylor, 1976) and the effect of capture time on infection prevalence (Field et al., 1998). Monthly samples 53 Chaptcr Tlircc - Immunoassay Diagnosis were taken between October 1999 and September 2000 (inclusive) for the assessment of Hematodinium infection prevalence using the pleopod diagnosis method of Field & Appleton (1995). Bi-monthly samples of lobster tissue were also taken from sub-samples of the catch over the same period and tested for Heniatodinium infection using an immunoassay based on Western blotting for parasite antigens as detailed below. Tlie animals used were stored alive in a cool, damp environment (usually on ice) following capture for transfer to the laboratory for analysis. Pleopods were viewed using low power light microscopy (x 40 magnification). Tle accumulation of agglutinated parasite and haemocyte material was used to assign the infection stage according to Field & Appleton (1995). Stage 0 is apparently uninfected and Stages I to 4 are stages of increasingly severe patent infection. Tissue preparation and immunoassay conditions. Tissue samples from a standard subset of 60 lobsters (30 each of male and fcmalc), diagnosed as Stage 0 by the pleopod method, were prepared for immunoassay diagnosis. Small blocks (c. 2 mg) of hepatopancreatic tissue were excised, placed into 200 gl SDS- sample buffer (62.5 mM Tris-Hcl pH 6.8,12.5 % glycerol, 1.25 % P-mercaptocthanol) and immediately heated in a water bath at 95-100'C for 4 min. The denatured samples were then frozen at -20'C until analysis (usually within I week). Samples were defrosted immediately before analysis and 10 RI aliquots were subjected to discontinuous SDS-PAGE, performed according to the method of Laemmli (1970) with a 12.5 % acrylamide separating gel and a4% acrylamide stacking gel. Electrophoresis running conditions were as described by Neil et aL (1993). Following separation of proteins, gels were transferred to a nitrocellulose membrane (Amersham Life Science) in a buffer of 20 mM Tris, 150 mM glycine and 20 % (v/v) methanol at 100 V for 45 min 54 Chaptcr TVee - Immunoassay Diagnosis (BioRad mini-transblotter). Membranes were blocked overnight at 4*C in 5% (w/v) milk powder in Tris-buffered saline (TBS) (20 mM Tris base pH 7.6,137 mM sodium chloride, 3.8 % (v/v) HC1 with 0.1 % Twccn 20 and 0.1 % gelatin). After blocking, membranes were rinsed in TBS (pH 7.6) with 0.1 % Twcen 20 and 0.1 % gelatin, following which the primary rabbit antibody (anti-Hematodinium lysate - for production protocol see Field & Appleton, 1996) was applied for 4h at a dilution of 1/1000 in TBS (as above). Blots were then washed at room temperature five times for 10 rain each, with vigorous shaking. The secondary antibody (anti-rabbit-HRP conjugate) was then applied at a concentration of 1/1000 in TBS (as above) for Ih at room temperature and developed using chemi- luminescence reagents (Pierce Co. ) for 5 min and standard developer and fixer (Kodak Co. ) onto ECL film (Amcrsharn Life Science). Known Hematodinium-positive and uninfected haemolymph samples were used as controls. Hematodinium-positive samples were recognised by the presence of multiple-bands or smears due to reaction of numerous parasite proteins with the polyclonal primary antibody. The immunoassay-derived estimate of sub-patcnt/latent (SPL) infection was used to re-calculate the pleopod-derived prevalence estimate for Hematodinium infection in N. norvegicus, of both sexes, in the original sub-samples. To test the detection sensitivity of the western blotting method, Hemalodinium parasite cell density in the haemolymph of an infected lobster was determined microscopically. Haemolymph was withdrawn from the base of the fifth pereiopod and then transferred into an Improved Neubauer counting chamber viewed with a digital video camera (JVC TK- C 13 8 1) through a standard light microscope. Digital images were captured using Q-Video ImageLabTM software and a count of parasite cells was made later according to the method of Baker et al. (1966). Samples of the same haemolymph were diluted to 10 % in SDS- sample buffer, serially two-fold diluted, and subjected to SDS-PAGE and Western blotting 55 Chapter Three - Immunoassay Diagnosis as described above. Tlie sensitivity of the method was defined as the minimal number of parasites that could be detected by the Western blotting technique. To determine whether the Western blotting method could be used to distinguish between latent (parasites only in tissues) and sub-patent (low level haemolymph parasitacmia) Hematodinium infections, haemolymph and hepatopancreas samples from a small number of animals scored as Stage 0 by the pleopod method were run alongside one another. Detection of antigens in the hcpatopancrcas material only was taken to indicate a latent infection, whereas detection of antigens in the hepatopancreas and in the haemolymph was taken to indicate a sub-patent infection. Results. Severity of infection through the year. As well as allowing Hematodinium infection prevalence to be assessed at the population level, the plcopod staging method allows assessment of infection severity within individual lobsters (Stages of infection I to 4). Ile data presented in Figure I show the percentage of infected lobsters at the different stages of infection over the whole study period. In the early season (October and November), all infected lobsters captured displayed only very light infections (Stage 1). However, as the season progressed an increasing proportion of the infected lobsters captured were more severely infected (Stages 2 and 34) so that by June and July all infected lobsters captured were severely infected. These data suggest that Hematodinium infection severity is progressive in individual lobsters. Peak infection prevalence occurred in April. Sensitivity of the Western blot method. The use of serial dilutions of infected haemolymph, containing a known density of parasites, allowed the sensitivity of the immunoassay to be determined. The sensitivity 56 Chaptcr Threc - Immunoassay Diagnosis limit was 204 parasites. mm3 of haemolymph (lane D, Fig. 2). When a greater number of parasites were present, numerous bands were detected with the polyclonal anti- Ilematodinium antibody (lancs B and Q, but with fewer parasites no bands were detected. There was also reaction to the 66 kDa protein (bovine serum albumin) and two other proteins (molecular masscs of > 200 kDa and c. 97 kDa) in the molecular weight (M, ) markcr protein mixture (lane A). By applying the same method to excised hepatopancreas samples from field caught lobsters, previously diagnosed as Stage 0 by the pleopod method, sub-patcnt and/or latent infections were detected. Here, infected samples appeared as heavy, multiple-band reactions (see lobster sample numbers. 29,33,36,43 and 44 in Fig. 3), while samples of apparcntly uninfected lobsters showed no reaction to the anti-Hematodinium antibody. Incorporation ofimmunoassay data into pleopodprevalence analysis. lmmunoassay-derivcd infection prevalence data for SPL infections were used to re- calculate p1copod-derivcd infection prevalence estimates for the corresponding month. The immunoassay allowed for much earlier detection of infection in female lobsters (October) than was possible with the pleopod method (first detected in December) (Fig. 4A). By December, the immunoassay-dcrivcd estimates showed that infection prevalence was dramatically greater (over 25 %) than the pleopod analysis had suggested (less than 5 %). A similar situation was revealed with the February samples. By April ('peak season' from pleopod diagnosis -> 30 % infection), there were fewer SPL infections. By June, pleopod prevalence had reduced to approximately 5 %, and there were no SPL infections. This suggests that following the peak of patent infection in April, there were either no new Hematodinium infections or ncwly-infected animals had parasite burdens below the limit of detection with the immunoassay method (Fig. 4A). 57 Chapter Tliree - Immunoassay Diagnosis A similar pattern in the immunoassay and pleopod-derived infection prevalence estimates was also seen for male lobsters (Fig. 413), though patent infections appeared earlier (October) and overall prevalence at the peak of infection (April) was approximately half that of females. When the data for immunoassay and pleopod-derived infection prevalence were plotted separately, it was possible to examine the epidemiological nature of SPL and patent Hematodinium infection in N. norvegicus. In females, the SPL prevalence rose to a peak in February and then began to fall (suggesting that all animals showing patent infection within a given season were infected by February). Following this peak, SPL infections advanced to a patent state, with this peaking in April. As shown in Figures 4A and 413, when patent infection prevalence was at its highest (April) the prevalence of SPL infection had reduced to very low levels. The decline in SPL infection to zero in June suggests that infections may not have been carried over from one season to the next, but were instead acquired at some point after late summer (shown by the low level of sub-patent infection in October) (Fig. 5A). SPL and patent infection in male lobsters showed a similar epidemiological pattern, though here, the overall prevalence was considerably lower than in female lobsters (Fig. 513). The time period between a given level of SPL infection prevalence and the same level of patent infection prevalence (shown by the 'phase-shifted' nature of the prevalence peaks), may indicate the approximate incubation time of the Hematodinium parasite from the SPL to patent state. Data presented here suggest that this time period may be between 2 and 4 months. It is important to note that the prevalence of SPL infection was approximately equal to the prevalence of patent infection that followed. 'I'llis suggests that lobsters carrying a sub-patent or latent infection will display the symptoms of patent infection within the same season (Fig. 5a and 5b). r% Chapter Tbree - Immunoassay Diagnosis Discriminating between latent and sub-patent infections. Detection of parasite antigens in the tissue and not in the haemolymph is suggestive of a latent Hematodinium infection, while presence of antigens within the tissues and haemolymph of Stage 0 animals (by the pleopod method) is suggestive of a sub-patent infection. By assaying the haemolymph and hepatopancreas of Stage 0 animals, it was shown that the Western blot method was able to discriminate between latently (lanes D' and D2, Fig. 6) and sub-patently (lanes B1 and B2, and C' and C2 , Fig. 6) infected lobsters. it was found that there were differences in the degree of antigen reactivity in the haemolymph of sub-patently infected lobsters (compare lanes B2 and C2 , Fig. 6). This is consistent with there being progressive colonization of the haemolymph by the parasites subsequent to initial invasion from the tissues. Discussion. in the current study, further development of the TAT described by Field & Appleton (1996) into a multi-sample immunoblotting assay has allowed for the detection of SPL Hematodinium infections in field-caught samples of N. norvegicus. The Western blotting assay detected parasite densities of 204 parasites. mm' and above. This method is therefore approximately ten times more sensitive than the pleopod diagnostic method, in which Stage I infected animals have burdens of over 2000 parasites MM3 (see Field et al., 1992. Field & Appleton, 1995). The technique worked well for both diluted haemolymph and for tissue samples, with there being no reaction of the polyclonal antibody to N. norvegicus material. Reactions between the anti-Hematodinium antibody and the proteins in the M, marker probably reflect the presence of antibody against components of the parasite's culture medium (which included fetal calf serum) which would have been present on the surface of injected Hematodinium parasites during the initial production of the antibody (see Field & Appleton, 1996). Co Chapter Three - Immunoassay Diagnosis Latent Hematodinium infections of N. norvegicus are thought to involve parasites residing in tissues such as the hepatopancreas and muscle (see Appleton & Vickerman, 1998), while in low-level sub-patent infections parasites are also present in the haemolymph (Field & Appleton, 1996). As the main purpose of the immunoassay in the current studies was to detect all infections (latent and sub-patent) not detectable by the pleopod staging method, hepatopancreas samples were used. Immunoblotting of these did not distinguish between latent and sub-patent infections. Hence no distinction was made between sub-patent and latent infection states for immunoassay-derived prevalence calculations. 'Me data showed that the pleopod diagnostic method considerably under-estimates the prevalence of Hematodinium infection during the early season, but also that the degree of under-estimation is reduced as the main infection season (February to April) progresses. This reduction is likely to be due to the development of latent and sub-patent and latent infections into pleopod-detectable patent infections. The similarity in the amplitude of the SPL (immunoassay-derived) and patent (pleopod-derived) infection peaks suggests that all animals with a SPL infection within a given season will also express a patent infection within the same season. In addition, data for average patent infection severity (derived from monthly pleopod diagnosis) showed that a steady increase in infection severity occurred as the season progressed. As such, most infected animals captured at the end of the season had considerably larger parasite burdens than those captured at the start of the season. Data for infection severity progression is suggestive of a relatively slow incubation of the parasite in the haemolymph, and reinforces previous data which showed that the Hematodinium parasite burden increased by 100-fold over a period of 100 days in the haemolymph of aquarium-held infected lobsters (Appleton et al., 1997). The slow incubation of patent Hematodinium infection in the field may increase the chance of parasite transmission via cannibalism or predation. The increase in parasite number 60 Chapter Three - Immunoassay Diagnosis correlates well with the progressive utilization of host tissue and haemolymph storage products (Stentiford et al., 1999 - Chapter 4,2000b - Chapter 5). Most studies on Hematodinium infections of decapod crustaceans have proposed a long, asymptornatic latency, followed by a shorter period of parasite proliferation and spore release (Hudson & Shields, 1994). Colonization of a new host may either occur shortly after ecdysis or parasites may remain in the host through the moult (Shields, 1992). If infection is acquired within one season and remains in a latent state until the following season, it should be possible to detect the same level of latent infection prevalence out of season as patent infection in the following season. In the current study, the increasing SPL prevalence up to February, suggests that either acquisition of infection occurs within the same season that full patency develops, or that the immunoblotting technique failed to diagnose very low burdens of parasites outside of the main infection season. Similarly, the reduction in the prevalence of sub-patent infections to zero in June suggests that either Stage 0 animals at this time are completely free from infection, or that very low-level latent infections are undetectable using this assay. Tle comparison of tissue and haemolymph samples from the same lobster showed that latent infections could be distinguished from sub-patent infections using the immunoblotting technique. However, it may be that the parasite burden within the tissues of latently infected lobsters also differs significantly between individuals and over time. Lobsters infected with very low tissue-based parasite burdens (below 204 parasites MM3) would not be detectable with the current immunoassay. Alternatively, upon initial infection, the Hematodinium parasite may reside in different tissues or organs to those tested (see Field & Appleton, 1995,1996). If acquisition of infection does occur within the same season as patent infection is expressed, transmission is unlikely to be occurring via lesions in the cuticle of newly moulted lobsters as the main moulting season for N. norvegicus is during the spring and early summer (Farmer, 1974). In this case, acquisition of parasites is more likely to be 61 Chapter Tlree - Immunoassay Diagnosis occurring by the direct ingestion of vegetative stages or spores of the Hematodinium parasite (Appleton & Vickerman, 1998; Stentiford et al., 2001b, in manuscript - Chapter 2). Sensitive diagnosis of Hematodinium infection by the use of the polymerase chain reaction (PCR) has been demonstrated (Hudson & Adlard, 1994,1996). However, no follow-up studies over longer periods have been carried out. Utilization of a sensitive PCR method for the detection of very low parasite burdens is required if issues relating to the latency and transmission of the Hematodinium parasite in N. norvegicus are to be resolved. In order to make reliable comparisons of Hematodinium infection prevalence between different European N. norvegicus fisheries, the diagnostic method must be standardized and reproducible in the hands of different operators. While the pleopod diagnostic method has some merit for estimating Hematodinium-associated mortality in N. norvegicus. the insensitivity of the method, in addition to the subjectivity of assessment between different scorers (Stentiford et al., 2001b, in preparation - Chapter 2), suggests that the pre-season measurement of SPL infections (by immunoassay) may provide an alternative means of estimating Hematodinium infection prevalence in the field. Additionally, due to the phase- shifted nature of the immunoassay-derived prevalence peak from the pleopod-derived prevalence peak (by a period of 24 months), it is likely that immunoassay-derived prevalence data could be used as an early warning signal for the level of patent infection level that will appear in the fishery. As such, the immunoblotting technique described here has furthered our knowledge on the epidemiology of Hematodinium infection in N. norvegicus and the development of this into a rapid, non-subjective enzyme-linked- immunosorbant-assay (ELISA) is being pursued. 62 Chaptcr Three - Immunoassay Diagnosis References. Appleton, P. L. & Vickerman, K. V. (1998). In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sp. ) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116,115-130. Appleton, P. L., Field, R. H., Vickerman, K., Atkinson, RIA., Taylor, A. C., Rogerson, A. & Neil, D. M. (1997). Mortality of Nephrops norvegicus on the west coast of Scotland. Report to MAFF CSG, March 1997. Atkinson, R. J. A. & Naylor, E. (1976). An endogenous rhythm and rhythmicity of catches of Nephrops norvegicus. Journal of Experimental Marine Biology & Ecology 25,95-108. Baker, F. J., Silverton, R-E. & Luckock, E. D. (1966). Introduction to medical laboratory technology (4h edition). Butterworth & Co Ltd., London. pp. 508-523. Bryant, M. S., Lee, R. P., Lester, RTG. & Whittington, R. J. (1999). Anti-immunoglobulin antisera, used in ELISA to detect antibodies in barrarnundi Lates calcarifer to Cryptocaryon irritans. Diseases of Aquatic Organisms 36,21-28. Fanner, A. S. D. (1974). Reproduction in Nephrops norvegicus (Decapoda: Nephropidae). Journal of Zoology 174,161-183. Field, R. H. & Appleton, P. L. (1995). A Hematodinium-like dinoflagellate infection of the Nonvay lobster Nephrops norvegicus: observations on pathology and progression of infection. Diseases of Aquatic Organisms 22,115-128. Field, R. H. & Appleton, P. L. (1996). An indirect fluorescent antibody technique for the diagnosis of Hematodinium sp. infection of the Norway lobster, Nephrops norvegicus. Diseases of Aquatic Organisms 24,199-204. Field, R. H., Chapman, C. J., Taylor, A. C., Neil, D. M. & Vickerman, K. (1992). Infection of the Norway lobster Nephrops norvegicus by a Hematodinium-like species of dinoflagel late on the west coast of Scotland. Diseases of Aquatic Organisms 13,1-15. Field. R. H.. Hills, J. M.. Atkinson, R. J. A., Magill, S. & Shanks, A. M. (1998). Distribution and seasonal prevalence of Hematodinium sp. infection of the Norway lobster (Nephrops norvegicus) around the west coast of Scotland. ICES Journal of Marine Science 55,846- 858. Figueiredo, M. J. & Thomas, H. J. (1967). Nephrops norvegicus (Linnaeus, 1758) Leach -a review. Oceanography & Marine Biology, Annual Review 5,371407. Griffiths, S. G., Liska, K. & Lynch, W. H. (1996). Comparison of kidney tissue and ovarian fluid from broodstock Atlantic salmon for detection of Renibacterium salmoninarum, and use of SKDM broth culture and Western blotting to increase detection in ovarian fluid. Diseases of Aquatic Organisms 24,3-9. Hsu, L. U., Wang, K. H., Yang, Y. H., Tung, M. C., Hu, C. H., Lo, C. F., Wang, C. H. & Tsu, T. (2000). Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies. Diseases of Aquatic Organsisms 40,93-99. 63 Chapter Three - Immunoassay Diagnosis Hudson, D. A. & Adlard, R. D. (1994). PCR-techniques applied to Hematodinium spp. and Hematodinium-like dinoflagellates in decapod crustaceans. Diseases of Aquatic Organisms 20,203-206. Hudson, D. A. & Adlard, R. D. (1996). Nucleotide sequence determination of the partial SSU rDNA gene and ITSI region of Hematodinium cf. perezi and Hematodinhim-like dinoflagellates. Diseases of Aquatic Organisms 24,55-60. Hudson, D. A. & Shields, J. D. (1994). Hematodinium australis n. sp., a parasitic dinoflagellate of the sand crab Portunus pelagicus from Moreton bay, Australia. Diseases of Aquatic Organisms 19,109-119. Hudson, D. A. (1995). Biochemical parameters of the serum of the sand crab, Portunits pelagicus, with reference to the parasitic dinoflagellate, Hematodinium australis. Bulletin of the European Journal of Fish Pathologists 15,202-205. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 227,680-5 Love, D., Thomas, R. & Moles, A. (1996). Bitter crab hemolymph studies: Indicators of host physiological condition. Alaska Sea Grant College Program. AK-SG-96-02. McAliskey, M. & Briggs, R. P. (1997). Hematodinium in Irish sea Nephrops. ICES Committee meeting, Baltimore, USA. CM-1997AHH: 25. Messick, G. A. (1994). Hematodinium perezi infections in adult and juvenile blue crabs Callinectes sapidus from coastal bays of Maryland and Virginia, USA. Diseases of Aquatic Organisms 19,77-82. Meyers, T. R., Koeneman, T. M., Botelho, C. & Short, S. (1987). Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan tanner crabs, Chionoecetes bairdi. Diseases of Aquatic Organisms 3,195-216. Muftoz, P., Sitji-Bobadilla, A. & Alvarez-Pellitero, P. (2000). Antigenic characterization of Sphaerospora dicentrarchi (Myxosporea: Bivalvulida), a parasite from European sea bass Dicentrarchus labrax (Teleostei: Serranidae). Diseases of Aquatic Organisms 40,117- 124. Neil, D. M., Fowler, W. S. & Tobasnick, G. (1993). Myofibrillar protein composition correlates with histochemistry in fibres of the abdominal flexor muscles of the Norway lobster Nephrops norvegicus. Journal of Experimental Biology 183,185-20 1. Poulos, B. T., Kibler, R., Bradley-Dunlop, D., Mohney, L. L. & Lightner, D. V. (1999). Production and use of antibodies for the detection of Taura syndrome virus in penaeid shrimp. Diseases of Aquatic Organisms 37,99-106. Shields, J. D. (1992). Parasites and symbionts of the crab Portunus pelagicus from Moreton bay, eastern Australia. Journal of Crustacean Biology 12,94-100. Chapter Threc - Immunoassay Diagnosis Shields, J. D. & Squyars, C. M. (2000). Mortality and hematology of blue crabs, Callinectes sapidus, experimentally infected with the parasitic dinoflagellate Hematodinium perezi. Fishery Bulletin 98,139-152. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (1999). Changes in the plasma free amino acid profile of the Norway lobster, Nephrops norvegicus at different stages of infection by a parasitic dinoflagellates (genus Hematodinium). Diseases of Aquatic Organisms 38,15 1. 157. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (2000b). Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Norway lobster, Nephrops norvegicus L. during infection by a parasitic dinoflagellate of the genus Hematodinium. Diseases of Aquatic Organisms (in press). Stentiford, G. D., Neil, D. M., Atkinson, R. J. A. & Bailey, N. (2000a). An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infected by a parasitic dinoflagellates of the genus Hematodinium. Journal of Experimental Marine Biology & Ecology 274,169-18 1. Stentiford, G. D., Neil, D. M. & Atkinson, RTA. (2001a). Alteration of burrow-related behaviour of the Norway lobster, Nephrops norvegicus (L. ) During infection by the parasitic dinoflagellate Hematodinium. Marine and Freshwater Behaviour & Physiology (submitted). Stentiford, G. D., Neil, D. M. & Atkinson, R. J. A. (2001b). The relationship of Hematodinium infection prevalence in a Scottish Nephrops norvegicus population to seasonality, moulting and sex. ICES Journal of Marine Science (submitted). Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The effects of Hematodinium sp. -infection on aspects of the respiratory physiology of the Norway lobster, Nephrops norvegicus (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228, Tuck, I. D., Chapman, CI, Atkinson, RIA., Bailey, N. & Smith, R. S. M. (1997). A comparison of methods for stock assessment of the Norway lobster, Nephrops norvegicus, in the Firth of Clyde. Fisheries Research 32,89-100. 65 Chapter Three - himinnoassay Diagnosis Fig. 1. Percentages of Hematodinium-infected N. norvegicus at each infection stage, as judged by the pleopod staging method, in monthly samples from the Little Cumbrae site between October 1999 and September 2000. Overall monthly infection prevalence (%) in the catch is given above each bar. L5 0.8 15 5.6 9.0 162 19.9 12,0 111o () 3() 0,80/1) 100"o , 90% 8000 7M. 1 9, -, I UUO U 40% -0 3 Mo 20% 100/0 U-0 I I I Oct-99 Nov-99 IXc-99 Jan-00 Feb-00 Mar-00 Apr-00 Mav-00 Jtiil--()() JuýOo ALIV-00 Sep-00 Capture date Z Stage 3&4 EI S tage 2 EI Stage 1 66 Chapter Three - Immunoassay Diagnosis Fig. 2. (Top) 12.5 % SDS-PAGE gel of serially two-fold diluted-plasma from a Hematodinhim-infected N. norvegicus. (Bottom) Western blot of the same serial ly-di lutcd infected plasma showing reaction of the separated proteins with anti-Hemolodinium primary antibody in lanes B (815 parasites. mm), C (408 parasites. rnm) and D (204 parasites. mm'). Lack of reaction in lanes E to H (< 204 parasites. mm') show that parasite proteins present were at concentrations below that detectable with the assay Procedure. Note the reaction of the primary antibody proteins in the molecular weight marker in lane A. Numbers on left of gel rcfcr to approximate relative molecular weight. Hey = plasma hacniocyanin sub-units. AB CD EFGH 67 Chapter Three - hninunoassay Diagnosis Fig. 3. Western blot of hepatopancreas samples from a typical sub-sample of pleopod- diagnosed Stage 0 lobsters. Note the multi-band or smear reaction of parasite proteins to the primary antibody in lanes 29,33,36,43 and 44. Reaction of the primarv antibodv to proteins in the molecular weight marker mixture (HMW) also occurred (see also Fig. 2). A B HMW 29 30 31 iow:. 32 33 34 35 HMW 37 38 39 40 41 42 43 36 44 68 Chapter Three - Immunoassay Diagnosis Fig. 4. Immunoassay-corrected Hematodinium infection prevalence estimates for female (A) and male (B) N. norvegicus from the Little Cumbrae site. Immunoassay data (grey bars) were collected bi-monthly (in October and December 1999 and February, April, June and August 2000). Pleopod data (white bars) were collected monthly. 45 40 ý 35 1 30 25 20 is 10 5 0 45 40 35 30 25 20 15 10 5 A- Feniale Nephrops G Sub-patentlated irLfectiDn (3 Patcnt i&ction 0 El ,, Oct-99 Now99 Dec-99 Jan-00 Fcb-00 Mar6OO Apr6OO MarOO Jun-00 JuWO Aug-00 Sep-00 A- Male Nephrops 13 Sub-patcnvlatcnt infectim 13 Patent infectim Oct-99 Nov-99 Dec-99 Jan-00 Feb-00 Mar-00 Apr6OO May-00 Jun-oo J&OO Aug-00 Scp-oo Capture date 69 Chapter Tlirce - Immunoassay Diagnosis Fig. 5. Immunoassay-derived (sub-patent/latent) and pleopod-derived (patent) Hematodinium infection prevalence for female (A) and male (B) X norvegicus. Immunoassay samples were taken bi-monthly (see Fig. 4A and B). Note the phase-shifted nature of the immunoassay-derived and pleopod-derived prevalence curves and the higher level of infection prevalence in female lobsters. - Patcnt iifýction Oct-99 Nov-99 Dec-99 Jan-00 Feb-00 Mar-00 Apr-00 May-00 Jusv-00 JuW Aug-00 Sep-00 Capture date Oct-99 Nov-99 Dcc-99 Jan-00 Feb-00 Mar-00 Apr-00 May-00 Jun-00 J&OO Aug-00 Sep-00 Capture date 70 Chapter Three - Immunoassay Diagnosis Fig. 6. Western blot of hepatopancreas (lanes B1, C' and D') and haemolymph (lanes B2, C2 and D 2) samples from lobsters diagnosed as Stage 0 by the pleopod method. Note the detection of parasite material in the haemolymph and hepatopancreas in samples B and C (suggesting sub-patent infections) and the detection of parasite material in only the hepatopancreas of sample D (suggesting a latent infection). Lane A= reaction of relative molecular weight marker proteins to the primary antibody (see Fig. 2). Bl B2 cl c2 D' D2 71 Chapter 4- Plasma Amino Acids Chapter 4 Changes in the plasma free amino acid profile of the Norway Lobster, Nephrops norvegicus at different stages of infection by a parasitic dinonagellate (Genus Hematodinium). Abstract. The haemolymph free amino acid (FAA) composition of Norway lobsters, Nephrops norvegicus, from the Clyde estuary, Scotland UK, at different stages of infection by the parasitic dinoflagellate Hematodinium sp. was determined by reverse phase High performance liquid chromatography (HPLQ. In uninfected animals the total detected haemolymph FAA concentration was 3.79 pmol. ml-1. Collectively, glycine, histidine/glutamine, alanine, serine, taurine and asparagine constituting 80% of the total. The concentrations of several FAAs, notably serine, were reduced in early infections (Stage 1), while at later Stages (24) several FAAs were increased in concentration. The most significant change was in taurine, which was increased by 13 fold (from 0.22 to 2.56 ýtmol ml-') in the final infection stage, when its relative contribution to the total FAA was 41.6%. Possible causes of these changes in the lobster haemolymph FAAs are the breakdown of host tissues including the hepatopancreas and muscle, haemocýle lysis, a host stress response and release of FAAs from the parasite cells. These results have implications for the biology of Nephrops, and indicate that the taurine: serine ratio in the haemolymph provides a sensitive diagnostic measure of patent Hematodinium infections. Published as: Stentiford, G. D., Neil D. M. &Coombs, G. H. (1999). Changes in the plasma free amino acid profile of the Norway Lobster, Nephrops norvegicus at different stages of infection by a parasitic dinoflagellate (Genus Hematodinium). Diseases of Aquatic Organisms 38,151-157. 72 Chapter 4- Plasma Amino Acids Introduction. One consequence of some parasitic infections of invertebrates is change to the free amino acid (FAA) profile in the plasma. Ibis occurs in oysters infected by Bucephalus sp. and Minchinia nelsoni (Feng et al., 1970), honey bees infected by the microsporidian Nosema apis (Wang & Moeller, 1970), and mosquitoes infected with Nosema algerae (Henn et al., 1998). 'Mere have been very few such studies on parasitized crustaceans, although alterations to the plasma FAA profile have been reported for the bacterium Acrococcus viridans infection in lobsters (Stewart, 1980) and the microsporidia Thelohania maenadis and Ameson michaelis infections in crabs (Vivares et al., 1980; Findley et al., 198 1). Populations of the Norway lobster (Nephrops norvegicus) from the coastal waters off the west coast of Scotland are infected with the dinoflagellate Hematodinium sp., with overall prevalence levels of over 70% in some years (Field et al., 1992). The parasite is systemic, invading most major tissues and organs, causes considerable histological disruption (Field & Appleton, 1995) and has a major effect on the appearance and properties of the haemolymph, including extensive loss of normal haemocytes (Taylor et al., 1996). However, there are no reports on the biochemical consequences of the infection. Nephrops is typical of marine crustaceans in having a large intracellular FAA pool (Robertson, 1961, Robertson et al., 1992), while extracellular concentrations are low. With such high tissue: haemolymph FAA ratios, the extensive pathological effects of Hematodinium infection on the tissues and organs of Nephrops may be expected to cause increases in the haemolymph concentrations of amino acids when released from damaged cells There may also be compensation for the parasite-induced changes to the haemolymph. Therefore this project -was undertaken to determine the haemolymph FAA composition during infection of Nephrops by Hematodinium, to elucidate the extent of such changes, to chart their time course in relation to the onset and progress of the infection, and to determine whether the host undergoes compensatory changes in its plasma FAA profile. 73 Chapter 4- Plasma Amino Acids Methods. Collection and treatment ofanimals. Norway lobsters (Nephrops norvegicus) were otter-trawled from a location south of Little Cumbrae in the Clyde Sea area, Scotland, UK, and subsequently maintained in a closed aquarium (IO'C, 33ppt salinity) in the Division of Environmental and Evolutionary Biology, University of Glasgow, Scotland, UK. Animals were fed ad libitum, once weekly on mussels (Mytilus edulis) but were not fed for at least five days prior to haemolymph collection. All animals were in the intermoult state (Aiken, 1980) and were assessed for Hematodinium infection by the pleopod staging method of Field & Appleton (1995). Sample collection. Haemolymph was drawn from the base of the fifth pereiopod into a sterile syringe and centrifuged immediately at 17,000 xg for 10 min. at IOT to remove cellular material and suspended debris. The clear plasma was deproteinized using ice cold methanol (four parts methanol to one part plasma) for 10 min. at 4'C. Protein precipitate was removed by centrifugation and the supernatant passed through a 0.22 m filter before storage at -70'C. High performance liquid chromatography (HPLQ analysis. HPLC grade methanol (MeOH), tetrahydrofuran (THF) and acetonitrile were obtained from BDH Laboratory Supplies, Leicestershire, UK. Crystalline amino acids (aspartate, glutamate, asparagine, serine, histidine, glutarnine, glycine, threonine, arginine, alanine, tyrosine, valine, methionine, tryptophan. phenylalanine, isoleucine and leucine). taurine. sodium acetate, mercaptoethanol (ME) and o-phthaldialdehyde (OPT) were from Sigma-Aldrich Co. (Poole, UK), Reagent grade Millipore-filtered water was used throughout. Deproteinized plasma samples were analysed by HPLC using a Rainin Dyn Microsorb C18 4.6 x 150 mm column (with Microsorb C18 guard column), a Gilson HPLC system (model 302 74 Chapter 4- Plasma Amino Acids Pumps, model 802C manometric module, model 811 dynamic solvent mixer and model 121 fluorometer) interfaced to a computer with 712 HPLC controller software. Amino acids were eluted after pre-column OPT-derivatization using the method of Zuo & Coombs (1995) by mixing equal volumes of plasma and OPT reagent. The method allowed for quantitation of 12 amino acids and taurine. Co-elution of valine/methionine and histidine/glutamine occurred in all runs and neither cysteine nor proline were detectable. Standard curves were constructed using an equimolar mixture of the above amino acids and taurine and plasma concentrations were derived from these curves. Comparison of FAA concentrations in the plasma of uninfected and infected Nephrops was performed either by one-way analysis of variance (ANOVA) for normally distributed data, or by a Kruskal-Wallace test for non-normal distributions. Between stage comparisons were made with a Tukey's pairwise analysis (normal distributions) and a Mann-Whitney test (non-normal distributions). Significance was considered to be at p<0.05. Results. Analysis of the FAA concentrations in Nephmpsplasma. A total of 81 lobsters were examined for infection by Hematodinium using the pleopod staging method of Field & Appleton (1995). Stage 0 denotes an uninfected animal, with patent infection progressing from Stage I to Stage 4. Plasma FAA concentrations (ýtmol. ml-) were derived from peak areas on the HPLC chromatograms. The OPT-derivatized FAA compounds identified in Nephrops plasma under different levels of infection are shown in Table 1. In uninfected Mephrops (Stage 0), the total detected FAA concentration was 3.79 ýLmol. ml-, with glycine, histidine/glutamine, alanine, serine, taurine and asparagine collectively constituting almost 80 % of this total. The total identified FAA concentration was highest in the plasma of Stage 3 animals (6.70 ptmol. ml"'), this being significantly greater than that of uninfected plasma (p < 0.001). 75 Chapter 4- Plasma Amino Acids The FAA profile of Hematodinium-infected Nephrops plasma was substantially altered through the course of the infection (Table 1). The concentrations of five FAA compounds were significantly less in the plasma of Stage I (early patent infection) animals compared with uninfected controls, resulting in a depression (though not significant) in the total concentration of detected FAA. Among the individual amino acids, the change of serine was the greatest, falling from 0.34 ýtmol. ml'l to 0.21 grnol. ml" (p < 0.001), while other FAA's showing reduced concentrations included asparagine (p < 0.05), valine-methionine (p < 0.01), tryptophan (p < 0.01) and isoleucine (p < 0.01). A different trend was apparent with lobsters at Stage 2 infection, in which several FAA's exhibited very significant increases in concentration. However, the most prominent increase was with taurine which increased from 0.22 gmol. ml" in stage 0 to 1.04 pmol. ml-1 in Stage 2 (p < 0.001). By Stage 3 of infection, taurine (at 2.20 ýtmol. ml-) was dominant in the plasma, and glutamate had also increased significantly from its Stage 2 concentration (p < 0.0 1). In contrast, serine, after its initial reduction, remained low in the later stages of infection and did not change significantly among successive levels of infection. Plasma arginine concentrations in Stage 3 animals were at three times the Stage 0 levels, although the increase in total plasma FAA in Stage 3 infected animals was due primarily to the increased taurine concentration, In Stage 4 animals, most plasma FAA's had returned to concentrations not significantly different from those of stage 0 animals. The only exceptions to this were taurine, which had increased approximately 13-fold (Fig. la), glutamate, with a 10-fold increase (Fig. lb), and scrine, which was at approximately one half of the concentration in uninfected animals (Fig. I C). The importance ofp1asma taurine in iqfection. Taurine accounted for the majority of the increase in total detected FAA during infection. It 76 Chapter 4- Plasma Amino Acids showed significant differences between Stages 0 and 2 (p < 0.001), 1 and 2 (p < 0.001) and 2 and 3 (p < 0.001), but not between Stages 3 and 4 (p > 0.05) (Fig. la). However, when expressed relative to total detected FAA concentration, the difference in taurine amounts between Stage 0 (5.8% of total FAA) and Stage 1 (13.7% of total FAA) are significant (p < 0.05) (Fig. 2). Additional to increasing absolutely from Stage 0 to Stage 4 (0.22 Wol. ml-' to 2.56 ýLmol. ml-'), taurine also made an increasing relative contribution to total FAA, rising from 5.8% in Stage 0 to 41.6% in Stage 4 plasma. The application of a ratiometric measure of increasing taurine concentration against decreasing serine concentration (Fig. 3) also revealed significant differences between Stages 0 and I (i. e. between animals that are apparently healthy and those patently infected). The taurine: serine ratio increased significantly between Stages 0 and I (p < 0.0 1), 1 and 2 (p < 0.0 1), and 2 and 3 (p < 0.00 1), but not between Stages 3 and 4 (p > 0.05). It should be noted that the difference in this ratio between Stage 0 and Stage I is more significant (p < 0.01) than the comparison of absolute concentration (p > 0.05) or relative contribution of taurine to the total (p < 0.05). Discussion. Several studies on invertebrates have considered alterations in FAA profile and non-protein nitrogen under conditions of infection, but few have considered changes in the FAA profile at different stages of infection (Henn et al., 1998). The ability to estimate the infection severity by a simple morphological index applied to the host greatly facilitated the current study of Hematodinium infection in Nephrops, allowing the changing pattern of plasma amino acid concentrations to be tracked through the course of infection. This provides a good basis for understanding the biochemical disturbances that occur during Hematodinium infection. 77 Chapter 4- Plasma Amino Acids Plasma FAA profile in unin ected and infected Nepbmps nonqi , s, Of the FAAs detected in normal plasma, glycine, histidine/glutamine, alanine, serine asparagine and taurine collectively constitute almost 80 % of the total concentration (3.79 ýtmol. ml-). It should be noted however that this excludes proline, which is known to be high in Nephrops abdominal muscle (Robertson et al., 1992), and cysteine, both of which are not derivatized by OPT. The total concentration in Nephrops haemolymph may then be similar to the 5.8 ýLmol. ml" reported for Homanis gammanis (Camien et al., 195 1). The decrease in the concentrations of some amino acids early in infection (Table 1) is consistent with parasite utilisation of these compounds as the haemolymph is colonised. A reduction in non-protein nitrogen also occurs during early infection by Aerococcus viridans in Homarus americanus (Stewart, 1980). The significant reduction in the concentration of free serine that occurred even in early infection (Fig. lc) could reflect preferential utilization of this compound by Hematodinium, or alternatively parasitic consumption of carbohydrate precursors or a disruption to the biosynthetic pathway of serine. Serine is considered an intermediate in the biosynthesis of taurine in lobster skeletal muscle (Finney, 1978); the observed depletion of serine may be linked to up-regulation of the synthesis of taurine during infection. Taurine is generally not metabolised, and hence the increased taurine concentration relative to total FAA (Fig. 2) could be due to a combination of increased biosynthesis and its non-utilisation relative to the other FAA compounds. The increased total FAA concentration that occurred in later stages of infection may have several causes. There is widespread disruption in all major organs and tissues during infection of Nephrops by Hematodinium, especially at the later stages (Field & Appleton, 1995). Intracellular compounds liberated by cellular damage would be likely to appear in the haemolymph and lead to the observed increase in concentration of total FAA. However, the elevated total FAA concentration is not due to an increase in all component FAA's, but rather to 78 Chapter 4- Plasma Amino Acids elevated levels of a few (see Table 1) which suggest either selective release or selective utilisation of released amino acids. The particular increase in plasma taurine may in part reflect the fact that its concentration is known to be high in the crustacean hepatopancreas (Van Marrewijk & Ravenstein, 1974) and skeletal muscle (Pochon-Masson et al., 1984), both of which undergo considerable disruption during Hematodinium infection (Field & Appleton, 1995). Another possible source of plasma FAAs is the haemolymph itself, which contains both proteins such as haernocyanin and various types of haemocytes. Haemolymph proteins serve as stores for amino acids (Pequeux et al., 1979), and host mobilisation or parasitic degradation of these proteins could lead to elevated levels of some FAA in the plasma. Taylor et al. (1996) reported a large reduction in plasma hacmocyanin in Hematodinium-infected Nephrops. Taurine, however, is not a component of proteins, and so the elevated concentrations of this amino sulphonic acid must have a source other than the catabolism of haemocyanin. The dramatic decline in the concentration of haernocytes in the host haemolymph during severe infection (Field & Appleton, 1995) suggests that haemocytes are destroyed by the parasite (although inhibition of their production may be a contributory factor), and lysis of haemocytes would result in their cellular contents being released into the haemolymph. Since taurine is concentrated within the haemocytes of certain animals (Thoroed & Fugelli, 1994), this cell lysis could also contribute to the observed elevation of the plasma taurine levels. Since the large FAA pool in tissues of marine invertebrates such as Nephrops is essential in osmoregulation (Claybrook, 1983; Robertson et al., 1992), the increased FAA concentration in the haemolymph of Nephrops during late infection could, in partý reflect an attempt by the host to compensate for osmolytes depleted by the parasite, as reported for the oyster Crassostrea virginica infected by Bucephalus sp. and Minchinia nelsoni (Feng et al., 1970). Taurine is an important osmolyte in various animal groups, being a relatively more expendable amino compound as it is non-essential (Chamberlain & Strange, 1989). However, the finding that the 79 Chaptcr 4- Plasma Amino Acids concentration of several other amino acids not thought to be principally involved in osmoregulation also increased during Hematodinium infection (Table 1) suggests that the changes in haemolymph FAA have multiple origins. Finally, the possibility cannot be excluded that the elevated concentrations of taurine and other compounds are of parasite origin. To determine this an analysis of the FAA composition of media from in vitro cultures of Hematodinium (Appleton & Vickerman, 1998) and of the parasites themselves is required. Implications ofp1asma FAA changes to the ecology of Mphmpx. A number of studies have implicated taurine as a neurotransmitter or neuro-modulator (Oja & Kontro, 1978; Kuriyama et al., 1978). In crustaceans, taurine mimics the inhibitory actions of gamma-aminobutyric acid (GABA) and affects cardiac rhythm (Zatta, 1987; Payen et al., 198 1). Glutamate also has pharmacological activity at low concentrations (Lin & Cohen, 1973), and is maintained at trace concentrations (0.04 ýimol. ml-) in the plasma of uninfected Nephrops (Fig. lb). However, during infection the concentration of free glutamate in Nephrops plasma increased to values that exceed the threshold for stimulating crayfish muscle (0.053 4mol. ml-1, see Lin & Cohen 1973). Due to the open nature of the crustacean circulatory system, these elevated concentrations of neuro-active compounds could affect the behaviour and locomotory ability of Hematodinium-infected Nephrops. This could significantly alter their ability to evade capture, both by predators and by trawlers, and so has both practical and economic consequences since Nephrops is currently the United Kingdom's most valuable shellfish resource. Animals with late stage infections are moribund. This could result from the actions of neuro-active compounds, although the increased respiratory demands due to the high parasitic burden will also contribute to this state (Taylor et al., 1996). 80 Chapter 4- Plasma Amino Acids Elevated levels of FAAs in infected plasma may also have implications for predator attraction. Amino acids play an important part in prey detection in crustaceans (Voigt et al., 1996) and many species are particularly sensitive to taurine, with behavioural responses being elicited at concentrations as low as 10-13 M (Johnson & Atema, 1986). T'he increased plasma concentration of taurine together with damage to the excretory antennal gland (Field & Appleton, 1995) implies that the excretion of taurine and other amino compounds is increased during infection. This could attract benthic scavengers, which rely on chemical cues (Wong & Moore, 1995). If this is confirmed, then the possibility should be investigated that these scavenging species can act as alternative hosts for Hematodinium. This may help to elucidate the full life cycle of Hematodinium (Appleton & Vickerman, 1998). Diagnosis of Hematodinium-infection in Nephrops. Infection staging by observing the pleopod under low-power microscopy (Field & Appleton, 1995) is currently the primary method for field diagnosis of patent Hematodinium infection, while the use of a polyclonal antibody allows for accurate detection of sub-patent and latent infections (Field & Appleton, 1996). The pleopod staging method requires laboratory facilities, is somewhat subjective and can result in mis-diagnoses of infection severity and failure to detect light infections. Another limitation is that it provides only a 'parasitic index', and not an assessment of host condition. Many studies on invertebrate responses to various stressors have suggested that the use of biochemical indices may provide more consistent and reproducible measures of host condition (Livingstone, 1982; Fossi et al., 1997). The current study has shown that the taurine concentration alone can diagnose infection from stage 2 onwards (Fig. I a). while the reduced concentration of serine (Fig. Ic), the increased percent contribution of taurine to the total FAA (Fig. 2), and particularly the taurine: serine ratio (Fig. 3) provide powerful measures of early patent infection. The ratiometric measure also demonstrates that the distinction between stage 3 and stage 4 infections is an artificial one. A measure based upon the 81 Chaptcr 4- Plasma Amino Acids haemolymph taurine-serine ratio may provide a better estimate of the progression of infection than the pleopod method, and could be a valuable tool for surveys of disease prevalence in commercial Nephrops stocks. The development of a rapid, biochemical field assay using information gained in this study may be particularly useful in mapping Hematodinium infection along the Scottish coastline and would provide fisheries managers with a better insight into disease epidemiology in the valuable Nephrops fishery. 'Me finding of the increased concentration of taurine during Hematodinium infection of Nephrops may also have relevance to other host-parasite interactions. As taurine is involved in stress responses and occurs at high concentrations in the tissues of many invertebrates, alterations in its plasma concentration may be both an important mediator and also a useful index in a number of infection pathologies and stress-related effects. 82 Chapter 4- Plasma Amino Acids References. Aiken, D. E. (1980). Moulting and growth. In: The Biology and Management of Lobsters. (eds. Cobb, J. S. & Phillips, B. F. ). Academic Press, New York, pp. 91-163. Appleton, P. L. & Vickerman, K. (1998). In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sP. ) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116,115-130. Camien, M. N., Sarlet, H., Duchiteau, G. & Florkin, M. (195 1). Non-protein amino acids in muscle and blood of marine and freshwater Crustacea. Journal of Biological Chemistry 193,881-885. Chamberlain, M. E. & Strange, K. (1989). Aniosmotic cell volume regulation :a comparative review. American Journal of Physiology 277,159-173. Claybrook, D. L. (1983). Nitrogen metabolism. In: The Biology of Crustacea Vol. 5. Internal Anatomy and Physiological Regulation. (ed. Mantel L. H). Academic Press, London, p. 163-202. Feng, S. V., Khairallah, E. A. & Canzonier, W. J. (1970). Hemolymph free amino acids and related nitrogenous compounds of Crassostrea virginica infected with Bucephahis sp. and Minchinia nelsoni. Comparative Biochemistry & Physiology 34,547-556. Field, R. H. & Appleton, P. L. (1995). A Hematodinium-like dinoflagellate infection of the Norway lobster Nephrops norvegicus: observations on pathology and progression of infection. Diseases of Aquatic Organisms 22,115-128. Field, R. H. & Appleton, P. L. (1996). An indirect fluorescent antibody technique for the diagnosis of Hematodinium sp. infection of the Norway lobster, Nephrops norvegicus. Diseases of Aquatic Organisms 24,199-204. Field, R. H., Chapman, C. J., Taylor, A. C., Neil, D. M. & Vickerman, K. (1992). Infection of the Norway lobster Nephrops norvegicus by a Hematodinium-like species of dinoflagellate on the west coast of Scotland. Diseases of Aquatic Organisms 13,1-15. Findley, A. M., Blakeney, E. W. Jr. & Weidner, E. H. (1981). Ameson michaefis (Microsporidia) in the blue crab, Callinectes sapidus : parasite-induced alterations in the biochemical composition of host tissues. Biological Bulletin 161,115-125. Finney, C. M. (1978). Isotopic labelling of taurine; implications for its synthesis in selected tissues of Homants americanus. Comparative Biochemistry & Physiology 61B, 409413. Fossi, M. C., Savelli, C., Casini, S., Franchi, E., Mattei, N. & Corsi, 1. (1997). Multi- response biomarker approach in the crab Carcinus aestuarii experimentally exposed to benzo(a)pyrene, polychlorobiphenyls and methyl-mercury. Biomarkers 2,311-319. Henn, M. W., Schopf, R., Maier, W. A. & Seitz, H. M. (1998). The amino acid composition of Anopheles stephensi (Diptera : Culicidae) infected with Nosema algerae (Microsporid -a Nosematidae). Journal of Invertebrate Pathology 71,42-47. 83 Chapter 4- Plasma Amino Acids Johnson, B. R. & Atema, J. (1986). Chemical stimulants for a component of feeding behaviour in the common gulf weed shrimp, Leander tenuicornis (Say). Biological Bulletin 170,1-10. Kuriyama, K., Muramatsu, M., Nakagawa, K. & Kakita, K. (1978). Modulating role of taurine on release of neurotransmitters and calcium transport in excitable tissues. In: Taurine and Neurological disorders. (eds. Barbeau, A. & Huxtable, R. J. ). Raven Press, New York, pp. 201-216. Lin, S. & Cohen, H. P. (1973). Crayfish ventral nerve chord and hemolymph: content of free amino acid and other metabolites. Comparative Biochemistry & Physiology 45B, 249- 263. Livingstone, D. R. (1982). General biochemical indicators of sublethal stress. Marine Pollution Bulletin 13,261-263. Oja, S. S. & Kontro, P. (1978). Neurotransmitter actions of taurine in the central nervous system. In: Taurine and Neurological disorders (eds. Barbeau, A. & Huxtable, R. J. ). Raven Press, New York, pp. 181-200. Payen, G. G., Pochon-Masson, J., Porterner, C. & Chattanger, F. (198 1). Effects of eyestalk ablation on the concentration of taurine in various organs of the shore crab Carcinus maenas (L. ) (Decapoda: Brachyura). Comparative Biochemistry & Physiology 69A, 571- 574. Pequeux, A., Vallota, A. C. & Gilles, R. (1979). Blood proteins as related to osmoregulation in Crustacea. Comparative Biochemistry & Physiology 64A, 433435. Pochon-Masson, J., Payen, G. G., Portemer, C. & Chattanger, F. (1984). Variations in taurine in the ovaries and hepatopancreas of the female crab Carcinus maenas L. (Decapoda : Brachyura) during the different phases of its genital activity. International Journal of Invertebrate Reproduction & Development 7,127-133. Robertson, J. D. (1961). Studies on the chemical composition of muscle tissue. 11. The abdominal flexor muscles of the lobster Nephrops norvegicus (L. ). Journal of Experimental Biology 38,707-728. Robertson, J. D., Cowey, C. B. & Leaf, G. (1992). The free amino acids in muscle of three marine invertebrates Nephrops norvegicus (L. ), Dmithis poývphemzls (L. ) and Eledone cirrhosa (Lamarck). Comparative Biochemistry & Physiology IOIA, 545-548. Stewart, J. E. (1980). Diseases. In: The Biology and Management of Lobsters (eds. Cobb, J. S. & Phillips, B. F. ). Academic Press, New York, pp. 301-342 Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The effects of Hematodinizim sp. -infection on aspects of the respiratory physiology of the Nonvay Lobster, Nephrops norvegicus (L. ). Journal of Experimental Marine Biology & Ecology h7,217-228. Thoroed, S. M. & Fugelli, K. (1994). Free amino compounds and cell volume regulation in erythrocytes from different marine fish species under hypoosmotic conditions: the role of a taurine channel. Journal of Comparative Physiology 164B, 1-10. 84 Chaptcr 4- Plasma Amino Acids Van Marrewijk, WIA. & Ravenstein, HIL. (1974). Amino acid metabolism of Astacus leptodactylus Esch. 1. Composition of the free and protein-bound amino acids in different organs of the Crayfish. Comparative Biochemistry & Physiology 47B, 531-542. Vivares, C. P., Cuq, J-L., Ceccaldi, H. J. & Richard, P. J. (1980). Influence d'une microsporidiose sur les acides aminds libres de Carcinus mediterraneus Czerniavsky, 1884 sournis A diverses salinitids et A des valeurs extr8mes de tempdrature. Journal of Experimental Marine Biology & Ecology 43,207-220. Voigt, K, Wennemer, H. & Atema, J. (1996). Spectral tuning properties of chemoreceptor cells in the front walking legs of the common spider crab Libinia emarginata. Marine & Freshwater Behaviour & Physiology 27,261-267. Wang, D-1. & Moeller, F. E. (1970). Comparison of free amino acid composition in the haemolymph of healthy and Nosema-infected female honey bees. Journal of Invertebrate Pathology 15,202-206. Wong, Y. M. & Moore, P. G. (1995). Biology of feeding in the scavenging isopod Natatolana borealis (Isopoda: Cirolanidae). Ophelia 3,181-196. Zatta, P. (1987). Calcium and taurine interaction in the heart of the crab Carcinus maenas. Canadian Journal of Fisheries & Aquaculture Science 44,1765-1768. Zuo, Z. & Coombs, G. H. (1995). Amino acid consumption by the parasitic amoeboid protists Entamoeba histolytica and Einvadens. FEMS Letters. 130,253-258. 85 Chapter 4- Plasma Amino Acids Stage 0 _(n = 23) Cone. 0.01 0.04 0.16 034 0.56 1.17 0.11 0.11 0.55 0.22 0.06 0.14 0.02 0.05 0.10 0.14 3.79 : ESE 0.002 0.007 0.01 0.03 0.04 0.15 0.01 0.007 0.05 0.03 0.006 0.009 0.003 0.005 0.007 0.008 0.28 emolymp Stage 1 Conc. 0.03 0.06 0.12* 0.21*** 0.52 0.93 0.10 0.12 0.41 0.42 0.06 0.10** 0.00*0 0.04 0.05** 0.11 3.29 h Free A (n = 11) ±SE 0.01 0.01 0.01 0.05 0.11 0.17 0.02 0.02 0.05 0.09 0.007 0.01 0.00 0.006 0.01 0.02 0.41 mino Ad Stage 2 Cone. 0.03 0.11*** 0.13* 0.17*** 0.42 ** 1.43 0.10 0.23 ** 0.52 1.04*** 0.08 0.14 0.01** 0.06 0.08* 0.14 4.68 d (Concen 28) +SE 0.008 0.02 0.01 0.01 0.04 0.14 0.01 0.03 0.05 0.1 0.008 0.01 0.003 0.006 0.007 0.01 0.33 trations i Stage 3 _(n = 13) Conc. 0.05 0.19*** 0.11** 0.18** 037** 1.90* 0.09 0.31 0.55 2.20*** 0.12* 0.20 0.03 0.11** 0.09 0.20* 6.70*** ýLmol. m. ±SE 0.02 0.03 0.03 0.03 0.07 0.23 0.02 0.03 0.07 0.21 0.02 0.05 0.01 0.02 0.01 0.03 0.60 1-1) Stage 4 Cone. I 0.06 (). 40*** 0.09 0.19* 0.44 1.24 0.06 0.16 0.60 2.56*** 0.07 0.15 0.02 0.08 0.09 0.20 6.41 * (n =6) -ISE 0.03 0.21 0.04 0.04 0.09 0.30 0.03 0.03 0.16 0.33 0.02 0.02 0.01 0.02 0.02 0.05 0.93 Amino acid Aspartate Glutamate Asparagine Serine Histidine/Glutamine Glycine Threonine Arginine Alanine Taurine Tyrosine Valine/Methionine Tryptophan Phenylalanine Isoleucine Leucine Total Identificd FAA Table 1. OPT-derivatized free amino acids from the hacmolymph of Nephrops norvegicus. Significant differences from Stage 0 concentrations are denoted by *(p < 0.05), * *(p < 0.0 1) and ***(p < 0.001). 86 Chapter 4- Plasma Amino Acids Figure la-c. Mean (±SE) plasma Taurine (a), Glutamate (b) and Serine (c) in the haemolymph of Nephrops norvegicus at different stages of infection by Hematodinizim. For statistical significance among groups see Table I and text. 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Fig. I a. Plasma Taurine I r-" I Stage 4 Stage 0 0.70- 0.60- 0.50- 0.40- 0.30- 0,20- 0.10- 0.00- 0.40- 0.35- 0.30- 0.25- 0.20- 0.15- 0.10- 0.051 0.00 Stage 0 Stage I Stage 2 Stage 3 Stage 4 Fig. Ic. Plasma Serine -E Stage I Fig. lb. Plasma Glutamate Stage 2 Stage 3 Stage 0 Stage I Stage 2 Stage 3 Stage 4 Infection stage I I I Q'7 Chapter 4- Plasma Amino Acids Figure 2. Mean (±SE) contribution of taurine to total detected FAA in the haernolymph of Nephrops norvegicus at different stages of infection by Hematodinium, plus between- group significance table. Mean taurine % contributions are given above each bar. Infection stage 1 2 3 4 0 P<0.05 p<0.001 p<0.001 p<0.001 p<0.01 p<0.001 p<0.001 2 p<0.00 1 p<0.00 1 P>0.05 50 40 30 20 5.8% 13.7% I 41.6% 22.4% -1 10 0 0 I 2 Infection Stage 3 3.61/c I 3 I 4 I 88 Chapter 4- Plasma Amino Acids Figure 3. Mean (±SE) taurine-serine concentration ratios in haemolymph of Nephrops norvegicus under different degrees of infection by Hematodinium, plus between-group significance table. Mean ratio ± standard error of mean given above each bar. Infection 1 2 3 4 Stage 0 p<0.01 p<0.001 p<0.00 1 p<0.001 p<0.01 p<0.001 p<0 01 . 2 p<0.001 p<0.01 3 . M P>0.05 VS. - i I g , 25 -1 20 t is ý loý 13.77±2.3 6.05±0.5 0.71±0.1 16.71±3.9 2.70±0.7 F'I 4 "I 0123 Infection Stage QQ Chapter Five - Muscle Biochemistry Chapter 5 Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Norway lobster, Nephrops norvegicus (L. ) during infection by a parasitic dinoflagellate of the genus Hematodinium. Abstract. Changes in various biochemical and ultrastructural characteristics of the deep abdominal flexor (DAF) muscles were studied in Norway lobster (Nephrops norvegicus) from the Clyde estuary, Scotland, UK, at different stages of infection by a parasitic dinoflagellate of the genus Hematodinium. Abdominal DAF muscles from infected lobsters showed slight, significant increases in total water content, along with greatly depleted glycogen reserves and an altered free amino acid profile. However, protein concentration and composition remained unchanged. Ultrastructurally, parasitic infection of DAF muscle fibres caused alterations in sarcolemmal structure, and localized disruption of myofibrillar bundles around the periphery, but not throughout the centre of the fibres. Overall, the reduction in swimming performance previously reported for N. norvegicus during Hematodinium infection reflect an alteration in carbohydrate supply to the active muscle and some subtle disruption of muscle structure. The altered carbohydrate titre could reflect the Hematodinium parasites acting as a carbohydrate sink in the haemolymph, a disruption of normal tissue glycogenesis, or some alteration in the host's hormonal regulation. These changes could also adversely affect the taste, texture and marketability of infected meat. Published as: Stentiford, G. D., Neil, D. M. & Coombs, G. H. (2000). Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Norway lobster, Nephrops norvegicus (L. ) during infection by a parasitic dinoflagellate of the genus Hematodinium. Diseases of Aquatic Organisms 42,133-141. 90 Chapter Five - Muscle Biochemistry Introduction. Damage to crustacean muscle has been widely reported from various infective agents, including microsporidians (Findley et al., 1981; Langdon, 1991; Kabre, 1992; Dennis & Munday, 1994; Childers et al., 1996), ciliates (Cawthorn, 1997), bacteria (Stewart, 1980) and dinoflagellates (Shields, 1994). Other studies have described muscle wastage with unknown etiology (Lindqvist & Mikkola, 1978), while host-induced (idiopathic) muscle necrosis can occur with natural or artificial stress (Akiyama et al., 1982; Anderson et al. 1990, Stentiford & Neil, unpublished data). Such changes in the biochemical composition of muscle tissue can have economic consequences, as in the case of Tanner crabs (Chionoecetes bairdi and C opilio) infected by a parasitic dinoflagellate (Hematodinium sp. ) which causes the so-called 'Bitter Crab disease'. In this case infected muscle tissue tastes bitter (Meyers et al., 1987) and is sometimes deemed to be unmarketable (Eaton et al., 1991; Taylor & Khan, 1995). Similar observations have also been reported for Hematodinium sp. infections of other commercially important crustaceans, including the sand crab Portunus pelagicus (Hudson, 1995) and the blue crab Callinectes sapidus (Messick, 1994). Populations of the Norway lobster (Nephrops norvegicus), from the wcst coast of Scotland are seasonally infected with a dinoflagellate of the genus Hematodinium, with prevalence levels of over 70% in some years (Field el al., 1992). Several studies have established the progression, diagnosis and pathology of Hematodinium infection in N. norvegicus (Field & Appleton, 1995,1996), and its effect on host physiology (Taylor et al., 1996) and haemolymph biochemistry (Stentiford et al., 1999 - Chapter 4). During late infections the deep abdominal flexor (DAF) muscles of X norvegicus, which are mainly responsible for the rapid abdominal flexion in 'tail flip' swimming in these lobsters (Newland & Neil, 1990), are partially invaded by the Hematodinium parasite (Field & Appleton, 1995). The abdominal musculature may also be altered in water content 91 Cliaptcr Five - Muscle Biochemistry during Hematodinium infection in N. norvegi . cus, similar to Hematodinium sp. infections in other commercially important crustaceans (Meyers et al., 1987; Messick, 1994; Hudson, 1995). Such changes in the structure and composition of DAF muscles could lead to altered contractile behaviour (Field et al., 1995). Deficits in the swimming performance have been measured in Hematodinium-infected animals (Stentiford et al., 2000 - Chapter 8). This has important consequences for the success of infected lobsters in escaping from predators (Neil & Ansell, 1995; Arnott et al., 1998) and in evading capture by trawl nets (Newland et al., 1992). Additionally, the texture and taste quality of the tail meat, the marketable product of a large Scottish fishery (07M in 1999) (FRS, 1999), may also be altered. Despite their potentially damaging effect, changes in the biochemistry and ultrastructure of the DAF muscles in Hematodinium-infected X norvegicus have never been studied systematically (Field et al., 1992,1995). Therefore in this study the water content, total protein, glycogen and free amino acids of DAF muscle fibres were measured in X norvegicus at different stages of Hematodinium infection, and related to changes in the ultrastructure of the muscle fibres. Materials and Methods. Animals. Norway lobsters (Nephrops norvegicus L. ) were caught by otter-trawl from a location south of Little Cumbrae in the Clyde Sea area, Scotland, UK, and subsequently maintained in a closed aquarium (IOC, 33 ppt salinity) in the Division of Environmental and Evolutionary Biology, University of Glasgow, Scotland, UK. Animals were fed ad libitum, once weekly on mussels (Mytilus edulis) but were not fed for at least five days prior to tissue collection. All animals were in the intermoult state (Aiken, 1980) and were assessed for Hematodinium infection by the pleopod staging method of Field and Appleton (1995). Individual pleopods were removed and viewed under low power microscopy. The 92 Chapter Five - Muscle Biochemistry accumulation of agglutinated parasite and haemocyte material was used to assign the appropriate stage, whereby Stage 0 is apparently uninfected and Stages I to 4 are patently infected (stages of the parasite evident in the haemolymph). Tissue removal and treatment. The deep abdominal flexor (DAF) from each of 128 lobsters was dissected out under physiological saline, blotted to remove excess moisture and weighed (wet weight). Muscle tissue was then frozen in liquid nitrogen and freeze-dried for 36 h (Edwards Modulyo freeze drier). Tissue was then re-weighed (dry weight) and samples were individually ground using a standard mortar and pestle. Ground samples were stored at -20'C until analysis. For SDS-PAGE analysis of the DAF muscle, individual muscle fibres were dissected out in calcium-free physiological saline and placed into 200 ýtl of SDS-sample buffer, denatured at 95*C for 4 min and stored at -20'C until electrophoretic protein separation (Neil et al. 1993). For electron microscopy, muscles were fixed for 2h at room temperature in a solution containing 4% glutaraldehyde and 2% paraformaidehyde in 0.1 M phosphate buffer, pH 7.4, with 2% sucrose and 1.5 % sodium chloride. Fixed samples were rinsed in 0.1 M phosphate buffer with 4% sucrose and post-fixed in I% osmiurn tetroxide in phosphate buffer for I h. Specimens were washed in several changes of distilled water and stained in 0.5 % uranyl acetate for I h. Following dehydration through an ethanol series, specimens were embedded in Spurr resin (Spurr, 1969). Thick sections (I gm) were stained with toluidine blue and suitable areas were thin-sectioned and mounted on uncoated copper/palladium grids and stained with uranyl acetate and lead citrate. Thin sections (60- 70 nm) were examined in a Zeiss 902 transmission electron microscope. Chemicals. 93 Chapter Five - Muscle Biochemistry For protein and glycogen analyses, sodium hydroxide (NaOH), trichloroacetic acid (TCA), potassium hydroxide (KOM, ethanol (EtOH) and the anthrone and Folin- Ciocalteu's phenol reagents were obtained from Sigma-Aldrich Co. (Poole, UK). For high performance liquid chromatography (HPLQ analysis, reagent-grade methanol (MeOH), tetrahydrofuran (THF) and acetonitrile were obtained from BDH Laboratory Supplies (Leicestershire, UK). Crystalline amino acids (aspartate, glutamate, asparagine, serine, histidine, glutamine, glycine, threonine, arginine, alanine, tyrosine, valine, methionine, tryptophan, phenylalanine, isoleucine and leucine), taurine, sodium acetate, mercaptoethanol (ME) and o-phthaldialdehyde (OPT) were also from Sigma-Aldrich Co. and reagent-grade Millipore-filtered water was used throughout. For SDS-PAGE, acrylamide-bis acrylamide (30%), sodium dodecylsulfate (SDS), ammonium persulfate (APS), N, N, N', N'- tetramethylethylenediamine (TEMED), brilliant-blue R250, TCA and molecular weight (M,. ) standards were purchased from Sigma-Aldrich Co. Muscle water and totalprotein assays. 'Me muscle water content was determined for 128 lobsters as the difference between the wet weight and dry weight measurements. For determination of total muscle protein, 10 mg of freeze-dried DAF from 117 lobsters was homogenised in 10 ml 0. IN NaOH, left for 12 h and centriffiged at 17,000 xg for 5 min. One ml of the supernatant was added to 2 ml of 10 % TCA and left for 12 h at 4*C to allow for complete precipitation of the protein. Samples were centrifuged as above and the protein pellet re-dissolved in I ml of 0.1 N NaOH. Aliquots of 0.5 ml were then assayed for total protein using the Folin-Ciocaltcu's phenol reagent (Lowry et al., 195 1). 94 Chapter Five - Muscle Biochemistry Muscle glycogen assay. For determination of muscle glycogen, 400 RI of 30 % KOH was added to each of 45 10 mg freeze-dried samples from lobsters at different stages of infection. The samples were boiled for 20 min in a water bath at 95-100*C, cooled and added to 700 ILI absolute ethanol before being placed on ice for 2 h. Following precipitation, samples were spun at 17,000 x g for 10 min and the supernatant discarded. One ml of distilled H20 was added to each sample before sonication, after which 50 RI was incubated at 95-100T in Iml of anthrone reagent before glycogen assessment (Carroll et al., 1956). Free amino acid (FAA) measurement ofmuscle. For determination of muscle FAA concentrations, each of 44 10 mg samples of freeze- dried DAF from lobsters at different stages of infection were sonicated in I ml McOH for I min (De Vooys, 1991). Sonicated samples were deproteinized for 30 min at 4T, following which the protein precipitate was removed by centrifugation at 17,000 xg for 10 min. The supernatant was passed through a 0.22 prn syringe filter before storage at -70'C until analysis. Reverse phase HPLC was carried out as described in Stentiford et al. (1999) using 20 ýd of the DAF extract derivatized with 180 ýd OPT. SDS-PAGE conditions. Samples of DAF muscle fibre were prepared as described above. Discontinuous SDS- PAGE was performed according to the method of Laemmli (1970) with 12.5 % acrylamide separating gel and a4% acrylamide stacking gel. Electrophoresis running conditions were as described by Neil et aL (1993), followed by overnight incubation in 10 % TCA and staining for 30 minutes with Coomassie-blue solution. Stained gels were later examined with an Appligene TMgeI imager. 95 Chapter Five - Muscle Biochemistry Data analysis. Comparisons of the water content, protein, glycogen and FAA concentrations in the DAF muscle of uninfected and infected N. norvegicus were performed either by one-way analysis of variance (ANOVA) for normally distributed data, or by a Kruskal-Wallace test for non-normal distributions. Comparisons between stages were made with a Tukey's pairwise analysis (normal distributions) and a Mann-Whitney test (non-normal distributions). Significance was considered to be at p<0.05. Results. Water assay qfDAF muscle. Percentage of water content in DAF muscle at all stages of infection showed small, but consistently elevated mean values, which were significantly different from the mean value for Stage 0 (76.66 %) for Stage 1 (77.57 %) (p < 0.05), Stage 2 (77.46 %) (p < 0.01) and Stage 3 (77.77 %) (p < 0.01). At Stage 4, an increase in DAF water was also observed, but was not significant (77.43 %) (p = 0.095) (Fig. 1). DAF muscle protein assay. The protein concentration (mg. g" dry weight) of DAF muscle showed no significant difference between uninfected (632.77 mg. g*l dry weight DAF) and Hematodinium- infected animals (p = 0.943). When expressed as mg. g-' wet weight, the p-value was reduced (p = 0.443), but differences between uninfected and infected animals were still not significant. DAF muscle SDS-PA GE analysis. Myofibrillar proteins from DAF muscle fibres were separated using standard SDS-PAGE procedures. Figure 2 shows that the protein banding pattern of Hematodinium-infected 96 Chapter Five - Muscle Biochemistry DAF is similar to that of the uninfected control, with no apparent qualitative differences in the relative content of the major myofibrillar proteins. Electrophoresis using high percentage acrylamide gels (20 %) confirmed this absence of extra bands in the low molecular weight region, while low percentage acrylarnide gels (5-7'%) revealed no loss of bands in the high molecular weight region (data not shown). DAF muscle glycogen assay. The glycogen concentration in the DAF muscle of uninfected N. norvegicus had a mean value of 13.59 mg. g-1 dry weight. This was progressively reduced in successive stages of Hematodinium infection to 8.36 mg. g" dry weight in Stage I (p < 0.001), 2.29 mg. g-1 dry weight (p < 0.00 1) in Stage 2,3.10 mg. g" dry weight (p < 0.00 1) in Stage 3 and 1.92 mg. g-' ' dry weight (p < 0.001) in Stage 4. When different infection stages were compared, significant differences were found between Stage I and all other stages (p < 0.001), but not between Stages 2 and 3 (p = 0.273) or Stages 3 and 4 (p = 0.194) (Fig. 3). FAA analysis qfDAF muscle. Concentrations (ýLmol. gfw *') of FAA in DAF muscle of N. norvegicus were derived from the areas of the various peaks on the HPLC chromatograms. The OPT-derivatizcd FAA compounds identified in the DAF muscle of animals with different levels of infection are shown in Table 1. In uninfected X norvegicus (Stage 0), the total FAA concentration was 410.64 l. Lmol. gfW-1, with glycine, arginine, alanine, taurine and histidine/glutamine collectively constituting over 90 %. The total FAA concentration were highest in late stage infections, but was significantly different from uninfected DAF muscle only for Stage 4 animals (486.39 limol. gfiv") (P < 0.05). 97 Chapter Five - Muscle Biochemistry The concentrations of several individual FAA compounds were altered significantly during Hematodinium-infection (Table 1). The histidine/glutamine concentration was reduced in early infection, while both aspartate and taurine reached their highest concentrations at Stage 2. Taurine is particularly noteworthy, being significantly higher than the control DAF at all stages of Hematodinium infection. Serine and phenylalanine shared a similar pattern, with increasing concentrations that peaked in Stage 3. Serine made the largest contribution to the increased total FAA concentration, changing from 5.99 ýLmol. gfw-' in uninfected animals to 24.06 ýLmol. gfw-' in Stage 4 of infection. Glycine, which accounted for over 70 % of the total detected FAA in uninfected DAF, rose in concentration during infection, although the changes within stages were not significant (Table 1). Ultrastructure qfDAFmusclefibres When infected N. norvegicus were examined ultrastructurally, Hematodinium parasites were often observed in close association with fibres of the DAF muscle (Fig. 4c). Compared to normal muscle fibres (Fig. 4a), the sarcolemma of Hematodinium-infected muscle fibres was often disrupted with apparent disorganisation at the fibre periphery, separation of myofibrillar bundles and expansion of the tubular systems (Fig. 4b). Ilie majority of the myofibrillar bundles located more centrally in Hematodinium-infectcd DAF muscle fibres appeared normal in sarcomeric organisation, tubular systems and organelles (nuclei and mitochondria). Discussion. There are considerable alterations in the biochemistry and some changes in the ultrastructure of the abdominal musculature of Nephrops norvegicus during Hemajodinium infection. There is a slight (up to 1.1 %), but significant increase in water content of DAF 98 Chapter Five - Muscle Biochemistry muscle during infection. The implications for this increase in DAF water for normal muscle function arc not known, though it is possible that the dynamics of normal muscle contraction are altered (Field et al. 1995). An alteration in the water content may also affect the cooked meat texture and nutritional quality of infected N. norvegicus muscle. Measurements of total DAF muscle protein (mg. g" dry weight) provide an indirect indication of gross alterations that may occur during Hematodinium infection in N. norvegicus. The data presented here show that, although there may be some association of the parasite with muscle bundles during heavy infections (Fig. 4c) (Field & Appleton, 1995), the total muscle protein concentration remains unaffected. The representation of the data as mg. g-' wet weight cmphasises the effect of the increase in DAF water on the total DAF protein concentration. The similarity between the total protein in the DAF muscle of infected and uninfected lobsters suggests that the general structure of this muscle is relatively unaffected during Hematodinium infection. SDS-PAGE is a convenient tool for studying the expression of different contractile protein isoforms in crustacean muscle (Mykles, 1985; Yoshinaka et al., 1989; Youlin & MykIes, 1990; Neil et al., 1993; Ishimoda-Takagi et al., 1997). Fast muscle contains characteristic isoforms of paramyosin, troponin-C, I and T (Mykles, 1985) and a 75kDa protein that is cross-reactive with tropornyosin antibodies (Mykles, 1997). In the current study, the DAF myofibrillar protein profile is largely unaltered during patent Hematodinium infection of N. norvegicus (Fig. 2) and additional bands, indicative of breakdown fragments of muscle proteins, are also absent. These SDS-PAGE results are consistent with the measures of total protein, indicating that there is no significant alteration in muscle protein content during patent Hematodinium infection. Ultrastructurally, most myofibrillar bundles in the interior of muscle fibres retain their normal organisation, even during heavy infection (Fig. 4a-c) and the contractile machinery may produce normal levels of force, if activated. Alternatively, it cannot be excluded that 99 Chapter Five - Muscle Biochemistry the extensive disruption of sarcolemmal membrane structure of the DAF fibres which occurs during infection could prevent normal activation by affecting the distribution of ions across the sarcolemma. Fibres with lowered membrane resting potential would have reduced ability to respond to synaptic transmission with membrane depolarisations, and the subsequent steps in excitation-contraction coupling could then be suppressed. Thus, infected DAF muscle fibres may fail to contract in a normal way, although their contractile machinery is intact. The presence of all major contractile proteins in Hematodinium-infected DAF fibres suggests that normal tail flexion (at least the part reliant on the proteins themselves) should still be possible. However, Stentiford et al. (2000) (Chapter 8) demonstrated that most parameters of the swimming performance of N. norvegicus (including number of flips to exhaustion, total distance travelled and flip velocity) are reduced during progressing Hematodinium infection. This reduction in swimming performance does not reflect gross change in the total amount of contractile protein present, or to the breakdown of contractile machinery of Hematodinium-infected DAF muscle, but is related to more subtle factors either within the muscle itself, or in the supply of energy to support muscle contraction. The most obvious change occurring in Hematodinium-infected DAF muscle is the reduction in the concentration of glycogen to approximately 15 % of its normal value in Stage 4 of infection. Similar reductions in muscle glycogen concentration have been noted with bacterial infection by Aerococcus viridans var. homall in the lobster, Homarus americanus (Stewart, 1980) and also with several nematode infections of insects; in locusts (Loclista migratoria and Schistocerca gregaria) (Jutsum & Goldsworthy, 1974 and Rutherford & Webster, 1978 respectively) and the mosquito (Cidex pipiens) (Schmidt & Platzer, 1980). In these cases, the parasites may become a carbohydrate sink by consuming haemolymph glucose (the precursor for glycogenesis). Perhaps the parasites inhibit glycogen synthesis in the host, thereby allowing them to consume the host's glucose 100 Chapter Five - Muscle Biochemistry reserves (Rutherford & Webster, 1978). Interestingly, Schmidt & Platzer (1980) have suggested that the condition of the host may establish the degree to which the parasites develop, with the growth of the parasites proceeding as the nutritional state of the host deteriorates. Such a feature in Hematodinium-infection in X norvegicus would explain why infection prevalence is maximal during the spring, when animals are reaching their peak physiological condition before the onset of the main moulting season, while infection prevalence during the summer and autumn months is low (Field et. al., 1998). 'Me reduction in the concentration of glycogen in DAF muscle during Hematodinium infection in N. norvegicus may reflect changes in normal regulatory hormone function. Hyperglycaemia is a response to various stresses, and in crustaceans is mediated, at least in part, by the crustacean hyperglycaernic hormone (CHM (Chang et al., 1999). CHH released by the X-organ/sinus gland complex acts on the main glycogen reserve tissues (hepatopancreas, abdominal muscle and possibly hemocytes) to stimulate glycogenolysis, which increases extracellular glucose availability (Santos & Keller, 1993). A disruption, or up-regulation of CHH release during Hematodinium infection may be partly responsible for depleting the DAF stores of muscle glycogen and liberating glucose, which is then utilized by the developing parasites. Depletion of the DAF muscle glycogen reserve may also impact upon the swimming performance of Hematodinium-infected N. norvegicus (Stcntiford et al., 2000 - Chapter 8), with the reduced carbohydrate reserve causing a shortfall in the re-supply of ATP to the contractile machinery. In this study, the main reduction in DAF glycogen was seen at Stage 2 of Hematodinium infection, and Stentiford et al. (2000) (Chapter 8) reported that the main reduction in swimming performance also occurred at Stage 2. Ille progressive reduction in carbohydrate reserves during Hematodinium infection may thus be the major factor in the reduced swimming performance. However, other features, such as the 101 Chapter Five - Muscle Biochemistry increased respiratory demand (Taylor et al., 1996) and loss of integrity of DAF muscle fibre membranes may also be important, especially during severe infections. Like many marine invertebrates, crustaceans maintain high free amino acid (FAA) concentrations within their cells for osmoregulation (Lange, 1963) and alterations in the FAA profiles of muscles occur in various parasitic infections (Vivares et al., 1980; Findley et al., 1981). The total FAA concentration reported in this study for the DAF muscle of uninfected N. norvegicus is comparable to that reported for the same species by Robertson et al. (1992), considering that proline, which was found by Robertson (1961) to be high in N. norvegicus muscle, was not derivatized using OPT in this study. During Hematodinium infection, the concentration of several FAA compounds was significantly altered. The largest changes were in serine and phenylaIanine, which increased during progressing infection, and in taurine, which made the largest single contribution to the increase in total DAF muscle FAA. We have previously reported that plasma taurine increases significantly with severity of Hematodinium infection in N. norvegicus (Stentiford et al., 1999 - Chapter 4). Up-regulation of taurine in the DAF muscle or a leakage of FAA compounds via the damaged sarcolemma of infected N. norvegicus may be partly responsible for this plasma increase (Finney, 1978). If up-regulation in taurine synthesis is occurring, it may be the result of some form of stress reaction due to the large plasma parasite burden (Stentiford et al., 1999 - Chapter 4). Free amino acids significantly affect the taste of crustacean meat, with differences in extractive components between species underlying their characteristic flavours (Hayashi et al., 198 1; Shirai et al., 1996). Changes in the composition of FAAs and other compounds (nucleotides, sugars, organic acids and minerals) in the DAF may be implicated in the anecdotal changes in taste of Hematodinium-infected N. norvegicus meat and also in the characteristic taste change accompanying 'Bitter Crab' infection in Tanner crabs (Chionoecetes bairdi) (Meyers et al. 1987). Detailed study of the extractive components 102 Chapter Five - Muscle Biochemistry from the muscles of N. norvegicus and Chionoecetes bairdi is necessary to identify the chemical basis of these taste alterations. References. Aiken, D. E. (1980). Moulting and growth. In: The Biology and Management of Lobsters (eds. Cobb, J. S. & Phillips, B. F. ). Academic Press, New York, pp. 91-163 Akiyama, D. M., Brock, J. A. & Haley, S. R (1982). Idiopathic muscle necrosis in the cultured freshwater prawn (Macrobrachium rosenbergii). Veterinary Medicine & Small Animal Clinician 77,1119-1121. Anderson, I. G., Nash, G. & Shariff, M. (1990). 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Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan tanner crabs, Chionoecetes bairdi. Diseases of Aquatic Organisms 3,195-216. Mykles, D. L. (1985). Multiple variants of myofibrillar proteins in single fibres of the lobster claw muscles; evidence for two types of slow fibre in the cutter closer muscle. Biological Bulletin 169,476483. Mykles, D. L. (1997). Crustacean muscle plasticity: molecular mechanisms determining mass and contractile properties. Comparative Biochemistry & Physiology 117B, 367-378. Neil, D. M. & Ansell, A. D. (1995). The orientation of tail-flip escape swimming in decapod and mysid crustaceans. Journal of the Marine Biological Association, UX 75,55-70. Neil, D. M., Fowler. W. S. & Tobasnick, G. (1993). Myofibrillar protein composition correlates with histochernistry in fibres of the abdominal flexor muscles of the Norway lobster Nephrops norvegicus. Journal of Experimental Biology 183,185-20 1. Newland, P. L. & Neil, D. M. (1990). Ile tail flip of the Norway lobster, Nephrops norvegicus. 1. Giant fibre activation in relation to swimming trajectories. Journal of Comparative Physiology 166A, 517-527. Newland, P. L., Neil. D. M. & Chapman, C. J. (1992). Escape swimming in the Norwaý Lobster. Journal of Crustacean Biology 12,342-353. Robertson, J. D. (1961). Studies on the chemical composition of muscle tissue. 11. The abdominal flexor muscles of the lobster Nephrops norvegicus (L. ). Journal of Experimental Biology 38,707-728. Robertson, J. D., Cowey, C. B. & Leaf, G. (1992). The free amino acids in muscle of three marine invertebrates Nephrops norvegicus (L. ), Limulus polyphemus (L. ) and Eledone cirrhosa (Lamarck). Comparative Biochemistry & Physiology 101 A, 545-548. Rutherford, T. A. & Webster. J. M. (1978). Some effects of Mermis nigrescens on the hemolymph of Schistocerca gregaria. Canadian Journal of Zoology 56,339-347. Santos, E. A. & Keller, R. 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Changes in the plasma free amino acid profile of the Norway lobster, Nephrops norvegicus at different stages of infection by the parasitic dinoflagellate (genus Hematodinium). Diseases of Aquatic Organisms 3 8,15 1- 157. Stentiford, G. D., Neil, D. M., Atkinson, R. J. A. & Bailey, N. (2000). An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infected by a parasitic dinoflagellate of the genus Hematodinium. Journal of Experimental Marine Biology & Ecology 247,169-18 1. Stewart, J. E. (1980). Diseases. In: Ile Biology and Management of Lobsters (eds. Cobb, J. S. & Phillips, B. F. ). Academic Press, New York, p. 301-342 Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The effects of Hemalodinium sp, -infection on aspects of the respiratory physiology of the Norway Lobster, Nephrops norvegicus (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228. Taylor, D. M. & Khan, R. A. (1995). Observations on the occurrence of Hematodinium sp. (Dinoflagellata : Syndinidae), the causative agent of bitter crab disease in Newfoundland snow crab (Chionoecetes opilio). Journal of Invertebrate Pathology 65,283-288. Vivares, C. P., Cuq, J-L, Ccccaldi, H. J. & Richard, P. J. (1980). Influence d'une microsporidiose sur les acides aminds libres de Carcinus mediterraneus Czcmiavsky, 1884 sournis A diverses salinitids et A des valeurs extr8mes de tempdrature. Journal of Experimental Marine Biology & Ecology 43,207-220. Yoshinaka, R., Sato, K., Itoh, Y., Nakajima, S. & Sato, M. (1989). Content and partial characterisation of collagen in crustacean muscle. Comparative Biochemistry & Physiology 94B, 219-223. Youlin, L. & Mykles, D. L. (1990). Analysis of myosins from lobster muscles : fast and slow isozymes differ in heavy chain composition. Journal of Experimental Zoology 255, 163-170. 106 Chapter Five - Muscle Biochemistry Stage:. 4 (a 5): Asp Glu Asn Ser flis/Gin Gly Thr Arg Ala Tau Tyr VaVMet Phe lie Sütge(n1O) 1.05 0.13 3.88 4.22 5.99 19.51 300-81 3.20 29.86 20.14 1.65 3.24 0.38 1.65 2.91 0.39 0.85 1.45 2.89 13.23 0.72 Z38 3.55 1.18 0.27 0.44 0.17 5.54 ** 3.98 14.57* 11.49* 361.55 3.49 19.00* 23.01 20.11* A 12.17 1.10 2.54 0.87* 1.62 0.42 ajo DAF FAA Con St1"(u : -. IO) SR 2.46*** 1 0.42 2.76 0.42 0.29 3.51 0.90 26.56 0.35 3.03 3.40 2.03 0.15 0.18 0.16 0.24 0.31 3.76 3.61 ILS3 9.18 ** 329.71 2.66 26.08 25.24 27.17*** 0.94* 2.26 0.58 0.83 2.18 a39 0.52 2.75 1.36 2630 0.62 3.47 zil 3.61 0.16 0.41 0.09 0.16 0.55 4.66 4.75 26.06** 11.82* 329.33 5.00 35.76 33.25* 24.91** 2.00 3.97 1.96** 2.66 3.95 0.74 0.60 5.01 1.45 39.21 0.82 3.01 4.60 3.13 O. J: 0.73 0.49 0.59 0.55 4.27 4.62 24.06** 13.40 336.97 3.54 35.40 29.14 19.15* 2.19 4.52 1.41 239 3.51 0.83 0.99 4.56 3.20 34.28 1.03 4.65 5.29 Z91 0.99 Loo 0.73 0.70 0.72 Table 1. Summary of free amino acid concentrations in the deep abdominal flexor muscle of uninfected (Stage 0) and Hematodinium-infected (Stages 14) Nephrops norvegicus. Kev-. * (p < 0.05), ** (p < 0.0 1), *** (p < 0.00 1). Full names of amino acids are given in methods scc'tion. 107 Chapter Five - Muscle Biochemistry Fig. l. Percentage of water content in deep abdominal flexor muscle from uninfected (Stage 0) and Hematodinium-infected (Stages 14) Nephrops norvegicus. Total N= 128 (Stage 0- n24, Stage I- n23, Stage 2- n6 1, Stage 3-n 15 and Stage 4- n5). 79.0- 78.5 - 78.0- 77.5 - 77.0 - 76.5 - 76.0- 75.5 - 75.04 0234 Infection Stage Fig. 2.12.5% SDS-PAGE gel showing separation of contractile proteins from DAF fibres from uninfected and Hematodinium-infected Nephrops norvegicus. Key; Lane A (Stage 0), B (Stage 1), C (Stage 2), D (Stage 3), E (Stage 4), F (molecular weight marker), MHC (myosin heavy chain), PI/2 (paramyosin I and 2), 75kDa (unnamed fast muscle protein), TN-T (troponin-T), A (actin), TM (tropomyosin), TN-l (troponin-I isoforms), Ma-LC (myosin alpha light chain). Numbers on right indicate molecular weights in kDa. ABCD E F 29 2.4 2o. 1 108 Chapter Five - Muscle Biochemistry Fig. 3. Concentrations of glycogen (mg. g-' dry weight) in deep abdominal flexor muscle from uninfected (Stage 0) and Hematodinium-infected (Stages 14) Nephrops norvegicus. Total N 45 (Stage 0-n 10, Stage I-n 10, Stage 2-n 10, Stage 3-n 10 and Stage 4- n5). 16 -1 14 12 A ca 0 10 ý% 0 02 Infr. cfion stalp 4 109 Chapter Five - Muscle Biochemistry Fig 4 (A-C). (A). DAF muscle fibre from uninfected X norvegicus showing an intact sarcolemma, tight junctions between adjacent myofibre bundles and a well-developed tubular system. Scale bar =I Wn. (B). DAF muscle from Hematodinium-infected N. norvegicus showing a disrupted sarcolemma and expansion of inter-bundle spaces in peripheral fibres. Scale bar = Iýtm. (C). Hematodinium parasite cell on the outer surface of a DAF muscle fibre. Note the disorganisation and fibrous appearance of the sarcolemma. Scale bar =I ýLrn. 110 Chapter Six - Carbohydrate dynamics Chapter 6 Carbohydrate Dynamics and the Crustacean Hyperglycaemic Hormone (CHH): Effects of Parasitic Infection in Lobsters Abstract. The effects of a dinoflagellate parasite (Hematodinium sp. ) on carbohydrate metabolism were examined in the Norway lobster Nephrops norvegicus. Five stages of infection were observed. These included uninfected (Stage 0), sub-patently infected, lightly infected (Stage 1), and moderately and heavily infected (Stages 2 and 34, respectively). We observed that lobsters that were in Stages 14 of infection had significantly lower levels of hemolymph glucose compared to uninfected or sub-patently infected animals. These results were accompanied by significantly lower levels of hepatopancreatic glycogen in Stages 24 compared to Stages 0-1. Due to the disruption of the normal feedback loops which control the release of crustacean hyperglycaernic hormone (CHH) from the sinus gland, plasma concentrations increase with infection severity. Ile increase in CHH concentrations occurs concomitantly with reduced concentrations of plasma glucose and tissue glycogen. We discuss these data with the perspective that the parasite places a heavy metabolic load on the host lobster. Published as: Stentiford, G. D., Chang, E. S., Chang, S. A. & Neil, D. M. (2000). Carbohydrate Dynamics and the Crustacean Hyperglycaernic Hormone (CHH): Effects of Parasitic Infection in Lobsters. General & Comparative Endocrinology (in press). III Chapter Six - Carbohydrate dynamics Introduction. Stress responses occur in all animals when regulated physiological systems are extended beyond their normal range by external stressors. Indicators of such stress responses may therefore be useful in assessing the short-term well-being or long-term health status of an animal (Fossi et al., 1997; Paterson and Spanoghe, 1997). Such indicators have received considerable attention in commercially important decapod crustacean species (Paterson and Spanoghe, 1997; Chang et al., 1999) which, following capture, often require transportation to market. Post-capture economic losses in the North American lobster (Homanis americanus) fishery are estimated to be between $50 and $75 million annually (Cawthorn, 1997). Every step in the capture and post-capture processes has the potential to impose stress on captive animals, and under extreme conditions loss of the homeostatic regulation, by failure of all or part of the integrated response, may lead to increasing physiological disturbance and ultimately death (Morris and Airriess, 1998). Another important stressor is the infection of an animal by parasites (see Thompson, 1983 for review). The Norway lobster (Nephrops norvegicus), which is the subject of an important commercial fishery in Scotland, is seasonally infected by a dinoflagellate of the genus Hematodinium, with prevalence levels reaching 70% in populations of N. norvegicus from the inshore waters off the west coast of Scotland (Field et al., 1992). A number of studies have established the basic characteristics of Hematodinium infection in N. norvegicus in terms of its progression, diagnosis and pathology (Field and Appleton, 1995, 1996), its effect on host physiology (Taylor et al., 1996), haernolymph and tissue biochemistry (Stentiford et al., 1999 - Chapter 4,2000b - Chapter 5) and locomotion (Stentiford et al., 2000a - Chapter 8). The ability to estimate the infection's severity by a simple morphological index (Field and Appleton, 1995) and by an immunoassay for sub- patent infection (Field and Appleton, 1996) has greatly facilitated these epidemiological studies of Hematodinium infection in N. norvegicus (see Stentiford et al., 1999 - Chapter 112 Chapter Six - Carbohydrate dynamics 4). It therefore offers a very useful model system in which to investigate the stresses imparted by parasitic infection in crustaceans. A number of researchers have suggested different methods for quantifying the stress reactions in crustaceans; these include the measurement of different hemocyte types in the haemolymph (Jussila et al., 1997), physiological, biochemical (Paterson and Spanoghe, 1997; Stentiford et al., 1999 - Chapter 4), and molecular changes in tissue and haemolymph (Fossi et al., 1997) and the elevated transcription of heat shock proteins during periods of stress (Chang et al., 1999). A response that has received recent attention is that of the change in the plasma titre of the crustacean hyperglycaernic hormone (CHH) in relation to imposed stressors (see Chang et al., 1999). The occurrence of some 'diabetogenic' factor from the eyestalks of crustaceans has been known for many years (Abramowitz et al., 1944), as has evidence for the elevation of plasma glucose levels in crustaceans undergoing stress (Telford, 1968). The CHHs from several species have now been well described and in some cases, their amino acid profiles sequenced (see Keller et al., 1985; Kegel et al., 1989,1991; Lacombe et al., 1999 for review). These 8-9 kDa neuropeptides produced by perikarya in the mcdulla-terminalis X. organ, can be selectively released from the sinus gland into the blood stream (Santos and Keller, 1993b) after which they are known to target the hepatopancreatic plasma membranes (Kummer and Keller, 1993), the abdominal musculature (Santos and Keller, 1993a) and the hemocytes (Santos and Stefanello, 1991), with the liberated glucose either moving to the extracellular fraction by diffusion, or being converted intraccliularly to lactate via glycolysis (Santos and Keller, 1993a). The advantage of such a system is not entirely understood, but due to the predictable elevation of plasma CHH during certain stresses (notably hNpoxia) in crustaceans (Santos and Keller, 1993a; Webster, 1996; Chang et al., 1999), it is envisaged that hyperglycaernia may be involved in the so-called 'flight or fright' response in these animals. An interesting feature of this process is that the lactate 113 Chapter Six - Carbohydrate dynamics resulting from enhanced glycolysis, released to the extracellular medium, could serve as a positive feedback mechanism for CHH release, with the increased CHH in turn stimulating glycogenolysis which then increases glucose availability (Santos and Keller, 1993b). Variations in blood glucose have been observed in crustaceans under several different environmental and physiological conditions (such as extremes of temperature, salinity, anoxia, starvation and emersion - see Hall and van Hain, 1998) and also during parasitism (Stewart, 1980), and it is presumed that these changes may be at least partly mediated by alterations in plasma CHH. It has been shown that the plasma CHH titre is consistently increased during emersion and hypoxia and that this leads to elevated plasma glucose concentration (Santos and Keller, 1993a; Webster, 1996; Chang et al., 1999). However, it has not yet been shown whether the plasma CHH titre is altered in relation to parasitism. Tberefore we have investigated the effect of progressive infection of the Norway lobster by Hematodinium parasites on the plasma CHH titre, as a possible sensitive indicator of stress, and assessed whether these changes may be implicated in the altered plasma glucose and tissue glycogen concentrations that often accompany these infestations. Materials and Methods. Collection and Treatment ofAnimals. Norway lobsters were collected with an otter trawl from a location south of Little Cumbrae in the Clyde Sea area, Scotland, UK. Lobsters were maintained in a closed aquarium (9'C, 33 ppt salinity) at the University of Glasgow. Animals were allowed to settle in the aquarium for one week and were fed ad libitum on squid (Loligo spp. ) and mussel (Mytilus spp. ) tissue. Animals were not fed for three days prior to the cxperimcnt to avoid any effects of differential feeding and all animals were in the intcrmoult state as defined by Aiken (1980). Lobsters were staged for patent Hematodinium infection using the pleopod staging method of Field and Appleton (1995). According to the plcopod 114 Chapter Six - Carbohydrate dynamics staging method, Stage I denotes a lightly infected animal, with infection progressing through to Stage 4 (late stage infection). Stages 3 and 4 (heavily infected) animals were grouped in all analyses as animals in these groups show very similar disease characteristics (see Stentiford et aL, 1999 - Chapter 4). For diagnosis of true Stage 0 (uninfected) and sub- patent/latent infection, samples of hepatopancreas from previously diagnosed Stage 0 animals underwent western blot analysis using a polyclonal antibody against the Nephrops norvegicus isolate of Hematodinium sp. (see Field & Appleton, 1996 for details of antibody production). All animals showing a positive reaction to the anti-Hematodinium antibody in hepatopancreas tissue were diagnosed as sub-patent (i. e. Hematodinium-infected, but below the limit of detection using the pleopod staging method). Animals showing no reaction to the antibody were diagnosed as uninfected (Stage 0). Chemicals. For glycogen analyses, potassium hydroxide (KOH), ethanol and the anthrone and Folin- Ciocalteu's phenol reagents were obtained from Sigma-Aldrich Co. (Poole, UK). For glucose analysis, perchloric acid (PCA), sodium phosphate (dibasic), glucose and orthophosphoric acid (85 %) were obtained from Sigma-Aldrich Co., while the glucose oxidase assay kit was obtained from Boehringer-Mannheim (kit no. 124028). For CHH analysis, bovine serum albumin (fraction V), EDTA, glycine ethyl ester, Tween 20, and 2,2'-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid were obtained from Sigma-Aldrich Co., while the streptavidin-peroxidase solution was obtained from Boehringer-Mannheim. Haemolymph and Tissue Preparation. A total of 100 haemolymph samples from uninfected and Hematodinium-infected lobsters were drawn from the base of the fifth perciopod into sterile syringes and centrifugcd immediately at 17,000 xg for 10 min at IOT to remove cellular material and suspended 115 Chapter Six - Carbohydrate dynamics debris. For the CHH assay, aliquots of plasma were frozen immediately in liquid nitrogen before freeze-drying for 24 h. Dried samples were stored at -20'C until transport to, and analysis at, the Bodega Marine Laboratory. For the glucose assay, the clear plasma was deproteinized using an equal volume of 0.6 M PCA for 10 min at 4T. Protein precipitate was removed by centrifugation and the supernatant was stored briefly at -20T until analysis at the University of Glasgow. The hepatopancreata, from 45 lobsters at different stages of Hematodinium infection were dissected out in physiological saline. Excised samples were blotted to remove excess moisture and weighed (wet weight) before rapid freezing in liquid nitrogen and freeze- drying for 36 h. Tissue was then re-weighed (dry weight) and samples were individually ground using a standard mortar and pestle. Ground samples were stored at -200C until analysis of total glycogen content at the University of Glasgow. Measurement ofPlasma Glucose Concentration. Glucose concentration in the haemolymph of uninfected and infected lobsters was measured using the glucose oxidase method (Bochringer-Mannheim) in a microplate method as described by Webster (1996). Briefly, 50 ýd samples of deproteiniscd plasma were added to 450 ýd of 0.2 M phosphate buffer (pH 7.4) and 100 ýd samples of this solution were used in the assay with 200 RI of the enzyme chromogcn reagent. Concentrations of plasma glucose were read from a standard curve constructed for glucose. Measurement ofPlasma CHH Titre. The production of an antibody against purified CHH-A from H. americanus has been described by Chang et al. (1998) and previous studies have shown that N. norvegicus CHH can be detected using this antibody (G. D. Stentiford and E. S. Chang, unpublished data). In the current study, freeze-dried plasma samples were re-suspended to their original volume 116 Chapter Six - Carbohydrate dynamics with ddH20 and assayed for CHH-A using the ELISA method of Chang et al. (1998). HPLC-purified CHH from the crayfish Orconectes limosus (Kegel et al., 1991) was used as the standard. Since purified N. norvegicus CHH was not available, it was not possible to quantify absolute concentrations of N. norvegicus CHH. The data were therefore presented as 0. limosus equivalents, which allow relative quantification. Measurement ofHepatopancreatic Glycogen Content. For determination of muscle and hepatopancreatic glycogen, 400 ýLl of 30 % KOH was added to 20 mg of the freeze-dried samples from lobsters at different stages of infection, following which samples were boiled for 20 min in a water bath maintained at 95-100'C. Samples were cooled and added to 700 ýd absolute ethanol before being placed on ice for 2 h. Following precipitation, samples were spun at 17,000 xg for 10 min and the supernatant discarded. One ml of ddH20 was added to each sample before sonication. Fifty ýd of each sonicated sample were incubated at 95-100T in I ml of anthrone reagent before measurement of total glycogen (see Carroll et al., 1956). Data Analysis. Comparisons of plasma glucose concentration, plasma CHH titre and hepatopancrcatic glycogen content of uninfected and infected X norvegicus were performed either by one- way analysis of variance (ANOVA) for normally distributed data, or by a Kruskal-Wal lacc test for non-normal distributions. Comparisons between stages were made with a Tukey's pairwise analysis (normal distributions) and a Mann-Whitney test (non-normal distributions). Significance was considered to be at p<0.05. 117 Chapter Six - Carbohydrate dynamics Results. Plasma Glucose Concentrations. The mean concentration of glucose in the plasma of uninfected N. norvegicus was 180.0 ýtg. ml" plasma. Ilie glucose concentration in sub-patently infected lobsters (180.1 gg. ml" plasma) was not significantly different to that of Stage 0 animals (p = 0.979), but patently infected animals of all stages showed significantly lower concentrations of plasma glucose than Stage 0; in Stage 1 (41.4 gg. ml-1 plasma) (p < 0.001), Stage 2 (47.8 gg. ml" plasma) (p < 0.001) and Stage 3-4 (25.3 gg. ml" plasma) (p < 0.001). All patently infected animals also showed significantly lower concentrations of plasma glucose than sub-patently infected lobsters (all p<0.00 1), but different stages of patently infected lobsters showed no significant difference in glucose concentration (all p>0.05) (Fig. 1). Hepatopancreatic Glycogen Concentrations. The mean glycogen concentration of the hepatopancreas of uninfected N. norvegicus at 16.19 mg. g" dry weight was slightly higher than that of the deep abdominal flexor muscle (Stentiford et al., 2000b - Chapter 5). Hepatopancreatic glycogen concentration was reduced during patent Hematodinium infection. The reduction was not significant in Stage I lobsters (14.71 mg. g" dry weight, p=0.574), but was highly significant in both Stage 2 (2.01 mg. g" dry weight, p<0.001) and Stage 3-4 (0.84 mg. g" dry weight, p<0.001) Hematodinium infection. When different stages of infection were compared, significant reductions in hepatopancreatic glycogen were found between Stage I and Stage 2 (p < 0.001) and Stage I and Stage 3-4 (p < 0.01), but not between Stage 2 and Stage 3-4 (p 0.098) (Fig. 2). Plasma CHH concentrations. 118 Chapter Six - Carbohydrate dynamics The mean concentration of CHH in the plasma of uninfected N. norvegicus was 32.2 finol. ml". The mean concentration of CHH in the plasma of sub-patently infected lobsters (107.65 fmol. ml-') was significantly higher than that of Stage 0 animals (p < 0.05), while at Stage I (light patent infection), the mean concentration was higher (though not significantly, p=0.057) than Stage 0, and lower (though not significantly, p=0.070) than sub-patently infected lobsters. In later stages of patent Hematodinium infection, the plasma CHH concentration was significantly higher than that of uninfected animals; in Stage 2 (77.2 finol. ml", p<0.001) and Stage 34 (106.6 fmol. ml-1, p<0.001). The significant increase in plasma CHH concentration between Stage I and Stage 34 animals (p < 0.05) and almost between Stage I and Stage 2 animals (p = 0.080) suggests that the titre of CHH increases with severity of patent infection. It is interesting to note however that the mean plasma CHH titre of sub-patently infected lobsters is not significantly different to that of Stage 34 lobsters (p = 0.997) (Fig. 3). Discussion. Carbohydrate Dynamics in HemaloShium-infected N. nomegiats. The current study has identified large alterations in the carbohydrate profile in the plasma and tissues, and in the crustacean hyperglycaemic hormone (CW titrc in the plasma, of N. norvegicus infected by the parasitic dinoflagellate Hematodinium sp. The concentration of glucose in the plasma remains relatively unchanged in sub-patcntly infected lobsters, but is significantly reduced during all stages of patent infection. In Stage I infected lobsters tile plasma glucose is already reduced to approximately 30 % of its normal value, and this reduction in early infection suggests that glucose provides a readily usable substrate for the growth of the Hematodinium parasite in the haemolymph of N. norvegicus. Similar reductions in the concentrations of simple carbohydrates in the host tissues have been reported for other parasitic infestations in crustaceans (Stewart, 1980, Camilorn, 1997) and 119 Chapter Six - Carbohydratc dynamics insects (Schmidt and Platzer, 1980), and in these cases it has been suggested that the parasites may be acting as a 'carbohydrate sink', absorbing haemolymph glucose and thereby forcing the host to re-supply glucose to the haemolymph to maintain carbohydrate homeostasis. Ile re-supply of glucose to the plasma occurs mainly via tissue-based glycogenolysis, and the main storage tissues for such polysaccharides in crustaceans are the hepatopancreas (Dall and Moriarty, 1983), the muscle (Scwoch, 1972) and the hemocytcs (Johnson et al., 1971). The dramatic decrease in the concentration of glycogen in the hepatopancreas that occurs between Stage I and Stage 2 of infection (Fig. 2) and the similar depletion found to occur in the deep abdominal flexor muscle of Hematodinium- infected N. norvegicus (Stentiford et al., 2000b - Chapter 5) are consistent with a response to an elevated burden of circulating parasites (Field et al., 1992). The control of carbohydrate dynamics in the haemolymph and tissues of crustaceans is known to be exerted mainly by circulating levels of CHH (Sedlmeier, 1985). The cellular reception of CHH neuropeptide molecules elicits the mobilization of glucose from intracellular glycogen stores via glycogenolysis (Santos and Keller, 1993b), with the liberated glucose either moving to the extracellular fraction by diff-usion, or being converted intracellularly to lactate via glycolysis (Santos and Keller, 1993a). in patently infected lobsters, the plasma CHH concentration shows a steady and significant increase in relation to infection severity (Fig. 3), which is mirrored by a progressive decrease in the concentration of plasma glucose (Fig. 1). The co-cxistence of a high plasma CHH concentration and a low plasma glucose concentration, together with the significant reduction in tissue glycogen stores noted both in this study (Fig 2) and a previous study (Stentiford et al., 2000b - Chapter 5), is the opposite of what may be expected in a normally-operating feedback systern and is probably due to a disruption of the normal carbohydrate feedback control loop by the parasites. As infection progresses, the developing parasites will consume the glucose being liberated from the tissue glycogen 120 Chapter Six - Carbohydrate dynamics reserves, lowering circulating glucose concentrations and thereby reducing the negative feedback on CHH release from the sinus glands (see step (1) in Fig. 4 and Santos and Keller, 1993a). This, in turn, will initiate an increased release of CHH (step (2) in Fig. 4), inducing progressive glycogen depletion of the tissues (step (3) in Fig. 4). As the parasite burden increases, a steadily-reducing plasma glucose concentration, a progressive depletion of tissue glycogen stores and an increasingly elevated plasma CHH titre are all to be expected. In addition to this change in negative-feedback control of CHH release by plasma glucose, the increased plasma lactate levels that are known to occur during Hematodinium infection (Taylor et al., 1996) may also stimulate CHH release, with consequent effects on glycogenolysis (Santos and Keller, 1993b). The most likely cause of these elevated plasma lactate levels is a switch to anaerobic metabolism, which Taylor et al. (1996) have recorded in terms of increased lactate and a lowered haemolymph pH, a situation which is exacerbated by a reduction in the oxygen carrying capacity of the haemolymph. At the cellular level, pyruvate, the end product of glycolysis, is converted under anaerobic conditions to lactate, which either accumulates within the cell or is moved to the extracellular medium (step (4) in Fig. 4). Potentially, tissue glycogen reserves could be replenished from the lactate by the process of gluconeogencsis, but this is unlikely to be significant since in invertebrates, these pathways are thought to be relatively inefficient compared with the Cori cycle in mammals (Schulman and Landau, 1992), and probably operate under aerobic conditions (Ellington, 1983). Moreover, gluconeogenesis is antagonized by CHH itself, which is known to activate cyclic nucleotide-dependent protein kinases, leading to a phosphorylation and therefore inhibition of glycogen synthase (Sedimeier, 1985; Santos and Keller, 1993a), and is also inhibited by hyperosmolarity (Li et al., 1992), which is known to occur in Hematodinium infection (Stentiford et al., 1999 - Chapter 4). It is most likely, therefore, 121 Chapter Six - Carbohydrate dynamics that the elevated plasma CHH concentration in patent infection is due primarily to a 'functional hypoxia' in the infected lobster, which elicits a cascade response similar to that seen during the 'environmental hypoxia' caused by emersion (Santos and Keller, 1993a; Webster, 1996; Chang et al., 1999). Implications of Altered Carbokydrate Dynamics during Hefflatodinium Infection. Studies on the physiological condition of crustaceans over the entire moulting cycle have shown that the carbohydrate reserves are generally highest in the late intermoult period (Icely and Nott, 1992) and are also dependent upon the reproductive cycle (Tuck et al., 1997). In terms of parasitic infection, Schmidt and Platzer (1980) note that the condition of the host may establish the degree to which the parasite develops, with hosts in the best physiological condition harbouring the largest parasite burdens. The prevalence of Hematodinium infection is known to be highest in N. norvegicus during the spring (Field and Appleton, 1995) and in females (G. D. Stentiford, unpublished data). Female crustaceans have relatively larger amounts of hepatopancreatic tissue than males as a means of supplying the nutritional requirements for egg rearing and spawning (Farmer, 1974a). In N. norvegicus, this egg-rearing period may last for up to 8 months of the year, during which time the female lobster remains for the most part, within the burrow (Farmer, 1974b). The higher incidence of Hematodinium infection in female lobsters may reflect some relative advantage of the female host to the parasite. Recently, it has been shown that the concentration of CHH in the plasma is increased by up to 100-fold in the hours leading up to moulting, with levels returning to normal following ecdysis (Chung et al., 1999). The spring peak of Hematodinium infection in Scottish N. norvegicus coincides with the onset of the main moulting period for this species (Field et al., 1998) and it is tempting to suggest that a greatly increased concentration of CHH in the plasma at this time may create haemolymph conditions that are suitable for 122 Chapter Six - Carbohydrate dynamics rapid growth of the parasite population. The significantly elevated concentration of CHH in the plasma of sub-patently infected lobsters found in this study could either be responsible for, or caused by, the appearance of Hematodinium parasites in the haemolymph following latent infection. Further studies on the effect of elevated CHH concentration on latently infected lobsters are necessary to investigate the role of this hormone in initiation of haemolymph infection in N. norvegicus. An increase in plasma CHH titre due to other stressors (such as seasonal hypoxia, temperature changes or pollution) could also be implicated in infection progression. As the main carbohydrate resources become exhausted, parasites may switch to alternative, and less cfficient forms of energy such as fatty acids, proteins and free amino acids (Schmidt and Platzer, 1980; Thompson and Dahlman, 1998). Studies of plasma and tissue free amino acid and protein dynamics during Hematodinium infection of N. norvegicus have shown that the normal profile of these compounds is considerably altered as infection severity increases (Taylor et al., 1996; Stentiford et al., 1999 - Chapter 4, 2000b - Chapter 5). It is likely that such changes occur following exhaustion of the most accessible and high-yielding forms of nutrition: the plasma and tissue carbohydrates. Under such conditions, the host will reach a state of physiological starvation from which it cannot recover, and at this stage the post-parasitic forms emerge and the host will die (Schmidt and Platzer, 1980). Aquarium observations of Hematodinium-infected N. norvegicus have identified an apparently similar response, where swarming stages of the parasite are extruded via the integumental membranes, gills and the mouth. Ibis leaves the host moribund and then dead within 24 h (Field and Appleton, 1995; Appleton and Vickerman, 1998). It may be suggested that sporulation of the Hematodinium parasite within, and exit from the host occurs when the parasite has exhausted the host's nutrient reserves. Host condition may then also play an important role in determining the timing of sporulation of Hematodinium in N. norvegicus and may explain why sporulation has been observed in 123 Chapter Six - Carbohydrate dynamics some lobsters displaying only Stage I and 2 infection (Appleton and Vickerman, 1998). During the terminal stages of Hematodinium infection, the arthrodial membranes appear swollen and the haemolymph volume is apparently increased (Appleton and Vickerman, 1998). Increased water uptake has been associated with high concentrations of CHH in the plasma of Carcinus maenas preceding ecdysis (Chung et al., 1999). It is possible to suggest therefore, that elevated levels of plasma CHH within, or preceding, the final stages of Hematodinium infection in N. norvegicus could be involved with the observed swelling of arthrodial membranes and may facilitate the exit of motile dinospores (sporulation) from the host lobster. Tle current study has, for the first time, identified a link between the disruption of carbohydrate handling during parasitic infection of crustaceans and alterations in the expression of the crustacean hyperglycemic hormone. Whether the increase in the plasma CHH titre is caused directly by parasitic disruption of the lobster's endocrine system, or indirectly, by interfering with the positive and negative feedback loops in the plasma, requires further investigation. However, future studies which consider plasma and tissue carbohydrate dynamics in crustaceans should take into account that the relatively large variations often seen in the hyperglycemic response (Hall and van Ham, 1998), while being related to the moult stage of the test animal, may also be due to its overall health status. 124 Chapter Six - Carbohydrate dynamics References Abramowitz, A., Hisaw, F., and Papandrea, D. (1944). The occurrence of a diabetogenic factor in the eyestalks of crustaceans. Biological Bulletin 86,1-4. Aiken, D. E. (1980). Moulting and growth. In: ' The Biology and Management of Lobsters. (eds. Cobb, J. S. & Phillips, B. F. ). Academic Press, New York, pp. 91-163. Appleton, P. L. & Vickerman, K. V. (1998). In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sp. ) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116,115-130. Carroll, N. V., Longley, R. W. & Roe, J. D. (1956). The determination of glycogen in liver and muscle by use of the anthrone reagent. Journal of Biological Chemistry 220,583-593 Cawthorn, R. J. (1997). Overview of "bumper car" disease - impact on the North American lobster fishery. International Journal of Parasitology 27,167-172. Chang, E. S., Chang, S. A., Keller, R., Reddy, P. S., Snyder, M. J. & Spees, JI. (1999). Quantification of stress in lobsters: crustacean hyperglycemic hormone, stress proteins and gene expression. American Zoologist 39,487-495. Chang, E. S., Keller, R. & Chang, S. A. (1998). Quantification of crustacean hyperglycemic hormone by ELISA in hemolymph of the lobster, Homarus americanus, following various stresses. General & Comparative Endocrinology 111,359-366. Chung, J. S., Dircksen, H. & Webster, S. G. (1999). A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas. Proceedings of the National Academy of Science, USA 96,13103- 13107. Dail, W., and Moriarty, DTW. (1983). Nutrition and digestion. In: The Biology of Crustacea Vol. 5 (ed. L. H. Mantel). Academic Press, New York. pp. 215-26 1. Ellington, W. R. (1983). The recovery from anaerobic metabolism in invertebrates. Journal of Experimental Zoology 228,431444. Farmer, A. S. D. (1974a). Reproduction in Nephrops norvegicus (Decapoda: Ncphropidae). Journal of Zoology 174,161-183. Farmer, A. S. D. (1974b). Burrowing behaviour of the Norway lobster, Nephrop. v norvegicils (L. ) (Decapoda: Nephropidae). Estuarine & Coastal Marine Science 2,49-58, Field, R. H. & Appleton, P. L. (1995). A Hemalodinium-like dinoflagellate infection of tile Norway lobster Nephrops norvegicus: observations on pathology and progression of infection. Diseases of Aquatic Organisms 22,115-128. 125 Chapter Six - Carbohydrate dynamics Field, R. H. & Appleton, P. L. (1996). An indirect fluorescent antibody technique for the diagnosis of Hematodinium sp. infection of the Norway lobster, Nephrops norvegicus. Diseases of Aquatic Organisms 24,199-204. Field, R. H., Chapman, C. J., Taylor, A. C., Neil, D. M. & Vickerman, K. (1992). Infection of the Norway lobster Nephrops norvegicus by a Hematodinium-like species of dinoflagellate on the west coast of Scotland. Diseases of Aquatic Organisms 13,1-15. Field, R. H., Hills, J. M., Atkinson, R. J. A., Magill, S. & Shanks, A. M. (1998). Distribution and seasonal prevalence of Hematodinium sp. infection of the Norway lobster (Nephrops norvegicus) around the west coast of Scotland. ICES Journal of Marine Science 55,846- 858. Fossi, M. C., Savelli, C., Casini, S., Franchi, E., Mattei, N. & Corsi, 1. (1997). Multi. response biomarker approach in the crab Carcinus aestuaru experimentally exposed to benzo(a)pyrene, polychlorobiphenyls and methyl-mercury. Biomarkers 2,311-319, Hall, MR & van Ham, E. H. (1998). The effects of different types of stress on blood glucose in the giant tiger prawn Penaeus monodon. Journal of the World Aquaculture Society 29,290-299. Icely, J. D. & Nott, J. A. (1992). Digestion and absorption: digestive system and associated organs. In: Microscopic anatomy of Invertebrates Vol. 10 (eds. F. W. Harrison, and A. G. Humes). Wiley-Liss, New York. pp. 147-201 Johnson, M. A., Davies, P. S. & Elder, H. Y. (1971). Possible hepatic function for crustacean blood cells. Nature 23 0,47 1. Jussila, J., Jago, J., Tsuetnenko, E., Dunstan, B. & Evans, L. H. (1997). Total and differential haemocyte counts in western rock lobsters (Panuhrus cygnus George) under post-harvest stress. Marine & Freshwater Research 48,863-867. Kegel, G., Reichwein, B., Tensen, C. P. & Keller, R. (1991). Amino acid sequence of crustacean hyperglycemic hormone (CHH) from the crayfish, Orconectes limosus: emergence of a novel neuropeptide fan-dly. Peptides 12,909-913. Kegel, G., Reichwein, B., Weese, S., Gaus, G., Peter-Katalinic, J. & Keller, R. (1989), Amino acid sequence of the crustacean hyperglycernic hormone (CHH) from the shore crab, Carcinus maenas. FEBS Letters 255,10-14. Keller, R., Jaros, P. P. & Kegel, G. (1985). Crustacean hypcrglycemic ncuropeptides. American Zoologist 25,207-22 1. Kummer, G. & Keller, R. (1993). High-affinity binding of crustaccan hypcrglyccmic hormone (CHH) to hcpatopancreatic plasma membranes of the crab, Carcinus maenas and the crayfish, Orconectes limosus. Peptides 14,103-108. Lacombe, C., Grdve, P. & Martin, G. (1999). Overview on the sub-grouping of the crustacean hyperglycernic hormone family. Neuropeptides 33,71-80. Li. J., Stillman, J. S., Clore, J. N. & Blackard, W. G. (1992). The effect of hýpcrosmolarity 126 Chaptcr Six - Carbohydratc dynamics on muscle glycogen accumulation. Hormone & Metabolism Research 24,99-102. Morris, S. & Airriess, C. N. (1998). Integration of physiological responses of crustaceans to environmental challenge. South African Journal of Zoology 33,87-106. Paterson, B. D. & Spanoghe, P. T. (1997). Stress indicators in marine decapod crustaceans, with particular reference to the grading of western rock lobsters (Panuhrus cygnus) during commercial handling. Marine & Freshwater Research 48,829-834. Santos, E. A. & Keller, P, (1993a). Effects of exposure to atmospheric air on blood glucose and lactate concentration in two crustacean species: a role of the crustacean hyperglycaernic hormone (CHH). Comparative Biochemistry & Physiology 106A, 343- 347. Santos, E. A. & Keller, R. (1993b). Regulation of circulating levels of crustacean hyperglycaernic hormone: evidence for a dual feedback control system. Journal of Comparative Physiology 163B, 374-379. Santos, E. A. & Stefanello, T. M. (1991). The hemolymph of Chasmagnathus granulata Dana, 1851 (Decapoda - Grapsidae) as a target tissue of the crustacean hyperglycemic hormone. Brazilian Journal of Medical & Biological Research 24,267-270. Schmidt, S. P. & Platzer, E. G. (1980). Changes in body tissues and hemolymph composition of Culex pipiens in response to infection by Romanomermis culicivorax. Journal of Invertebrate Pathology 36,240-254. Schulman, G. I. & Landau, B. R. (1992). Pathways of glycogen repletion. Physiological Reviews 72,1019-1035 Scwoch, G. (1972). Some studies on biosynthesis and function of trehalose in the crayfish Orconectes limosus Rafmesque. Comparative Biochemistry & Physiology 43B, 905-917. Sedlmeier, D. (1985). Mode of action of the crustacean hyperglycemic hormone. American Zoologist 25,223-232. Stentiford. G. D., Neil, D. M., Atkinson, RTA. & Bailey, N. (2000a). An analysis of swimming performance in the Norway lobster, Alephrops norvegicus L. infected by a parasitic dinoflagellates of the genus ýIematodinizim. Journal of Experimental Marine Biology & Ecology 274,169-18 1. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (1999). Changes in the plasma free amino acid profile of the Norway lobster, Nephrops norvegicus at different stages of infection by a parasitic dinoflagellates (genus Hematodinium). Diseases of Aquatic Organisms 3 8,15 1- 157. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (2000b). Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Norway lobster, Nephrop. v norvegicus L. during infection by a parasitic dinoflagellate of the genus Hematodinium. Diseases of Aquatic Organisms (in press). 127 Chaptcr Six - Carbohydratc dynamics Stewart, J. E. (1980). Diseases. In: The Biology and Management of Lobsters Vol. I (eds. J. S. Cobb & B. F. Phillips). Academic Press, New York. pp. 301-342. Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The effects of Hemalodinium sp. -infection on aspects of the respiratory physiology of the Norway lobster, Nephrops norvegicus (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228. Telford, M. (1968). The effects of stress on blood sugar composition of the lobster, Homarus americanus. Canadian Journal of Zoology 46,819-826. Tbompson, S. N. (1983). Biochemical and physiological effects of metazoan endoparasites on their host species. Comparative Biochemistry & Physiology 74B, 183-211. Thompson, S. N. & Dahlman, D. L. (1998). Aberrant nutritional regulation of carbohydrate synthesis by parasitized Manduca sexta L. Journal of Insect Physiology 44,745-753. Tuck, I. D., Taylor, A. C., Atkinson, R. J. A., Gramitto, M. E. & Smith, C. (1997). Biochemical composition of Nephrops norvegicus: changes associated with ovary maturation. Marine Biology 129,505-511. Webster, S. G. (1996). Measurement of crustacean hyperglycaemic hormone levels in the edible crab Cancer pagurus during emersion stress. Journal of Experimental Biology 199, 1579-1585. 128 Chapter Six - Carbohydrate dynamics Fig. 1. Concentration of glucose (ýtg. ml_) in the plasma of uninfected (Stage 0) and Hematodinium-infected (sub-patent and Stages 1,2.34) N norvegicus. 250 200 .0 iso u I- loo 0 0 u .2 50 01 Stage 0 Sub-P Stage I Stage 2 Stage 34 Infection status Fig. 2. Concentration of glycogen (mg. g-1 dry weight) in the hepatopancreas of uninfected (Stage 0) and Hematodinium-infected (Stages 1,2 34) X norvegicus. 20 18 16 - 14 12 to - 8- 6 4 2 0 0 2 Infection stage 3&4 129 Chapter Six - Carbohydratc dynamics Fig. 3. Concentration of the crustacean hyperglycaernic hormone (CF" (frnol. ml") in the plasma of uninfected (Stage 0) and Hematodinium-infected (sub-patent and Stages 1,2,34) N. norvegicus. 160- 140- 120 - 100 1 80 ý 60, 40 ý 20 ý sub-p 12 3&4 Status 130 Chapter Six - Carbohydrate dynainics (2) CHII 111111M Membrane cyclaws Glycogen 1 Glucose 6-phosphate Haemolymph (Low02) Glucose Pyruvate I Krebs cycle Lactate Tissue (e. g. hepatopancreas/muscle) Fig. 4. Schematic diagram of the changing relationship between tissue glycogen, plasma glucose and CHH release in the presence of Hematodinium parasites in the plasma of N. norvegicus (adapted from Santos & Keller, 1993a). For description see discussion text. 131 Cbapter Seven - Muscle Necrosis Chapter 7 A rapid onset, post-capture muscle necrosis in the Norway lobster, Nephrops norvegicus (L. ) from the west coast of Scotland, United Kingdom. Abstract. A post-capture, abdominal muscle necrosis of rapid onset has been identified in Norway lobsters (Nephrops norvegicus) captured off the west coast of Scotland, United Kingdom. Economic losses, due to mortality of these animals in transport, were encountered by Scottish wholesalers during the summer and autumn of 1999. Affected animals show a chamcteristic whitening of individual muscle fibres and fibre bundles of the abdomen within hours of capture, with a progression towards complete opacity of the abdominal musculature within a number of days. The pathology causes a loss of the normal function of the abdomen, thus preventing the normal 'tail flip' swimming. Electron microscopy failed to reveal any obvious causative agent but showed that affected tissue displayed a progressive disruption of sarcomere organization, loss of Z-Iine material, condensation of myofibrils and infiltration of necrotic regions by granulocytes. SDS-PAGE of affected muscle tissue showed that there was a great reduction of most of the major contractile proteins. The condition most closely resembles idiopathic or spontaneous muscle necrosis, a pathology previously reported from both wild and cultured crustaceans. Damage to the integument in conjunction with exposure to various stressors during and immediately following capture is the most likely cause of the pathology. The rapid onset of the pathology has implications for the post-capture handling procedure for N. norvegicus and their subsequent vivier transport to market. It may also be partially responsible for the high mortality rate of undersized N. norvegicus returned to the sea after capture and aerial emersion. Published as: Stentiford, G. D. & Neil, D. M. (2000). A rapid onset, post-capture muscle necrosis in the Norway lobster, Nephrops norvegicus (L. ) from the wcst coast of Scotland, United Kingdom. Journal of Fish Diseases (in press). 132 Chapter Seven - Muscle Necrosis Introduction. Pathological damage to crustacean muscle has been widely reported and is known to have various causative agents, including microsporidians (Findley et al., 198 1; Langdon, 199 1; Kabre, 1992; Dennis & Munday, 1994; Childers et al., 1996), ciliates (Cawthorn, 1997), bacteria (Stewart, 1980), dinoflageflates (Shields, 1994; Stentiford et al., 2000 - Chapter 5) and viruses (Arcier et al., 1999). A number of other studies have described muscle wastage with unknown etiology (Lindqvist & Mikkola, 1978), while host-induced (idiopathic) muscle necrosis has been reported to occur in conditions of natural or artificial stress, both in the wild and under aquaculture situations (Akiyama et al., 1982; Momoyama & Matsuzato, 1987; Nash et al., 1987; Anderson et al., 1990; Evans et al. 1999). Idiopathic muscle necrosis (also known as 4tail rot', muscle opacity, muscle necrosis, and spontaneous muscle necrosis) is characterized by focal to gross opaque lesions in the striated musculature of the abdomen. The lesions are composed of necrotic muscle cells with disorganized myofibrils, loss of sarcomeres, loss and atrophy of mitochondria and damage to the sarcoplasmic reticulum (Akiyama et al. 1982). Late stage necrosis is characterized by fragmentation and condensation of myofibrillar bundles (Nash et al., 1987). Hemocýlic infiltration of the damaged musculature is also commonly observed, these participating in aggregation, encapsulation and, in the late stages of necrosis, myophagia (Momoyama & Matsuzato, 1987, Nash et al., 1987 and Anderson et al., 1990). Nephrops norvegicus supports a major fishery in the United Kingdom, with the Scottish fishery contributing over 76% of this (07 million in 1999 - FRS, 1999). The majority of the landings are from trawler capture, with the animals being 'tailed' for sale as 'scampi'. Larger animals are also captured by baited creels and are often exported to continental Europe by special ly-designed 'vivier' vehicles. In order to ensure sale, exported live lobsters must be in good condition on arrival at market. 133 Chapter Seven - Muscle Necrosis During the summer and autumn of 1999, creel-caught lobsters from the Sound of Jura (west Scotland) and other west coast sites were reported to be dying or moribund during vivier transport, with some catches being refused at market due to the opaque appearance of their tail meat (Mr. G. Goldsworthy, personal communication). Signs of this condition were also noted in animals taken directly from creels, and in trawl-caught animals from the Clyde sea area (west Scotland) which were subsequently maintained in aquaria at the University of Glasgow. This study was carried out to establish the epidemiology of this condition in the Scottish west coast Nephrops norvegicus fishery and to describe its pathology and progression by using histological and biochemical techniques. Methods. Norway lobsters showing symptoms of the described condition (opaque abdominal musculature and inability to flex the abdomen) when caught in creels in the Sound of Jura (see Fig. 1) were collected from Loch Fyne Fisheries Co., Tarbert, west Scotland, UK. Trawl-caught lobsters from the Clyde sea area (Fig. 1), were maintained post-capture in the aquarium at the Division of Environmental and Evolutionary Biology, University of Glasgow, Scotland, UK. Clyde lobsters were fed ad libitum on mussels (Mytilus edulis) until preparation of abdominal muscle for histology and SDS-PAGE, while muscle tissue from the Sound of Jura lobsters was immediately prepared for histology. In order to establish the rate of progression of this condition, 200 lobsters captured in the Clyde sea area in January 2000 by standard otter trawling (Fig. 1) were selected randomly from a sub-sample and assessed for signs of muscle opacity immediately following capture, and again four hours post-capture, after holding them on the deck in a standard fish box covered with a damp sack. In those showing symptoms of the condition, individual segments of the abdomen were visually assessed for the location of muscle opacity. In 134 Chapter Seven - Muscle Necrosis order to investigate possible reasons for the onset of the condition within the abdomen, any damage to the integument in the vicinity of the necrosis was recorded by probing the area with a blunt dissection needle. All lobsters were also staged for infection by the dinoflagellate parasite Hematodinium sp., which is known to affect N. norvegicus in these regions (Field & Appleton, 1995). All lobsters were diagnosed as Stage 0 (uninfected). Histological samples from these animals were compared to previously collected muscle samples from Hematodinium-infected lobsters. Histology The deep flexor and medial superficial flexor muscles from the abdomen of N. norvegicus were exposed by removal of the abdominal cuticle and underlying extensor muscles. Small blocks of opaque muscle and muscle fibre bundles showing opacity along part of their length were removed under a dissecting microscope. Muscle tissue samples were also taken from animals diagnosed with Hematodinjum infection. Muscles were fixed for two hours in a solution containing 4% glutaraldehyde and 2% paraformaldchyde in 0.1 M phosphate buffer, pH 7.4, with 2% sucrose and 1.5 % sodium chloride for 2 hours at room temperature. Fixed samples were then rinsed in 0.1 M phosphate buffer with 4% sucrose and post-fixed in I% osmiurn tetroxide in phosphate buffer for I hour. Specimens were washed in several changes of distilled water and block stained in 0.5 % u=yl acetate for I hour. Following dehydration through an ethanol series, specimens were embedded in Spurr resin (Spurr, 1969). Thick sections (I ýim) were stained with toluidine blue for viewing with a light microscope and suitable areas were cut and mounted on uncoated copper/palladium grids and stained with uranyl acetate and lead citrate. Thin sections (60- 70 nm) were examined in a Zeiss 902 transmission electron microscope. 135 Chapter Seven - Muscle Necrosis SDS-PAGE For SDS-PAGE analysis of deep abdominal flexor muscle from lobsters showing symptoms of muscle opacity, individual muscle fibres were dissected out under calcium-free Nephrops physiological saline and placed into 200 ýtl of SDS-sample buffer, denatured at 95C for 4 minutes and stored at -20'C until electrophoretic protein separation (see Neil el al. 1993). Discontinuous SDS-PAGE was performed according to the method of Laemmli (1970) with gels containing 12.5 % acrylamide separating gel and 4% acrylamide stacking gel. Electrophoresis running conditions were as for those described by Neil et al, (1993), followed by overnight incubation in 10% TCA and staining for 30 minutes in Coomassie-blue solution. Stained gels were later imaged using an Appligene TM gel imager. Opaque muscle fibres were compared to fibres from lobsters showing no symptoms of muscle opacity. Results. In the early stages of the condition in Nephrops norvegicus, individual muscle fibres of the abdomen appeared opaque when viewed through the ventral membrane, with the opacity progressing to whole muscle fibre bundles and eventually the whole abdomen. In advanced cases, the abdomen did not respond to normal physical stimuli and thus affected lobsters could not initiate a normal 'tail-flip' response, while the thoracic limb system appeared to function normally. Progression of the condition was rapid, leading to death of affected lobsters within days. Light microscopy revealed that in the abdominal superficial flexor muscles of animals showing early stages of the pathology (individual opaque muscle fibres), the peripheral areas of the muscle appeared damaged, with disruption to the sarcolemma and infiltration of the damaged regions by granulocytes (Fig. 2a). However, other areas contained intact myofibrillar structure, with regular sarcomere arrangements. The deep abdominal flexor 136 Chapter Seven - Muscle Necrosis muscles, which form the major muscle mass within the abdomen, were also progressively affected by the necrosis, and in severe cases there was a complete loss of sarcomeric structure and a condensation of myofibrillar bundles (Fig. 2b). In some instances, the pathology was clearly progressing along individual myofibrillar bundles, with areas of semi-intact muscle being found immediately adjacent to areas of complete muscle degeneration, the latter being heavily infiltrated by granulocytes (Fig. 2c). Examination of the necrotic tissue using electron microcopy revealed that the normal sarcomcric structure observed in unaffected animals (Fig. 3a) was initially altered by expansion of the space occupied by the tubular system and the appearance of granular material in the cytoplasm (Fig. 3b). Regions of the muscle with a fairly intact myofibrillar structure also displayed some erosion of the sarcomcrcs, with degeneration originating in the vicinity of the Z-line (Fig. 3c). Penetrating between the myofibre interstices of relatively unaffected bundles were sinuous structures, containing filaments (Fig. 2d and 4a). In regions of pathological progression, the junctions between intact and degenerated muscle (see also Fig. 2b) were characterized by a dissolution of myofibrillar bundles into granular material (Fig. 4b). In areas of severe muscle necrosis in N. norvegicus, there was a partial to complete loss of myofibrillar and sarcolemmal structure, with components of the myofibrillar membrane system often the only recognizable feature in the granular cytoplasm separating islands of densely-staining, condensed myofibrillar bundles (Fig. 5a). Necrotic regions of muscle were also characterized by aggregations of atrophied mitochondria within the granular cytoplasm (Fig. 5b), the appearance of myelin figures (Fig. 5c) and the infiltration of the damaged tissue by granulocytes (Fig. 2c and 5d). Such areas occasionally contained unidentified inclusions measuring 100-150nm in diameter (Fig. 6). A certain degree of muscle breakdown was also found to occur in the abdominal muscles of N. norvegicus heavily infected by the dinoflagellatc parasite Hematodinium (Fig. 3d). 137 Chapter Seven - Muscle Necrosis However, in contrast to the effects of necrosis, this mainly involved erosion of groups of myofilaments within the sarcomeres, with the Z-Iine region remaining relatively unaffected. SDS-PAGE analysis of the necrotic condition revealed a major loss of contractile proteins from opaque deep abdominal flexor muscle fibres compared with their amounts in unaffected fibres (Fig. 7). 'I'lie myosin heavy chain was completely absent, while paramyosin, troponin-T, actin, tropomyosin, troponin-I isoforms and the myosin (x-light chain were significantly reduced in quantity. in a study of trawled N. norvegicus, immediately following capture the onset of the necrotic pathology was seen in at least one abdominal muscle fibre bundle in 8% of the 200 lobsters. When these animals were re-examined 4 hours later, after holding them in a covered fish box on deck, this prevalence had increased to 29 % (Fig. 8a). In the affected lobsters, opacity was found in various abdominal segments, with those adjacent to the ccphalothorax being the most commonly affected (Fig. 8b). It is perhaps significant that visible damage to the abdominal integument (tearing or puncturing) was observed in the vicinity of opaque lesions in 46.5 % of the lobsters with symptoms of muscle necrosis. Small, undetectable lesions or minor damage to the main carapace may have been present in other animals, though this was not investigated. Discussion. Lack ofcausative agent The current study has identified a post-capture, abdominal muscle necrosis of rapid onset in the Norway lobster, Nephrops norvegicus captured from the west coast of Scotland, Externally, the condition resembles that of a microspordian infection. These arc common pathogens of crustacean tissue, often causing severe softening, opacity and general degeneration of the musculature (Breed & Olson, 1977; Findley et al., 1981; Olson & Laman, 1984; Langdon, 138 Chapter Seven - Muscle Necrosis 1991; Dennis & Munday, 1994; Childers et al., 1996). However, a number of features of the observed necrosis are counter-indicative of microsporidian involvement. Firstly, progression of microsporidiosis tends to be much slower than that observed for the condition in N. norvegicus. Breed & Olson (1977) report that progression from a light to a heavy microsporidian infection takes approximately 120 days in crangonid shrimps. The condition observed in N. norvegi . cus in this study had a much shorter time course, progressing significantly over a period of 4 hours following trawl capture, and leading to death within a further 34 days (a feature also noted for spontaneous muscle necrosis in P. aztecus - Rigdon & Baxter, 1970). Secondly, no microsporidian life stages were visible in any of the histological sections of abdominal muscle tissue, examined by either the light- or electron-microscopy. Indeed, no other pathogenic agents were detected within the abdominal musculature, although in certain specimens some unidentified bodies of approximately 150nm in diameter were detected within severely damaged regions of muscle (see Fig. 6). The possibility that these bodies are viruses cannot be ruled out at present, especially as viruses are known to cause a similar necrosis condition in post-larval stages of the freshwater prawn, M rosenbergil (Arcicr et al., 1999). The identity of these bodies and their possible involvement in the muscle necrosis in N. norvegicus requires ftirther investigation. There is also no evidence for any bacterial involvement in the pathology seen in N. norvegicus, though bacteria have also been implicated in a similar muscle necrosis and rapid death syndrome in M rosenbergii (Chcng & Chen, 1998) and may also be a secondary consequence of necrosis in the same species (Nash et al., 1987). The pathology ofnecrosis Due to absence of any obvious causative agent, the seasonal nature of the condition in crcel- caught animals (appearing in the summer and autumn) and the propcnsity for trawled animals to become affected even during the winter months, the opaque muscle condition noted in N. norvegicus resembles the spontaneous, idiopathic muscle necrosis notcd in a number of 139 Chapter Seven - Muscle Necrosis crustacean species; in Cherax terminatus (Evans eral, 1999), Mrosenbergii (Delves-Broughton & Poupard, 1976; Akiyama et al., 1982; Nash et al., 1987; Anderson et al., 1990), Penaeus aztecus (Rigdon & Baxter, 1970, Lakshmi et al. 1978), Penaeus Japonicus (Momoyama & Matsuzato, 1987), Procambarus clarkii (Linqvist & Mikkola, 1978) and also in the fish Stizostedion vitreum (Holloway & Smith, 1982). Indeed, histological features of the condition in X norvegicus, including a general loss of myofibrillar and sarcomeric structure, coupled with differential reaction of necrotic muscle tissue to histological staining, are similar to those reported in muscle necrosis of P. Japonicus (Momoyama & Matsuzato, 1987) and P. aztecus (Rigdon & Baxter, 1970). Ilie appearance of sinuous filaments with a fibrous structure is similar to that noted during muscle necrosis in P. japonicus, where the production of collagenous fibres was extensive in mid to late progression of the condition (Momoyama & Matsuzato, 1987). The presence of myelin figures and atrophied mitochondria in conjunction with infiltration of degenerated areas of tissue by granulocytes is consistent with the features ascribed to idiopathic muscle necrosis in M. rosenbergii (Nash et al. 1987). Loss of Z-Iine material, which is a prominent feature of necrosis in N. norvegicus, is reported to occur in a number of pathological and physiological conditions (Kumudavalli Reddy el al., 1975) and also represents an early step in pre-moult muscle atrophy in crustaceans (Mykies & Skinner, 1990a). The calcium-dependent proteases involved in pre-moult atrophy are localized in the sarcoplasm (Mykles & Skinner, 1990b) and it is conceivable that the necrotic condition observed in N. norvegicus involves activation of these proteascs, leading to the initial breakdown of Z-Iine material. Interestingly, the breakdown of abdominal muscle fibres induced by Hematodinium infection in N. norvegicus does not disrupt the Z-line, but rather involves the erosion of groups of myofilaments within the sarcomeres. This suggests that the protcolytic processes that occur in this parasitic infection are different to those occurring in necrosis. 140 Chapter Seven - Muscle Necrosis Factors which induce the onset ofnecrosis. This study has shown that muscle opacity can be observed in all segments of the abdomen, but with a tendency for the condition to occur in the segments adjacent to the cephalothorax. A similar feature was noted in idiopathic muscle necrosis in Penaeus Japonicus (Momoyama & Matsuzato, 1987) and C tenuimanus (Evans et al., 1999), while others have reported the necrotic condition to be associated with the more distal abdominal segments in P. aztecus (Rjgdon & Baxter, 1970, Lakshmi et al., 1978) and M rosenbergii (Akiyama et al., 1982, Nash et al., 1987). The mechanism underlying the location of lesions is not known, but it is possible that breaches in the integument (seen in almost half of those lobsters exhibiting symptoms of the pathology) may provide foci for initiation of necrosis, the progression of which is exacerbated by subsequently imposed stressors. it is well known that crustaceans are exposed to a considerable array of stressors during and after capture (including crowding, air exposure, light exposure, heat exposure and mechanical damage) and a number of studies have considered the expression of biochemical and molecular stress indicators during commercial handling of crustaceans (see Jussila et al., 1997, Paterson & Spanoghe, 1997 and Chang et al., 1999). N. norvegicus will certainly be exposed to such stresses during trawl capture. Most studies of spontaneous muscle necrosis in crustaceans also report an induction of hyperactivity in animals prior to the onset of necrosis. Such hyperactivity in animals which are habitually sedentary is a common response to a number of stressors, and is followed by exhaustion and elevated levels in hacmolymph L-lactatc, which can remain elevated for 24 to 48 hours after the stressor has been removed (see Nash et OL, 1987). Extreme exertion in strongly glycolytic muscles such as the deep abdominal flexor muscle also causes rapid utilization of glycogen, generating local heat and lactic acid. both of which arc known to have degenerative effects on the affected and the surrounding muscle fibres (Hulland. 1985). It is therefore conceivable that during capture and handling of N. norvegicus. the repetitive 'tail flipping' that is commonly induced could be causing these types of effect. Initiation of the 141 Chaptcr Seven - Muscle Necrosis condition, possibly caused by damage to the abdominal integument, may then be exacerbated by further stressful post-capture holding conditions. Once the condition in N. norvegicus muscle is established and begins to progress, it does so extremely rapidly, with complete necrosis of the tail muscle and death within days. It has been suggested that rapid progression of necrosis may be linked to the infiltration of the muscle by activated granulocytes and their subsequent production of superoxide radicals, which induce lipid peroxidation, damage membranes and kill cells (Fridovich, 1978; Di Giulio et al., 1989. Nash et al., 1987). However, the role of 02- and other reactive oxygen intermediates in the necrosis of N. norvegicus muscle remains to be established. It has been noted in P. aztecus that the appearance of muscle necrosis can be reversed if the environmental stressors; are removed soon after onset of the condition (Rigdon & Baxter, 1970, Lakshmi et al., 1978). The data presented in this study for the prevalence of muscle opacity immediately after trawl capture. and 4 hours later (Fig. 8a) reinforce the theorv that the holding conditions of the animals in the period immediately following capture are crucial in determining whether the necrotic condition develops or regresses. This has important consequences both for the vivier transport of live N. norvegicus, and the quality of the meat in 'tailed' lobsters. It may also contribute to the high mortality of discarded N. norvegicus which arc returned to the sea after trawl capture and several hours of cmersion (Ulmestrand et al., 1998). The apparent effect of damage to the integument and the reported reversal of the similar necrotic condition in A aztecus requires further investigation in N. norvegicus and may lead to advisory measures for the post-capture handling of this commercially important species. 142 Chapter Seven - Muscle Necrosis References. Akiyama, D. M., Brock, J. A. & Haley, S. R. (1982). Idiopathic muscle necrosis in the cultured freshwater prawn (Macrobrachium rosenbergh). Veterinary Medicine and Small Animal Clinician 77,1119-112 1. Anderson, I. G., Nash, G. & Shariff, M. (1990). Mass larval mortalities in giant freshwater prawn, Macrobrachium rosenbergii de Man, cultured in Malaysian modified static 'green water' systems. Journal of Fish Diseases 13,127-134. Arcier, J-M., Herman, F., Lightner, D. V., Redman, R. M., Mari, J. & Bonami, J-R. (1999). A viral disease associated with mortalities of hatchery-reared postlarvae of the giant freshwater prawn Macrobrachium rosenbergii. Diseases of Aquatic Organisms 3 8,177-18 1. Breed, G. M. & Olson, R. E. (1977). Biology of the microsporidian parasite Pleistophora crangonj n. sp. in three species of crangonid sand shrimps. Journal of Invertebrate Pathology 3 0, 387405. Cawthorn, R. J. (1997). Overview of "bumper car" disease - impact on the north American lobster fishery. International Journal of Parasitology 27 (167-172). Chang, E. S., Chang, S. A., Keller, R., Sreenivasula Reddy, P., Snyder, M. J. & Spees, J. L. (1999). Quantification of stress in lobsters : crustacean hyperglycaernic hormone, stress proteins and gene expression. American Zoologist 39,487-495. Cheng, W. & Chen, J-C. (1998). Isolation and characterization of an Enterococcus-like bacterium casing muscle necrosis and mortality in Macrobrachlum rosenbergit in Taiwan. Diseases of Aquatic Organisms 34,93-10 1. Childers, R. K., Reno, P. W. & Olson, R. E. (1996). Prevalence and geographic range of Nadelspora canceri (Microspora) in dungeness crab Cancer magister. Diseases of Aquatic Organisms 24,135-142. Delves-Broughton, I& Poupard, C. W. (1976). Disease problems of prawns in recirculation systems in the U. K. Aquaculture 7,201-217. Dennis, D. M. & Munday, B. L. (1994). Microsporidiosis of palinurid lobsters from Australian waters. Bulletin of the European Association of Fish Pathologists 14,16-18. Di Giulio, R. T., Washburn, P. C., Werining, R. J., Winston, G. W. & Jewell, C. S. (1989). Biochemical responses in aquatic animals: a review of determinants of oxidative stress. Environmental Toxicology & Chemistry 8,1103-1123. Evans, L. H., Fotedar, S., Fan, A. & Jones, B. (1999). Investigation of idiopathic muscle necrosis and circulating hemocytes in the freshwater crayfish Cherax tcnuimanus exposed to acute and chronic stressors. Freshwater Crayfish 12 (in press). Field, R. H. & Appleton, P. L. (1995). A Hematodinium-like dinotlagcllate infection of the Norway lobster Nephrops norvegicus : observations on pathology and progression of infection. Diseases of Aquatic Organisms 22,115-128. 143 Chapter Seven - Muscle Necrosis Findley, A. M., Blakeney, Jr. E. W. & Weidner, E. H. (1981). Ameson michaefis (Microsporidia) in the blue crab, Callinectes sapidus: parasite induced alterations in the biochemical composition of host tissues. Biological Bulletin 161,115-125. Fridovich. 1. (1978). The biology of oxygen radicals. Science 201.875-880. FRS (1999). Nephrops stocks around Scotland. Fisheries Research Service (FRS) fish stocks information leaflet; December 1999 Holloway, Jr. H. L. & Smith, C. E. (1982). A myopathy in North Dakota walleye, Stizostedion vitreum (Mitchill). Journal of Fish Diseases 5,527-530. Hulland, T. J. (1985). Muscles and tendons. In: Pathology of domestic animals, Vol. I (eds. Jubb, K. V. F., Kennedy, P. C. & Palmer, N. ). Academic press, London. pp. 139-199 Jussila, J.. Jago, J., Tsuetnenko, E., Dunstan, B. & Evans, L. H. (1997). Total and differential haemocyte counts in western rock lobsters (Panulints cygnits George) under post-harvest stress. Marine & Freshwater Research 48,863-7. Kabre, G. (1992). Effects of the microsporidian Thelohania conlejeani on the metabolism of total muscular and hepatopancreatic phospholipids in the crayfish Austropotamobius pallipes, Le Reboulet, 1858. Annales des Sciences Naturelles, Zoologie 13,97-102. Kumudavalli Reddy, M., Edinger, J. D., Rabinowitz, M. & Fischman, D. A. (1975). Removal of Z-Iines and oc-actinin from isolated myofibrils by a calcium-activated neutral protease. Journal of Biological Chemistry 250,42784284. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 227,180-5. Lakshmi, G. J., Venkataramiah, A. & Howse, H. D. (1978). Effect of salinity and temperature changes on spontaneous muscle necrosis in Penaeus aztecus Ives. Aquaculture 13,35-43. Langdon, J. S. (1991). Microsporidiosis due to a pIcistophorid in marron, Cherax tenulmanus (Smith) (Decapoda: Parastacidae). Journal of Fish Diseases 14,3344. Lindqvist, O. V- & Mikkola, H. (1978). On the etiology of the muscle wasting disease in Procambarus clarkii in Kenya. Freshwater Crayfish 4,363-372. Momoyama, K. & Matsuzato, T. (1987). Muscle necrosis of culture kuruma shrimp Ulenaeus japonicus). Fish Pathology 22,69-75. Mykles, D. L. & Skinner, D. M. (1990a). Calcium-dependent proteinases in crustaceans. In Mellgren R. L. & Murachi T. (eds). Intracellular Calcium-Dependent Protcolysis. Boca Raton: CRC Press pp. 139-154. Mykles, D. L. & Skinner, D. M. (1990b). Atrophy of crustacean somatic muscle and the proteinases that do the-job. A review. Journal of Crustacean Biology 10,577-594. 144 Cliaptcr Sc%, cn - Muscic Nccrosis Nash, G., Chinabut, S. & Limsuwan, C. (1987). Idiopathic muscle necrosis in the freshwater prawn, Macrobrachium rosenbergii de Man, cultured in Thailand. Journal of Fish Diseases 10, 109-120. Neil, D. M., Fowler, W. S. & Tobasnick, G. (1993). Myofibrillar protein composition correlates with histochernistry in fibres of the abdominal flexor muscles of the Norwav lobster Nephrops norvegicus. Journal of Experimental Biology 183,185-20 1. Olson, R. E. & Laman, C. N. (1984). Prevalence of microsporidian infection in commercially caught pink shrimp, Pandalusjordani. Journal of Invertebrate Pathology 43,407413. Paterson, B. D. & Spanoghe, P. T. (1997). Stress indicators in marine decapod crustaceans, with particular reference to the grading of western rock lobsters (Panulints cygnus) during commercial handling. Marine & Freshwater Research 48,829-834. Rigdon, R. H. & Baxter, K. N. (1970). Spontaneous necroses in muscles of brown shrimp Penaeus aztecus Ives. Transactions of the American Fisheries Society 3,583-7. Shields, J. D. (1994). The parasitic dinoflagellates of marine crustaceans. Annual Review of Fish Diseases 4,241-27 1. Spurr, A. R. (1969). A low viscosity epoxy embedding resin for electron microscopy. Journal of Ultrastuctural Research 26,3143. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (2000). Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Nonvay lobster, Nephrops norvegicus L. during infection by a parasitic dinoflagellatc of the genus Hematodinium. Diseases of Aquatic Organisms (in press). Stewart, J. E. (1980). Diseases. In: Cobb, J. S. & Phillips, B. F. (eds. ) The Biology and Management of Lobsters. Academic Press, New York, pp. 301-342. Ulmestrand, M., Valentinsson, D., Sangster, G. I., Bova, D., Kynoch, R. J., Brccn, M., Graham, G. N., Soldal, AN., Cruikshank, 0., Moth-Poulson, T. & Lowry, N. (1998). Nephrops survival after escape from commercial fishing gear or discarded from dcck. ICES Nephrops life history study group meeting. La Corufla, Spain. 1998. 145 Chapter Seven - Muscle Necrosis Fig. l. Map of western Scotland showing the capture sites in the Sound of Jura (A) and the Clyde sea area (B). The University Marine Biological Station Millport is situated on the Isle of Cumbrae in the Clyde estuary. SrN 56*N SSON 146 Chapter Seven - Muscle Necrosis Fig. 2 (A-D). (A) Superficial flexor muscle from lobster showing early symptoms of pathology. Note the intact sarcomeres; and adjacent region of necrotic muscle showing infiltration by granulocytes. Scale bar = 40 ýLin (B). Deep flexor muscle from lobster showing late stage necrosis. Note the junction between relatively intact and disorganized muscle (arrow), condensed myofibre bundles and granulocyte infiltration Scale bar = 80 ýLm (see also Fig. 21)). (C) Fibrous processes at the junction between intact and disorganized muscle (arrow) Scale bar = 30 ýtm (D). Infiltration of granulocytes into necrotic regions of DAF muscle. Scale bar = 20 ýLm. Chapter Seven - Muscle Necrosis Fig. 3 (A-D). (A). Electron micrograph of normal DAF muscle showing regular sarcomeric structure, clear Z-line (arrow) and well-developed tubular system (T). Scale bar =I pm (B). Electron micrograph of DAF muscle from lobster showing early symptoms of muscle necrosis, Note the expansion of the spaces between adjacent fibre bundles, disruption of the tubular system and appearance of granular material in the cytoplasm (arrow). Scale bar =I pm (Q. Electron micrograph from lobster showing progressing signs of necrosis. Note the disorganized appearance of myofibre bundles, expansion of the inter-bundle spaces and degeneration originating at the Z-line (arrows). Scale bar =I pm (D). Electron micrograph of superficial flexor muscle from Hematodinium-infected lobster. Note the clear erosion of material from within the sarcomere (E) and the intact Z-lines (arrows) Scale bar =I pm. 148 Cliaptcr Seven - Muscle Nccrosis Fig. 4 (A, B) (A). Electron micrograph showing fibrous protrusion in DAF musclc of lobster showing early symptoms of necrosis. Scale bar =I ýtrn. (B). Electron micrograph of junction between relativeiN, intact and severelv necrotic DAF muscle. Note tile loss of normal sarconicric organization and appearance of granular material in the region of the Junction (arrows). Scale bar =I [tm. 149 Chapter Sewn - Mtisclc Nccrosis Fig. 5 (A-D). (A). Electron micrograph of densely staining DAF muscle fi-orn lobsters shoxving severe symptoms of necrosis. Note the erosion of bundles (E) and the presence of granular material (arroxv). Scale bar =I ýtm (B). Electron micrograph of atrophied 1111tocliondria \\ ithin the granular cNloplasm of heavily necrotic DAF muscle. Scale bar =I ýuii (C) Electron micrograph of myelin figures within the granular cytoplasm of licavily necrotic DAF 11111scic Scale bar =I ýtm. (D). Electron micrograph of a large (L) and a small (S) granulocyte ill the cytoplasm of heavily necrotic DAF muscle. Scale bar =I ýim. 150 Chapter Seven - Muscle Necrosis Fig. 6. Electron micrograph of unidentified bodies in the cytoplasm of heaviIN nccrotic DAF muscle. Scale bar =I ýtm. 151 Chapter Seven - Muscle Nccrosis Fig. 7.12.5% SDS-PAGE gel of contractile proteins from an unaffected muscle fibre (lane B) and a necrotic muscle fibre (lane Q from N. norvegicus. Lane C contains a molecular weight marker (weights in kDa given on left of gel). Kev: MHC (nivosin heavv chain). P (paramyosin 1 2), HP (liacmolyniph proteins), TN-T (troponin-T), A (Actin), TM (tropornyosin), TN- I (troponin-1) and Ma-LC (myosin ct-light chain). A 205 116 97.4 66 45 B mlic p lip TN-T A I IM 29 TN-1 (4) M(x-L(' C li2 Chapter Seven - Muscle Necrosis Fig. 8 (A, B). (A). Chart showing the prevalence of muscle opacity symptoms in the abdomens qf N. norvegicus immediately following (0 hour) and 4 hours following trawl capture. (B). Chart showing the percentage of N. norvegicus exhibiting muscle opacity in different abdominal segments 4 hours following trawl capture. Addition of percentages to above 100% indicates that lobsters regularly showed muscle opacity in more than one abdominal segment. Male B 60 50 ý 40 ý 0 30 20 10 0-' Female Total 2nd 3rd Ah 5th 6di 153 Chapter Eight - Swimming performance Chapter 8 An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infected by a parasitic dinoflagellate of the genus Hematodinium. ' Abstract. Various components of swimming performance were measured in uninfected Norway lobsters (Nephrops norvegicus) and compared to animals at different stages of infection by a parasitic dinoflagellate (Hematodinium sp. ). Animals showed a progressive decline in overall swimming performance as infection severity increased, with reductions in the number of tail-flips performed, the number of swimming bouts and the total distance travelled by swimming. The velocity of the first (giant-fibre mediated) tail flip and average velocity over the swimming bout were also significantly reduced in infected lobsters. Possible reasons for this decreased swimming performance are suggested and the implications of this for predator avoidance of infected lobsters in the benthic habitat, and for capture by Nephrops by trawl rigs are discussed. Published as: Stentiford, G. D., Neil, D. M., Atkinson, R. J. A. & Bailey, N. (2000). An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infected by a parasitic dinoflagellate of the genus Hematodinium. Journal of Experimental Marine Biology & Ecology 247,169-18 1. 154 Chapter Eight - Swimming performance Introduction. 7ne Norway lobster Nephrops norvegicus, hereafter referred to by genus alone, like other species of lobster and like crayfish, can react to a visual or tactile stimulus by undertaking a series of 'tail flips' which propel the animal backwards through the water (Neil & Ansell, 1995). The main function of such a response is to remove the animal from a threatening situation, such as a predator. However, in the case of Nephrops, the response may facilitate avoidance of capture by trawlers (Newland & Chapman, 1989). 'Me general 'tail flip' response of Nephrops has been well studied: upon abrupt mechanical stimulation of the rostrum or telson, Nephrops performs a series of tail flips, the first of which is preceded by giant fibre activity in the abdominal nerve cord (Newland & Neil, 1990a, 1990b). This provides one of the best known examples of a stereotyped motor act (Newland et al., 1992). Such information has been used to show that the ability of Nephrops to evade trawler capture depends strongly on its ability to initiate a successful 'tail flip' response, although other factors such as its orientation to the trawl gear, proximity to the burrow and its ability to endure prolonged swimming are also important (Newland & Chapman, 1989). Nephrops supports a major fishery in the United Kingdom, with the Scottish fishery contributing over 76 % of this (07 million in 1999 - FRS, 1999). Recently, stocks of Nephrops off the west coast of Scotland have been shown to harbour a seasonal infection by a parasitic dinoflagellate of the genus Hematodinium (Field et al., 1992) which is responsible for a disease syndrome resembling the 'Bitter Crab disease' (BCD) reported in Alaskan tanner crabs (Chionoeceles bairdi) (Meycrs et al., 1987,1990) and also Hematodinium infections in other decapod hosts (Newman & Johnson, 1975. Wilhelm & Boulo, 1988; Latrouite et al., 1988; Shields, 1992; Hudson et al., 1993). Nephrops showing symptoms of patent infection are seen in catches taken in spring and early summer (Field & Appleton, 1995). A number of studies have established the basic characteristics of Hematodinium-infection 155 Chapter Eight - Swimming pcrformance (Field et al. 1992), its progression, diagnosis and pathology (Field & Appleton, 1995, 1996) and its effect on host physiology (Taylor et al., 1996). It has been noted that the parasite partially invades the main flexor muscles of the abdomen (Field & Appleton, 1995), suggesting that there may be some reduction in its ability to produce tail flips, which could in turn lead to altered swimming performance during patent infection. Reduced swimming performance during infection may alter the ability of Nephrops to evade capture by trawlers and predators and thus has obvious commercial and ecological implications for affected populations. Resultant adjustment of catchability, expressed as fishing mortality (F) per unit of fishing effort (E) (i. e. F/E) also has implications for the quantitative assessment of populations exhibiting significant disease prevalence. The present study was therefore carried out to determine whether changes can be observed in the swimming performance of Nephrops when infected by Hematodinium. Data are discussed in relation to the capture of infected animals. Methods. Collection and treatment ofanimals. Norway lobsters were collected with an otter trawl from a location south of Little Cumbrae in the Clyde Sea area, Scotland, UK. Lobsters were maintained in a closed aquarium (90C. 33 ppt salinity) in the Division of Environmental and Evolutionary Biology, University of Glasgow, before transportation to holding tanks (9"C, 33 ppt salinity) in the Fish Behaviour Unit (FBU) at the Fisheries Research Services Marine Laboratory Aberdeen (MLA), Scotland, UK. Animals were fed ad libitum on squid (Loligo spp. ) on arrival at MLA and again three days later. The carapace length of animals used was between 25 and 30 mm, to avoid known sizc-specific differences in swimming performance (Newland et al., 1988). Nephrops of both sexes were used as sex-specific differences in swimming performance are not seen unless the female is ovigerous (Newland et al., 1988). Animals were not fed for three days prior to the experiment to 156 Chaptcr Eight - Swinuning pcrformancc avoid any effects of differential feeding. All animals Nvere in the intermoult state as defined by Aiken (1980). Experimental set-up. Experiments designed to examine the effect of Hematodinium infection on various parameters of swimming performance were carried out in a 12m dumbbell-shaped tank housed in the FBU of MLA (see Fig. 1). A frame carrying three video cameras (D 1, D2 and D3) (Panasonic WV- CL350) fitted with 6.5mm auto-iris lenses was constructed along the length of tank area D (Fig. 1). A hand-held integrator switch (Primebridge PVS-I) allowed for switching between the different cameras, thereby keeping the animal in the field of view. The integrator output was linked to a time and date generator (Mitsibushi TDG-IOB) before being recorded on a standard VHS video recorder. A distance marker (10cm intervals) was placed along the length of tank area D and the tank was illuminated with standard halogen lighting fitted with dimmer switches. Experimental protocoL Animals were housed in tank area B (Fig. 1) prior to being individually removed in a net and placed into tank area C (settlement area) for 10 minutes. After settlement, each animal was admitted to tank area D (recording run) and subjected to front-end stimulation (rostrum taps) using a plastic rod. The three video cameras and switching box allowed the entire swimming sequence to be recorded. At the end of a trial, as determined by a failure to respond to three successive taps to the rostrum, each animal was removed from the run, measured, sexed and staged for Hematodinium infection using the pleopod staging method of Field & Appleton (1995). According to this method, Stage 0 denotes an apparently uninfected animal, with patent infection progressing from Stage I through to Stage 4. Stage 3 and 4 (heavily infected) animals were grouped in all analyses as animals in these groups show very similar disease characteristics (see Stentiford et al., 1999- Chapter 4). A 'bout' 157 Chaptcr Eight - Swimming pcrfonnancc of swimming is defined as the series of full flexions and extensions between tactile stimulation and rest and a 'flip' as one cycle of flexion and extension of the abdominal segments (Newland el al., 1988) Video Analysis. Recordings of Nephrops swimming performance (73 animals) were analyzed at the Division of Environmental and Evolutionary Biology at the University of Glasgow. The total number of tail flips, total number of bouts, number of flips per bout, total distance covered, overall swimming velocity, velocity of the giant and subsequent flip and flip frequency (Hz) were measured using a Panasonic AG-5700 VCR linked to a Panasonic AG-570 single-frame editing facility controller and a Panasonic WV-CM1000 colour monitor. Measurements of tail-flip velocity and flip frequency were made on a total of 46 animals with different stages of Hematodinium infection. An acetate overlay was placed on tile monitor screen and the position of the rostrum at the start position and at the initiation points of subsequent tail flips (tail extension following full flexion) were marked (see Amott et al., 1998). The number of frames (20 milliseconds) between each rc-extension was recorded and the distance covered between marked points was calculated from the distance marker on the tank floor. Overall velocity was measured over one complete bout within the first three bouts of the swimming sequence and from this, the velocity of the first flip (giant-fibre mediated) and subsequent flip (non-giant fibre mediated) was calculated. Tail flip frequency (Hz) was calculated by dividing the number of flips in one bout by the time taken up to the initiation of the final flip of a given bout. Data Anaývsis. Comparisons of swimming parameters from uninfected and infected Nephrops were 158 Chapter Eight - Swinuning performance performed either by one-way analysis of variance (ANOVA) followed by between stage comparisons with a Tukey's pairwise analysis (for normal distributed data), or by a Mann- Whitney test (for non-normal distributions). Tests were considered significant if p<0.05. Results. Video recordings from a total of 73 uninfected and infected lobsters were analysed for total number of tail flips, total number of bouts, number of tail flips per bout and total distance travelled, while swims by 46 animals were analysed for overall mean vclocit%-. velocity of the giant flip, number of non-giant flips per bout and flip frequency. The mean number of flips performed by animals in response to the standard stimulus regime was reduced in relation to the severity of infection (Fig. 2). Uninfected animals flipped a mean of 140.2 times, with a reduction to 26.2 times in Stage 3-4 infection (p < 0.001). There were significant differences between all stages of infection: Stage I and 2 (p < 0.001) and Stage 2 and 3-4 (p < 0.05). The mean number of bouts was also reduced in relation to the severity of infection (Fig, 3), from 57.9 in uninfected animals to 12.2 in Stage 3-4 infection (p < 0.001). Differences in this measure were significant between Stage 1 (43.9) and 2 (27.9) (p < 0.01) and Stage 2 and 3-4 (p < 0.01). Owing to these similar trends. the number of flips per bout changed less, although there was still a significant difference between uninfected (2.6 flips. bout*') and Stage 3-4 animals (1.8 flips. bout")(p > 0.05) and between Stage I animals (2.7 flips. bout"') and Stage 3-4 (p < 0.05). The total distance travelled in the tail flip sequences also reduced with infection (Fig. 4), from 24.05m in uninfected animals to 3.24m in heavily infected (Stage 3-4) animals (p < 0.001). The distance travelled was not significantly reduced in Stage I infection (20.56m) when compared with uninfected animals (p = 0.065), but was significantly reduced in Stage 2 (10.13m) (p < 0.00 1) and in Stage 34 (3.24m) (p < 0.00 1). 159 Chapter Eight - Swimming performance There was also a highly significant reduction in distance travelled between Stage I and 2 (p < 0.001) and between Stage 2 and 34 (p < 0.01). Figure 5 shows a typical bout velocity profile from an uninfected (Stage 0) and a heavilv infected (Stage 34) Nephrops. From the start position, both animals reach peak velocity with the first (giant-fibre mediated) flip. This velocity is then maintained, albeit slightly reduced, until the final flip, following which the animal glides to rest (one bout). The bout duration and velocity profile of infected animals is almost identical to that of uninfected animals, but the flip velocity of both the giant and subsequent flips, is greatly reduced b% the infection. The overall mean velocity of a swimming bout (Fig. 6) was not significantly reduced in Stage I infection (0.48 m. sec"') when compared with uninfected animals (0.56 m. sec") (p > 0.05). However, in Stage 2 (0.35 m. sec") and 34 (0.24 m. sec"'), overall velocity of the bout was reduced significantly (both p<0.001). There was also a significant decrease in overall velocity between Stage I and 2 (p < 0.0 1) and between Stage 2 and 34 (p < 0.05). Figure 7 shows the mean velocities of the giant flip and second (non-giant) flip for uninfected and infected animals. The mean giant flip velocity shows similar trends to that for overall mean velocity, with no significant differences between Stage I velocity (0.66 m. sec") and Stage 0 velocity (0.75 m. sec*') (p > 0.05) but highly significant differences between Stage 0 and Stage 2 (0.52 m. scc") (p < 0.01) and Stage 34 (0.32 m. scc*') (p < o. 001). Tile velocity of the subsequent flip, which is less than that of the giant flip in all cases, is also progressively reduced with the severity of infection, so that the relationship between the giant flip velocity and subsequent flip velocity remains unchanged (data not shown). Any change in the ratio of the number of non-giant (NG) flips to giant (G) flips is likely to influence the overall velocity (Fig. 6) since the NG velocity has been shown to be less than that of G flip velocity (Fig. 7). However, the number of NG flips per bout is not 160 Chapter Eight - Sivinuning performance significantly different, even in heavily infected animals (1.76 versus 1.59 in uninfectcd animals) (p > 0.05). The frequency of tail flips measures the rate at which repeated full tail flexions can be performed. When single bouts were analyzed, flip frequency was approximately 3 Hz in all stages of infection (Fig. 8) and no significant differences in flip frequency (Hz) were found between uninfected and infected Nephrops. Discussion. . fe Swimming performance in relation to Hematodinium in ction. The mean swimming speed of uninfected Nephrops measured in the present stud), (0.56 m. sec-1) is in good agreement with previously published measures made both in the laboratory and in the field (Newland & Chapman, 1989, Newland et al., 1988; Newland & Neil, 1990ab). However, Hematodinium infection causes significant reductions in the overall swimming capacity of Nephrops, which is most clearly expressed in a reduction in the total distance travelled before animals cease to respond. This progressive decline in swimming performance with increasing parasite burden involves a reduction in both the velocity of individual tail flips and the total number of flips performed, and may reflect damage to the muscle contractile proteins, a reduced ability to use available energy or a depletion of energy reserves. However, infected lobsters are still capable of initiating a giant tail flip at the start of each bout, and of performing as many subsequent non-giant flips as uninfected animals at an equivalent frequency, which suggests that the ricuronal circuitry controlling both giant fibre activation and non-giant pattern generation is unaffected by the infection. An alteration in normal muscle contraction could arise as a consequence of the parasite- induced damage of muscle fibres, as described by Field el al. (1992) and Field and Appleton (1995), with parasites penetrating the fibre interstices in late infection (Stage 3 161 Chapter Eight - Swinuning performance and 4) and possibly lysing the peripheral regions of the fibres. A reduced swimming performance would be expected as a result of such loss of contractile machinery, since giant-fibre mediated tail flipping maximally excites these muscles, causing them to operate close to their maximum isometric condition, with little or no reserve capacity. Muscle damage of a similar nature has also been reported as a symptom of Hematodinium. infections of other decapods (Meyers et al., 1987, Hudson & Shields, 1994 and Wilhelm & Mialhe, 1996). Muscle fibre damage could also affect the rate of muscle contraction bv disrupting the steps in excitation-contraction coupling. Although tail-flip frequency remained unchanged in infection, no separate measures of flexion duration were made, and therefore it cannot be excluded that a decrease in the contraction rate of abdominal flexor muscles also contributed to the decrease in propulsive swimming thrusts. Combined measurements of the movements of the abdomen during tail flipping and the propulsive forces that are produced, as performed in the studies of Newland and Neil (1990a, b) and Baden and Neil (1998), are required to resolve these issues. The lower number of tail flips may represent a reduced swimming endurance with progressing Hematodinium infection, since it has been shown that this infection is accompanied by changes in blood composition (Field and Appleton, 1995) and an increased respiratory demand on the host imposed by the large burden of Hematodinium parasites (Taylor et al., 1996). Infected animals may therefore be in a state of persistent partial exhaustion, which would restrict their capacity to respond to the increased respiratory demands of swimming. Elevated levels of muscle and plasma L-lactate are also known to cause a cessation of tail flipping in spiny lobsters (Vermeer, 1987), and in Nephrops recovery of full swimming ability is linked to recovery of near normal haemolymph L-lactate concentrations (Field et al., 1991; Newland et al., 1992). Heavily infected Nephrops have considerably elevated plasma L-lactate concentrations (Taylor et al., 1996), but apparently unaltered muscle L- 162 Chapter Eight - Swimming performance lactate concentrations (Field, 1992), and it is therefore possible that this increased plasma L-lactate is a factor in reducing the swimming endurance of infected animals. Depletion of the metabolic reserves used for active swimming may also play a part in the reduced swimming endurance during Hematodinium infection. Although the concentration and re- synthesis of ATP in abdominal muscle appear not to limit escape swimming endurance in uninfected Nephrops (Field et al., 199 1) in the manner reported for the scallop (Grieshaber, 1978), it is possible that the reduced muscle glycogen concentrations measured during Hematodinium infection (unpublished observations) cause a shortfall in the re-supply of ATP to the contractile machinery, thereby degrading swimming performance. The observed decline in the number of tail flips performed by Nephrops infected by Hematodinium is consistent with an increased rate of habituation in the giant fibre systems. Habituation of giant-fibre systems is a common feature of tail flip swimming in many crustacean species (Neil & Ansell, 1995; Arnott et al., 1998; reviewed by Edwards et al., 1999) including Nephrops (Newland et al., 1988), and is known to be mediated by the neurotransmitter GABA (Krasne & Roberts, 1967; Vu & Krasnc, 1993; Krasne & Tcshiba, 1995). One observation that may link Hematodinium infection to an alteration in the action of a neuronal pathway for habituation mediated by GABA is that free amino acid concentrations in the plasma of Nephrops are changed during infection (Stcntiford et aL. 1999- Chapter 4). Of these changes, an approximately 12-fold increase in the circulating taurine concentration is the most significant, and since taurine is known to have a neuromodulatory action (Oja & Kontro, 1978. Kuriyama et al., 1978), and in crustaceans is known to mimic the inhibitory actions of GABA (Zatta, 1987; Payen et al., 1981), it may contribute to a greater suppression of repeated giant fibre activation, and thus the reduction of tail flipping. 163 Chapter Eight - Swimming performance Reduced swimming performance in relation to trawl capture. Commercial Nephrops trawls are normally towed at speeds of between I and 1.6 m see*' (2-3 knots) and it is suggested that a high proportion of animals directly in line with the mouth of the net will be captured. Due to the observed reduction in swimming performance, it may be suggested that Hematodinium-infected Nephrops at the net periphery and within the net itself may be less likely to escape capture, while some of those directly in-line with the ground gear may evade capture by not swimming up and over the ground gear. Differences in the swimming performance of Nephrops at different stages of infection may also lead to stage-specific alterations in the probability of trawl capture. Most infected animals captured in trawls are either Stage I or 2 (unpublished observations). It is therefore possible that the behaviour of animals in more advanced stages of infection reduces their vulnerability to capture. The use of creel sampling for Hematodinium-infection assessment has shown that infected animals are rarely found within the creels (Pers. Obs. Dr. R. J. A. Atkinson). However, in the Clyde sea area, only shallow waters (with larger Nephrops) can be fished with creels, while the deeper grounds, which tend to contain Nephrops within the size range commonly infected by Hematodinium (30-35mm carapace) are fished by trawlers. As a result, a direct comparison of trawl and creel prevalence (see Wilhelm & Mialhe, 1996) would be misleading. However, both the present work and other studies of Nephrops swimming performance (Newland et al., 1992), suggest that infected animals are more likely to be captured by standard Nephrops trawling rigs than their non-infected counterparts. Tlius, the prevalence of Hematodinium reported for sites off the west coast of Scotland (Field et al., 1998) may be an overestimate of the true level of infection in these populations. The same decrease in swimming performance may also make infected animals more prone to predation, causing further imbalance in proportions of uninfected and infected animals. Early attempts to incorporate the high observed prevalence of Hematodinium infection 164 Chapter Eight - Swinuning pcrformance into analytical stock assessments of the Clyde sea area (ICES, 1997), modelled the effect as an additional loading on the natural mortality rate (since animals showing symptoms of patent infection usually die). However, the exercise was not entirely successful, leading, as it did, to unrealistic estimates of recruits entering the fishery. The observations above suggest that a more subtle approach is necessary when examining the cffect of the disease on the population. Reduced swimming performance implies increased catchability (F/E); in other words, a given unit of fishing effort would result in a higher increased mortality rate in populations with high prevalence of the disease. Furthermore, if infected animals are likely to be more susceptible to predation through reduced ability to escape, then the disease may not necessarily add to overall natural mortality but rather, replace a proportion of it. 165 Chaptcr Eight - Swimming pcrformancc References. Aiken, D. E. (1980). Moulting and growth. In: The Biology and Management of Lobsters. (eds. Cobb, J. S. & Phillips, B. F. ). Academic Press, New York, pp. 91-163. Arnott, S. A., Neil, D. M. & Ansell, A. D. (1998). Tail-flip mechanism and size-dependent kinematics of escape swimming in the brown shrimp Crangon crangon. Journal of Experimental Biology 201,1771-1784. Baden, S. P. & Neil, D. M. (1998). Accumulation of manganese in the haemolYmph. nerve and muscle tissue of Nephrops norvegicus (L. ) and its effect on neuromuscular performance. Comparative Biochemistry & Physiology 119A, 351-359. Edwards, D. H., Heitler, W. J. & Krasne, F. B. (1999). Fifty years of a command neuron: the neurobiology of escape behaviour in the crayfish. Trends in Neuroscience 22,153-16 1. Field, R. H. & Appleton, P. L. (1995). A Hematodinium-like dinoflagellate infection of the Norway lobster Nephrops norvegicus: observations on pathology and progression of infection. Diseases of Aquatic Organisms 22,115-128. Field, R. H. & Appleton, P. L. (1996). An indirect fluorescent antibody technique for the diagnosis of Hematodinium sp. infection of the Norway lobster, Nephrops norvegicus, Diseases of Aquatic Organisms 24,199-204. Field, R. H., Chapman, C. J., Taylor, A. C., Neil, D. M. & Vickerman, K. (1992). Infection of the Norway lobster Nephrops norvegicus by a Hematodinium-likc species of dinoflagcllate on the west coast of Scotland. Diseases of Aquatic Organisms 13,1-15. Field, R. H. (1992). The control of escape behaviour in, and the histopathology of, the Norway lobster, Nephrops norvegicus (L. ). PhD Thesis, University of Glasgow, 1992. Field, R. H., Hills, J. M., Atkinson, R. J. A., Magill, S. & Shanks, A. M. (1998). Distribution and seasonal prevalence of Hematodinizim sp. infection of the Norwav lobster (Nephrops norvegicus) around the west coast of Scotland. ICES Journal of Marine Science 55,846- 858. Field, R. H., Taylor, A. C. & Neil, D. M. (1991). Factors affecting swimming ability and its recovery in the Norway lobster (Nephrops norvegicus). Journal of the Marine Biological Association, U. K. 71,707-742. FRS (1999). Nephrops stocks around Scotland. Fisheries Research Service (FRS) fish stocks information leaflet; December 1999. Grieshaber. M. (1978). Breakdown and formation of high cnergy phosphates and octopine in the adductor muscle of the scallop. Chlamys opercularis (L. ) during escape s%%imming and rccovery. Journal of Comparative Physiology 126.269-276. Hudson, D. A. & Shields, J. D. (1994). Hematodinium austrafis n. sp., a parasitic dinoflagellate of the sand crab Portunits pelagicus from Morcton bay, Australia. Dis. Aquat. Org. 19,109-119. 166 Chapter Eight - Swinuning pcrformancc Hudson, D. A., Hudson, N. B. & Shields, J. D. (1993). Infection of Trapezia spp. (Decapoda: Xanthidae) by Hematodinium sp. (Duboscquodinida : Syndinidae): a new family record. Journal of Fish Diseases 16,273-276. ICES (1997). Report of the working group on Nephrops stocks. ICES CM 1997/Assess: 9 Krasne, F. B. & Teshiba, T. M. (1995). Habituation of an invertebrate escape rcflcx due to modulation by higher centres rather than local events. Proceedings of the National Academy of Sciences USA 92,3362-3366. Krasne, F. B. & Roberts, A. (1967). Habituation of the cra ish escape response du ng Yfi ri release from inhibition induced by picrotoxin. Nature 215,769-770. Kuriyama, K., Muramatsu, M., Nakagawa, K. & Kakita, K. (1978). Modulating role of taurine on release of neurotransmitters and calcium transport in excitable tissues. In: Taurine and Neurological disorders (eds. Barbeau, A. & Huxtable, R. J. ). Ravcn Press, New York, p. 201-216. Latrouite, D., Morizur, Y., Nodl, P., Chagot, D. & Wilhelm, G. (1988). Mortalite du tourteau Cancer pagurus provoquee par le dinoflagelle parasite: Hematodinium sp. ICES Committee Meeting CM. 1988/K: 32. Meyers, T. R., Bothelo, C., Koeneman, T. M., Short, S. & Imamura, K. (1990). Distribution of bitter crab dinoflagellate syndrome in southeast Alaskan tanner crabs Chionoecetay bairdi. Diseases of Aquatic Organisms 9,3743. Meyers, T. R., Koeneman, T. M., Bothelo, C. & Short, S. (1987). Bitter crab disease :a fatal dinoflagellate infection and marketing problem for Alaskan tanner crabs, Chlonocceles bairdi. Diseases of Aquatic Organisms 3,195-216. Neil, D. M. & Ansell, A. D. (1995). The orientation of tail-flip escape swimming in dccapod and mysid crustaceans. Journal of the Marine Biological Association, U. K. 75,55-70. Newland, P. L. & Chapman, C. J. (1989). The swimming and orientation behaviour of the Nonvay lobster, Nephrops norvegicus (L. ), in relation to trawling. Fisheries Research 8. 63-80. Newland, P. L. & Neil, D. M. (1990a). The tail flip of the Norway lobster, Nephrops norvegicus. 1. Giant fibre activation in relation to swimming trajectories. Journal Of Comparative Physiology 166A, 517-527. Newland, P. L. & Neil, D. M. (1990b). The tail flip of the Norway lobster, Nephrops norvegicus. IL Dynamic righting reactions induced by body tilt. Journal of Comparative Physiology 166A, 529-536. Newland, P. L., Chapman. CI & Neil, D. M. (1988), Swimming performance and endurance of the Norway lobster Nephrops norvegicits. Marine Biology 98,345-350. Newland, P. L., Neil, D. M. & Chapman, CT (1992). Escape swimming in the Norway Lobster. Journal of Crustacean Biology 12,342-353. 167 Chapter Eight - Swimming pcrfonnancc Newman, M. W. & Johnson, C. A. (1975). A disease of blue crabs (Callinectes sapidus) caused by a parasitic dinoflagellate, Hematodinium sp. Journal of Parasitology 64,158. 160. Oja, S. S. & Kontro, P. (1978). Neurotransmitter actions of taurine in the central nervous system. In: Taurine and Neurological disorders (eds. Barbeau, A. & Huxtable, R. J. ). Raven Press, New York, p. 181-200. Payen, G. G., Pochon-Masson, J., Portemer, C. & Chattanger, F. (198 1). Effects of cycstalk ablation on the concentration of taurine in various organs of the shore crab Carcinus maenas (L. ) (Decapoda : Brachyura). Comparative Biochemistry & Physiology 69A, 571. 574. Shields, J. D. (1992). Parasites and symbionts of the crab Portunus pelagicus from Moreton bay, eastern Australia. Journal of Crustacean Biology 12,94-100. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (1999). Changes in the plasma free amino acid profile of the Norway lobster, Nephrops norvegicus at different stages of infection by the parasitic dinoflagellate (genus Hematodinium). Diseases of Aquatic Organisms 3i, 151-157. Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The effects of Hematodinillm sp. -infection on aspects of the respiratory physiology of the Norway Lobster, Nephrops norvegicus (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228. -Vermeer, G. K. (1987). Effects of air exposure on desiccation rate, haemolymph chemistry, and escape behaviour of the spiny lobster, Palinurus argus. Fishery Bulletin, U. S. 85,45- 51. Vu, E. T. & Krasne, F. B. (1993). Crayfish tonic inhibition: prolonged modulation of behavioural excitability by classical GABAergic inhibition. Journal of Ncuroscicnce 13, 4394-4402. Wilhelm, G. & Boulo, V. (1988). Infection de I'ctrille Docarcinus puber (L. ) par un dinoflagelle parasite de type Hematodinium. ICES Committee Meeting CM. 1988/K: 32. Wilhelm, G. & Mialhe, E. (1996). Dinoflagellate infection associated with the decline of Necora puber crab populations in France. Diseases of Aquatic Organisms 26,213-219. Zatta, P. (1987). Calcium and taurine interaction in the heart of the crab Carcinus macnas. Canadian Journal of Fisheries & Aquatic Science 44,1765-1768. 168 Chapter Eight - Swinuning pcrfonnancc Fig. 1. Plan view of dumbbell tank at Marine Laboratory Aberdeen. Key : Area A (post-run animals), Area B (pre-run animals), Area C (pre-run settlement area) and Area D (rccordcd swimming run), divided into DI (camera I field), D2 (camera 2 field) and D3 (camera 3 field). iiaiseaviewing rea 169 Chapter Eight - Swimming performance Figs. 2,3 and 4. (2). Mean total number of tail-flips performed over entire swimming sequence (± standard error) by infection stage. (3). Mean total number of swimming bouts performed over the entire swimming sequence (± standard error) by infection stage. (4). Mean total distance travelled (m) by swimming, over the entire sequence (± standard error) by infection stage. Stage 0 denotes uninfected animals, while Stage 1,2 and 3-4 denote patent Hematodinium infection. 140 "I I 120 100 80 60 40 20 0 I I 0 70- 60- 50- 40- 30- 20 10 0 30 -1 25 20 10 0 0 12 Infection stage F-'--l 3&4 2 3&4 Infection stage 2 Infection stage 3&4 170 Chapter Eight - Swimming perfomlance Fig. 5. Typical bout velocity profile for an uninfectcd (Stage 0) and heavily infected (Stage 34) Nephrops. Key: S (start point - zero velocity), S to G (point of tail extension at end of giant flip), G to 1 (tail extension at end of flip following giant flip), 1 to 2 (tail extension of 2'd flip after giant), 2 to 3 (tail extension of Yd flip after giant), 3 to 4 (tail extension of 4h flip after giant), 4+ Glide (velocity of glide following fourth flip after giant). 0.9 0.8 0.7 0.6 - E5 0.5 - > 0.2 0.1 0 --0- Stage 0 SS to GG to I Ito 22 to 33 to 4 4+ glide glide to rest Portion of bout Fig. 6. Mean overall velocity (m. sec") of an individual swimming bout (± standard error) by infection stage. Stage 0 denotes uninfected animals, while Stage 1,2 and 3-4 denote patent Hematodinium infection. 0.7 - 0.6 - vý 0.5 - 0.4 0.3 0 0.1 0 02 3&4 Infection stage 171 Chapter Eight - Swinuning pcrfonnancc Fig. 7. Mean giant tail-flip and second tail-flip velocity comparisons (m. scc") (± standard error) by infection stage. Stage 0 denotes uninfectcd animals. while Stage 1.2 and 3-4 denote patent Hematodinium infection. 13 Giant flip vcolcity 13 Second flip velocity Infection stage Fig. 8. Mean tail flip frequency (Hz) over a swimming bout (± standard error) by infection stage. Stage 0 denotes uninfected animals, while Stage 1,2 and 34 denote patent Hematodinium infection. 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 0 12 Infection Stage 3&4 172 Chaptcr Nine - Swimming Endurance Chapter 9 Cessation of swimming and subsequent recovery in the Norway lobster, Nephrops norvegicus (L. ), and changes associated with infection by a parasitic dinoflagellate (genus Hematodinium). Abstract. The current study describes the pattern of tail flipping in the Norway lobster, Nephrops norvegicus (L. ) over a complete swimming sequence, and the subsequent performance following 4h of recovery. Data is compared to Hematedinium-infected N. norvegicus. The escape swimming response of uninfected N. norvegicus appears to be divided into two contiguous phases with distinct characteristics. The first phase comprises 80 to 100 relatively consistent, high power tail flips, while the variable number of second phase flips are less powerful and precede complete cessation of swimming. Lightly infected IV norvegicus appeared to show the same two-phase pattern, though the number of flips comprising the second phase was much less, leading to shorter swimming sequences. Heavily infected X norvegicus did not show this pattern but instead produced a smallcr number of weaker flips before cessation of swimming. Following 4h recovery, uninfcctcd N. norvegicus produced 84.3 % of the flips produced in the initial trial, this reduction being due to a smaller number of Phase 2 tail flips. Infected N. norvegicus showed a lower recovery rate than uninfected animals, this being proportional to the severity of infcction. The metabolic basis of the two phase swimming pattern and the reason for Icss Phase 2 tail flips being produced during Hematodinium infection is discussed in relation to the catchability of the weakest uninfected and of infected N. norvegicus by trawlers and predators, especially on fishing grounds where the trawling frequency is high. Submitted as: Stcritiford, G. D.. Neil. D. M. & Bailey. N. (2001). Cessation of swimming and subsequent recovery in the Nonvay lobster. Nephrops nonegicus (L. ), and changes associated %%-ith infection by a parasitic dinoflagellatc (genus Heinalodinium). Journal of Experimental Marine Biology & Ecology (submitted). 173 Chapter Nine - Swimn-dng Endurance Introduction. The ability of many crustacean species to perform both sustainable and exhaustive locomotion has been well studied (for reviews see Hartnoll, 1971, Hargreaves, 1980). Aerobic respiration typically supplies the energy needed for prolonged exercise, as seen in crustacean species undergoing extensive locomotion (Full & Herreid, 1983) and long. distance migrations (Houlihan et al, 1985). Anaerobic metabolism may also supplement the energy required during the migration of certain species (Booth et al., 1984), although these pathways are more generally associated with escape responses (Phillips et al., 1977). Exercise of this type is usually rapidly exhaustive, leading to increased tissue and haemolymph lactate titres, lowered tissue glycogen concentrations, reduced muscle ATP stores, alterations to acid-base status and a profound disturbance of ionic, osmotic and fluid volume homeostasis (Wood, 1991). The mechanisms involved in recovery from exhaustive exercise in crustaceans have also received considerable attention. Tley include the restoration of tissue phosphagen and ATP pools, the clearance of lactate and other anaerobic end products from active tissues, the recovery of intracellular and cxtraccllular pH to pre-exercise values and the replenishment of tissue glycogen (for review see Ellington, 1983). Recovery from exhaustion in crustaceans is generally slow (up to 24 hr) (Gade et al., 1986) and it is only when energy reserves have been replenished that locomotory performance recovers to its normal level. The Norway lobster (Nephrops norvegicus) is the subject of an important commercial fishery in Scotland and typifies a normally sedentary crustacean species which utilizes anaerobically-driven muscle contraction in order to elicit a rapid escape reaction in response to impending danger (Field et al., 199 1). This escape reaction in Nephrops. which comprises a series of rapid tail flexions, has been shown to be important in the avoidance of capture by trawlers (Newland & Chapman, 1989) and may also be used during aggressive conflict and escape from predators (see Amott et al., 1998). 174 Chapter Nine - SAimn*dng Endurance Stocks of Nephrops off the west coast of Scotland have been shown to harbour a seasonal infection by a parasitic dinoflagellate of the genus Hematodinium (Field et al., 1992), with animals displaying the symptoms of patent infection in catches taken during spring and early summer (Field & Appleton, 1995). Recently, it has been shown that many of the major features of swimming in Nephrops are altered during patent infection, and that this reduced swimming performance may alter the ability of infected lobsters to evade capture by trawlers and predators (Stentiford et aL, 2000a - Chapter 8). Any change in catchability also has implications for the quantitative assessment of populations exhibiting significant infection prevalence. I'he current study was carried out to establish if Hematodinium infection, as well as reducing the escape swimming performance in Nephrops (Stentiford et al., 2000a - Chapter 8) also affects its endurance and recovery rate. Data are discussed in relation to the factors that may limit the recovery of escape swimming, and to the consequences of recurrent challenge for the catchability of infected lobsters by trawlers and predators on heavily fished grounds. Materials and Methods. Collection and treatment ofanimals. Nephrops were collected with an otter trawl from a location south of Little Cumbrac in the Clyde Sea area (55.41'N, 4.56W), Scotland and were maintained in a closed aquarium at the University of Glasgow, before transportation to holding tanks in the Fish Behaviour Unit at the Marine Laboratory Aberdeen. Conditions in both systems were the same (9"C, 33 ppt salinity) and squid (Loligo spp. ) was provided ad libitum as food on arrival at MLA and again three days later. The carapace length of animals used was between 25 and 30 mm, to avoid known size. specific differences in swimming performance (Newland et al., 1988). Nephrops of both sexes were used since sex-specific differences in swimming performance are not sccn unless the 175 Chapter Nine - Swimming Endurance female is ovigerous (Newland el al., 1988). Animals were not fed for three days prior to the experiment to avoid any effects of differential feeding. All animals were in the intermoult state as defined by Aiken (1980). Experimental set-zip. All experiments were carried out in a 12m dumbbell-shaped tank as described by Stentiford et al. (2000a) (Chapter 8). Briefly, a frame carrying three video cameras (Panasonic WV-CL350) fitted with 6.5mm auto-iris lenses was constructed along the length of the tank. A hand-held integrator switch (Primebridge PVS-I) allowed for switching between the different cameras, thereby keeping the animal in the field of view. The integrator output was linked to a time and date generator (Mitsibushi TDG- I OB) before being recorded on a standard VHS video recorder. A distance marker (10cm intervals) was placed along the length of the tank floor, which was illuminated with standard halogen lighting fitted with dimmer switches. Experimental protocoL For the first swimming sequence, Nephrops were removed individually from the holding end of the tank with a net and placed into a settlement area for 10 min. After settlement, the animal was admitted to the main tank and subjected to mechanical stimulation (rostrum taps) using a plastic rod. The three video cameras and switching box allowcd the entire swimming sequence to be recorded. At the end of the swimming sequence, as detcrmincd by a failure to respond to three successive taps to the rostrum, the animal was removed from the main tank. measured. sexed, numbered with permanent ink and returned to the holding area (see Stentiford et a/. 2000a - Chapter 8). It was then allowed to recover for 4 h, after which time it was again removed from the holding area, and subjected to the same stimulus protocol as in the first session. Assessment for Hematodinium infection followed termination of this second swimming sequence using the pleopod staging method of Field 176 Chapter Nine - Swimming Endurance & Appleton (1995), according to which, Stage 0 denotes an apparently uninfectcd animal, with patent infection progressing from Stage I through to Stage 4. Stage 3 and 4 (heavily infected) animals were grouped in all analyses since animals at these stages show very similar pathologies (see Stentiford et aL, 1999 - Chapter 4). The swimming performance of a total of 73 Nephrops, both uninfected and Hematodinium-infected, was recorded in this way. Video Analysis. The recordings of Nephrops swimming performance were analyzed at the Division of Environmental and Evolutionary Biology at the University of Glasgow. The total number of tail flips and total distance covered were measured using a Panasonic AG-5700 VCR linked to a Panasonic AG-570 single-frame editing facility controller and a Panasonic WV- CM 1000 colour monitor. In the current study, a tail 'flip' is defined as one cycle of flexion and extension of the abdominal segments (Newland et al., 1988). Mean distance per flip was calculated by dividing the total number of flips by the total distance travelled during active swimming. Individual flip distances over an entire swimming sequence were recorded for a representative range of uninfected lobsters. For this, an acetate overlay was placed on the monitor screen and the position of the rostrum at the start position and at the initiation points of subsequent tail flips (tail extension following full flexion) were marked (see Arnott et al., 1998). Data were compared to that from Nephrops at each stage of infection. These data were used to determine any changes in the swimming performance over the course of a whole swimming sequence. Data Analysis. Comparisons of distance per flip in rested Nephrops were made bct%vccn uninfectcd and infected individuals by using a general linear regression model, followed by comparisons of 177 Chapter Ninc - S%vimirdng Endurancc the slope and elevation of regression curves using analysis of covariance (ANCOVA). Tliese data were then compared to those obtained after 4h of recovery following the initial swimming sequence, using a Mest to analyze the paired differences between observations from individual animals. Comparisons between the mean percentage recovery in the number of tail flips in different stages of infection were performed using paired t-tests. All tests were considered significant if p<0.05. Results. The swimming pattern of rested Mphrops. The swimming sequences of uninfected Nephrops in the rested state ranged from 90 to 180 tail flips, and were composed of two distinct phases. During Phase 1, which comprised the first 80 to 100 flips, the distance travelled in successive flips was relatively constant, with a mean of 18.97 ± 0.42 cm (n=6 individuals). After this, animals produced a variable number of less powerful flips (Phase 2), before the swimming sequence terminated. Figure la-f shows this two-phase pattern of swimming for the range of six uninfected animals, and illustrates the variation in the overall length of the swimming sequence, but the relative constancy of the number of flips comprising Phase I (indicated by the dashed line), When swimming sequences of representative individual Hematodinium-infectcd Nephrops were assessed, those with Stage 1 infection displayed a similar two phase pattern of swimming, but there was a reduction in the total number of tail flips performed (Fig. 2b). This was due almost entirely to a reduction in the number of Phase 2 flips, whcreas the number of Phase I flips (80 - 90) remained similar to that of Stage 0 animals. Stage 2 Hematodinium infection resulted in a ffirther reduction in the number of tail flips in a swimming sequence, and most individuals failed to reach the number representing the Phase I/ Phase 2 transition of uninfected animals (Fig. 2c and Fig. 3). However, comparing the 178 CMpter Nine - Swimming Endurance first 50 flips performed in each case, the mean distance per flip in Stage 2 Nephrops (18.86± 0.56 cm; n=6) was not significantly different to that of Stage 0 lobsters (18.97 ± 0.42; n=6) (p, > 0.05), indicating that these flips were equally powerful. This suggests that light infections primarily affect the number of flips performed, but have virtually no affect on either the transition between Phase I and Phase 2 swimming, or on the thrust produced by Phase I flips. In the advanced stages of infection (Stage 34) the swimming response of Nephrops differed more substantially from that of uninfected animals (Fig. 2d). The total number of flips in a swimming sequence was further reduced to less than 40, and in addition the distance covered in a swimming sequence was also considerably lower, yielding a mean value for distance per flip of 9.4 ± 1.7 cm. (n=1 1). 71"his indicates that all of the tail flips made by heavily infected animals are reduced in power, compared to both uninfected or lightly-infected animals. In uninfected animals the total distance travelled in a swimming sequence was proportional to the number of flips performed (Fig. 3 Stage 0), and the spread of values reflects the underlying variability seen in Fig. 1. This variability in the length of swimming sequences was also a feature of the data obtained from animals at different stages of infection (Fig. 3, Stages 14). For animals that performed only Phase I swimming (Stage 2& Stage 34), regression lines through their data sets have slopes approximately equal to their Phase I mean values (Stage 2-y=0.2103x - 1.8773; Stage 34 -y=0.182x - 0.2729) (Fig. 3). However, for animals that exhibited both Phase I and Phase 2 swimming (Stage 0 and Stage 1), the distance covered over the whole swimming sequence is the average of the Phase I and Phase 2 contributions, which is lower than for Phase I alone. Since this average decreases as the number of Phase 2 flips performed increases, the regression lines for these data sets have 179 Chapter Nine - Swimming Endurance progressively lower slopes as the length of the swimming sequence increases (i. e. from Stage 2 to Stage I to Stage 0) (Fig. 3). The lower elevation of the regression line for Stage 34 animals (y = 0.1388 - 0.3882), however, reflects the reduced distance travelled per flip achieved in the later stages of infection. The swimming pattern during recovery. The stimulation regime applied to rested animals was repeated after 4 hours recovery from the initial swimming sequence. The major change, compared with rested animals, was in the total number of flips performed, which reduced from mean values of 140.2 to 117.9 (p < 0.01) for uninfected animals, from 114.5 to 79.3 flips (p < 0.01) for Stage I animals, from 57.1 to 38.3 flips (p < 0.05) for Stage 2 animals and from 26.2 to 15.0 flips (p < 0,01) for stage 3-4 animals. This represents a partial recovery of the number of flips performed towards the values obtained in the rested state: 84.3 % recovery for Stage 0 animals, and significantly lower values of 69.8 %, (p < 0.0 1) for Stage 1,66.2 %, (P < 0.0 1) for Stage 2 and 36.8 % (p < 0.001) for Stage 34 infections (Fig. 4). These changes caused proportional reductions in the overall length of the swimming sequences (Fig. 5). In contrast, the power of the flips performed by the recovering animals. reflected in a plot of distance travelled vs number of flips (Fig. 6), showed little change from that of the rested animals. This can be seen in the regression curves, which follow the predicted trends as the swimming sequences shorten: i. e. increasing in slope in uninfected and Stage I animals (due to the smaller number of Phase 2 flips being performed during recovery), but remaining virtually the same in Stages 2 and Stage 34 animals (which performed only Stage I flips), Discussion. The current study has described the pattern of tail flipping in Nephrops over a complete swimming sequence, and the subsequent performance following 4h of recovery. it has also 180 Chapter Nine - Swimining Endurance shown that infection by Hematodinium leads to a reduction in the recovery rate of normal swimming ability and that this reduction is proportional to the plasma parasite burden. The exhaustion response and subsequent recovery. The escape swimming response of uninfected Nephrops appears to be divided into two contiguous phases with distinct characteristics. The first phase comprises 80 to 100 relatively consistent, high power tail flips, while the variable number of second phase flips are less powerful and precede complete cessation of swimming. A very similar escape response with two distinct phases has been observed in the Australian yabby Cherax destructor (Edwards & Baldwin, 1983). Compared to Stage I and to ý Stage 2 Hematodinium-infected Nephrops, uninfected animals had a relatively higher proportion of their swimming sequences contained within the Phase 2 period of swimming, where distance travelled per flip is less. The incorporation of a higher number of weaker tail flips into the overall swimming sequence of uninfected animals leads to a decreased regression curve slope relative to Stage I and 2 infected animals (which made fewer Phase 2 tail flips). Stage 34 Nephrops showed a reduced distance per flip, which is consistent with data presented by Stentiford et al. (2000a) (Chapter 8) which showed a significant reduction in flip velocity during the later stages of Hematodinium infection. The tail flip in crayfish and nephropid lobsters is essentially a 'single oar' propulsion system, whereby the inertial forces generated are largely attributed to movements of the tai I fan (Webb, 1979, Neil & Ansell, 1995). It is also known that an additional and significant thrust is created by 'squeeze forces' at the end of the tail flexion as the abdomen is pressed against the cephalothorax. ejecting trapped water (Daniel & Mcyh6fer, 1989). In the current study, the tail flips of Nephrops at late stage infection appeared to involve flexion of the tail fan alone, with little or no movement of the main abdomen. The reduccd velocity 181 Chapter Nine - SvAmraing Endurance reported by Stentiford et al. (2000a) (Chapter 8) therefore seems due to both a reduction in initial contraction power (see Stentiford el al., 2000b - Chapter 5) and an absence of the final 'squeeze forces' described by Daniel & Meyh6fer (1989). Metabolic energetics ofswimming. The rapid tail flexions involved in escape swimming in Nephrops are produced by the deep abdominal muscles, which are phasic in type (Neil et al., 1993), with high ATPase activity and little capacity for oxidative phosphorylation (Field et al., 199 1). These muscles are provided with high levels of phosphagen and a high glycogenolytic capacity (Gruschczyk & Kamp, 1990). It has been reported that glycogen is the principal fuel for exercise in crustaceans, with arginine phosphate being an important fuel for burst swimming (Onnen & Zebe, 1983; Raffin et al., 1988; Adamczewska & Morris, 1994). In crustaceans, the main end product of glycolysis, formed from the anaerobic reduction of pyruvate, is lactate (Phillips et al., 1977; Gdde et al., 1986; Lallier & Walsh, 1992. Henr%- et al., 1994; de Wachter et al., 1997), which accounts for most of the tissue acidosis associated with exhaustive swimming (Booth et al., 1984; Adamczewska & Morris, 1994; Kinsey & Ellington, 1996). Elevated levels of haemolymph and tissue lactate (Vermccr, 1987), depletion of the main tissue energy supplies (Henry et al., 1994) and habituation of the neuronal system (Neil & Ansell, 1995; Arnott et al., 1998; Edwards et al., 1999) have all been implicated in the cessation of exercise in crustaceans, and Field et al. (1991) showed that the absolute cessation of swimming, or 'functional exhaustion' in Nephrops is probably influenced by both. metabolic and neuronal factors. Due to the importance of lactate in the cessation of tail flipping (Vermeer, 1987, Field et al., 1991), the slow clearance of lactate from the haemolymph and tissue (Phillips et al., 1977; Forster et al., 1989. Booth et al., 1984; Henry et al., 1994) and the fact that muscle contraction function has also been shown to recover from a fatigued state more slowly in an acidic extracellular 182 Chapter Nine - Swimming Endurance environment (Renaud & Mainwood, 1985), it is probable that lactate accumulation is a major feature underlying the cessation of the swimming response in uninfected Nephrops. The elevated concentrations of haemolymph lactate which accompany Hematodinium infection of this species (Taylor et. aL, 1996) may ftirther reduce the swimming performance of Hematodinium-infected animals. In addition to the absolute cessation of tail flip swimming, the two-phase swimming pattern recorded in uninfected animal in the current study is suggestive of a subtle shift in energy substrate being utilized by the active muscle before complete cessation occurs. In the escape swimming in Cherax destructor, which demonstrates a similar two-phase swimming pattern, arginine phosphate-derived ATP was found to fiiel the initial burst of swimming (c. 30 flips), while anaerobic glycolysis was not used until the second phase of less powerful flips (England & Baldwin, 1983). It is conceivable that the onset of second phase swimming observed in Nephrops is controlled by similar mechanisms and that the transition point between Phase I and 2 swimming represents a depletion of muscle phospho-arginine reserves. It also suggests that the response to functional exhaustion in Arephrops is a progressive, stepwise process. The concentration of glycogen in the deep abdominal flexor muscle of Nephrops is known to be reduced in proportion to the severity of Hematodinium infection (Stentiford et al., 2000b - Chapter 5). As Stage I and Stage 2 Hemalodinium-infectCd individuals performed progressively fewer Phase 2 tail flips than their uninfected counterparts, it is likely that it is the energy substrate fiielling anaerobic glycolysis (principally glycogen) that limits the duration of the swimming response, rather than a depletion in the concentration of muscle phospho-arginine. Further studies on the concentrations of phospho-arginine in the abdominal flexor muscles of Hematodinium-infectcd animals would resolve this issue. 183 Chapter Nine _ Swimming Endurancc . In Stage 34 Hematodinium-infected Nephrops, additional physical and biochemical disturbances to the abdominal flexor muscles may be superimposed upon these more subtle alterations in energy metabolism, degrading the swimming response further (Stentiford el al., 2000b - Chapter 5). Repletion of muscle metabolites during recovery. Although recovery in the muscles powering escape responses might be expected to be relatively rapid (Ellington, 1983), a number of studies have shown that in crustaceans, removal of tissue and haemolymph lactate following exhaustive swimming is rather slow, with clearance times of between 3 and 24 h (Phillips et al., 1977; Forster et al., 1989; Booth et al., 1984; Henry et al., 1994). Although crustaceans do appear to possess the capacity for gluconeogenesis from lactate (Gdde et al., 1986, Lallier & Walsh, 1992, Henry et al., 1994), the system is regarded to be considerably less efficient than the Cori cycle described in mammals (see Schulman & Landau, 1992). Replenishment of glycogen reserves in the abdominal muscles of tail flipping crustaceans is important for the restoration of normal muscle fiinction, since it allows phosphagcn repletion (Ellington, 1983; Gruschczyk & Kamp, 1990; Shulman & Landau, 1992). The importance of glycogen in this tissue is probably a consequence of the relatively poor blood supply, which prevents regeneration of arginine phosphate by aerobic mechanisms (Gruschczyk & Kamp, 1990, Milligan & Girard, 1993). However, the recover%, of muscle from active exercise in invertebrates occurs in conjunction with a period of supranormal 01 consumption, indicating that at least some elements of recovery arc aerobic processes (Ellington, 1983). During recovery from exhaustive sivimming. replenishment of abdominal muscle phospho-arginine from intracellular glycogen stores is probably very efficient in uninfected and lightly-infectcd Nephrops. However, as phospho-arginine is replenished at the expense 184 Chapter Nine - Swimrning Endurance of glycogen, less glycogen will be available at this time as a substrate for Phase 2 swimming (which relies on glycolytic production of ATP). Such a response would explain why uninfected and lightly-infected animals do not perform as many Phase 2 flips during the recovery period. Stentiford et al. (2000a) (Chapter 8) have noted that during Hematodinium infection, animals may be in a state of persistent partial exhaustion, which restricts their capacity to respond to the increased respiratory demands of swimming. In heavily infected animals a 50% reduction in the 02 carrying capacity of the haemolymph leads to an internal hypoxia. with some tissues resorting to anaerobic metabolism, even when the animal is at rest (Taylor et al., 1996). A shift towards anaerobic metabolism (Baden et al., 1994), coupled with the increasing metabolic demands of the parasite is responsible for the reduction the tissue glycogen stores reported in Hematodinium-infectcd lobsters (Stcntiford el OL, 2000b - Chapter 5). A combination of reduced tissue glycogen and parasitc-induccd oxygen depiction make it unlikely that sufficient energy will be available for prolonged escape swimming in heavily-infectcd Nephrops. Under such conditions, the cfficicnt repletion of muscle phospho-argi nine and glycogen following periods of exertion, is doubtful. Additionally, the replenishment of tissue glycogen is significantly reduced during periods of starvation (Wood, 199 1) and under conditions of hypcr-osmolarity. due to mitochondrial shrinkage and a reduction in fatty acid oxidation (leading to increased glycolysis and a reduction in glycogen) (Li el al., 1992). 'Me large changes in the free amino acid composition of the hacmolymph and muscle reported to occur during Ilematodinium infection (Stentiford et al., 1999 - Chapter 4,2000b - Chapter 3) may be causing similar effects on the accumulation of glycogen in abdominal muscle of Nephrops. 183 Chapter Nine - Swimming Endurance The swimming response in relation to catchability. Swimming data presented for Hematodinizim-infectcd Nephrops in the current study, in conjunction with that previously reported by Stentiford et al. (2000a) (Chapter 8) and other studies on the swimming performance of Nephrops (Newland et al., 1992), suggest that infected animals are more likely to be captured by standard trawling rigs than their non. infected counterparts. The variation in swimming endurance (number of flips performed) and the two-phase swimming pattern seen in the absence of infection also suggests that capture by trawlers may not only be dependent on the position of the individual in relation to the net mouth (Newland & Chapman, 1989), but also on physiological status - with those Nephrops performing the least flips being the most likely to be overtaken and captured by the trawl net. Furthermore, the reduced swimming performance may make Hematodinium-infected (and those uninfcctcd individuals with the lowest swimming endurance) most prone to predation. The lower swimming performance in uninfcctcd Nephrops after 4h recovery suggests that this period was not sufficient to allow complete restoration of the normal swimming response. Recovery from exhaustion in crustaceans is generally slow (up to 24 hr) (Gadc et al., 1986) and it is only when energy reserves have been replenished that locomotory performance recovers. To some extent, the slow recovery time in Nephrops may be mitigated by the fact that the species constructs a burrow and is able to retreat to this when threatened by using the escape response. Once within the burrow, recovery from the swimming burst could take place with rclatively little interruption. In areas %%-here Nephrops exhibits crcpuscular emergence. a period in excess of four hours would be available bct%%-ccn forays from the burrow. The ability to remain within shelter. however, would depend on energy intake requirements being met and eventually animals have to emerge. On heavily fishcd grounds therefore, whcrc trawl frcqucncies = be as high as once every 24 h (Marrs et al.. 2000), animals which have recently evaded trawl capture may be more likely to be captured by subsequent trawls. 71is 186 Chaptcr Ninc - S%imming Endurancc effect may be enhanced in Hematodinium-infected Arephrops, which showed impaired recovery in the 4h period and are also known to have higher energy requirements owing to the parasite loading (Taylor et al., 1996); which may necessitate more frequent feeding activity. A detailed study of the metabolic limitations of escape swimming in uninfcctcd and Hematodinium infected Nephrops is required to resolve these issues. 187 Chapter Ninc - Swimming Endurancc References. Adamczewska, A. M. & Morris, S. (1994). Exercise in the terrestrial Christmas Island red crab Gecarcoidea natafts. 11. Energetics and locomotion. Journal of Experimental Biology 188,257-274. Aiken, D. E. 1980. Moulting and gro%Nth. In: Cobb, J. S. & Phillips, B. F. 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(1985). Energetics of swimming in Calfincacs sapidus and walking in Homarus americanus. Comparative Biochemistry & Physiology 82A, 267-279. Kinsey, S. T. & Ellington, W. R. (1996). 1 H- and 31 P-nuclear magnctic resonance studies of I-lactate transport in isolated muscle fibres from spiny lobstcr Panuhnis argus. Journal of Experimental Biology 199,2225-2234. Lallicr, F. H. & Walsh, P. J. (1992). Metabolism of isolatcd hcpatopancrcas cclls from the blue crab (Callinecles sapidus) under simulated post-cxcrcisc and hypoxic conditions. Physiological Zoology 65,712-723. Li. J., Stillman, J. S., Clore, J. N. & Blackard, W. G. (1992). The effect of h)Pcrosmolarity on muscle glycogen accumulation. flormonc and Metabolism Research 24,99-102. Marrs, S. J.. Tuck, I. D., Amcri, E., Atkinson, R. J. A., Santojanni. A. & Stevenson, T. D. I. (2000). Improvement of Nephrops stock assessment by use of micro-scale mapping of cffort and landings. Final Report to the European Commission. ltcfcrcncc 97/100, Study Project in support of the Common Fisheries Policy. 202pp +6 appendices. Milligan, C. L. & Girard, S. S. (1993). Lactate metabolism in rainbo%v trout. Jounial or Experimental Biology 180.175-193. Neil, D. M., Fowler, W. S. and Tobasnick, G. (1993). Nlyoribrillar protein composition correlates with histochcmistry in fibres of the abdominal flexor muscles of the Nonvay 189 Chapter Nine - Swimrning Endurance lobster Nephrops norvegicus. Journal of Experimental Biology 183,185-20 1. Neil, D. M. & Ansell, A. D. (1995). The orientation of tail-flip escape swimming in decapod and mysid crustaceans. Journal of the Marine Biological Association, U. K. 75,55-70. Newland, P. L. & Chapman, CI (1989). The swimming and orientation behaviour of the Norway lobster, Nephrops norvegicus (L. ), in relation to trawling. Fisheries Research 8, 63-80. Newland, P. L., Chapman, C. J. & Neil, D. M. (1988). Swimming performance and endurance of the Norway lobstcrNephrops norvegicus. Marine Biology 98,345-350. Onnen, T. & Zebe, E. (1983). Energy metabolism in the tail muscles of the shrimp Crangon crangon during work and subsequent recovery. Comparative Biochemistry & Physiology 74A, 833-838. Phillips, J. W., McKinney, R. J. W., Hird, FIR. & MacMillan, D. L. (1977). Lactic acid formation in crustaceans and the liver function of the midgut gland qucstioncd. Comparative Biochemistry & Physiology 56B, 427433. Raffin, J. P., 116bault, M. T. & Lc Gall, J. Y. (1988). Changes in phosphomctabolitcs and intraccllular pH in the tail muscle of the prawn Palaemon serratus as shown by in vivo 31P-NMP, Journal of Comparative Physiology 15813,223-228. Renaud, J. M. & Mainwood, G. W. (1985). The cffects of pli on the kinctics of fatigue and recovery in frog sartorius muscle. Canadian Journal of Physiology & Pharmacology 63, 1435-1443. Schulman, G. I. & Landau, B. R. (1992). Pathways of glycogen repletion. Physiological Reviews 72,1019-1035 Stentiford, G. D., Neil, D. M., Atkinson, R. J. A. & Bailey, N. (2000a). An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infcctcd by a parasitic dinoflagellate of the genus Hemalodinium. Journal of Expcrimcntal Marine Biology & Ecology 247,169-18 1. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (1999). Changes in the plasma free amino acid profile of the Nonvay lobster, Nephrops norvegicus at dlffcrcnt stages of infection bv the parasitic dinoflagellate (gcnus Ikmatodinhim). Diseases of Aquatic Organisms 3i, 151-157. Stcntiford, G. D., Ncil, D. M. & Coombs, G. 11. (2000b), Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Non%zy lobster, Nephrops norvegicus (L. ) during infection by a parasitic dinoflagcllatc of the gcnus Ikmatodinium, Diseases of Aquatic Organisms 42,133-14 1. Taylor, A. C., Field, R. 11. & Parslow-Williams, P. J. (1996). 111c cffccts of Ik"latodinlism sp. -infcction on aspects of the respiratory physiology of the Nonvay Lobstcr. Nel)hrol)s norvegicus (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228, 190 Chapter Nine _ Swimn-dng Endurance Vermeer, G. K. (1987). Effects of air exposure on desiccation rate, haemolymph chemistry, and escape behaviour of the spiny lobster; Palinurus argus. Fishery Bulletin, U. S. 85,45- 51. Webb, P. W. (1979). Mechanics of escape responses in crayfish (Orconectes virilis). Journal of Experimental Biology 79,245-263. Wood, C. M. (1991). Acid-base and ion balance, metabolism, and their interactions after exhaustive exercise in fish. Journal of Experimental Biology 160,285-308. 191 Cbapter Nine - Swinurdng Endurance Fig. la-E Distance travelled by individual tails flips, up to cessation of Swimming in a range of uninfectcd (Stage 0) N. norvegicus. T'he suggested point of transition to the 4 second phase' of swimming (see text) is represented by a dashed line. Animl 3 30 25 13 20- loý 5, 30 25 20 is 10 5 6 0 so 100 150 Aninul 31 200 0 50 Anýwl 55 100 150 200 Aninal 9 I 06 0 30 25 20 . in 10 0 0 06 50 100 150 200 0 so Aninul 11 100 Fhp number 150 200 I U 0 50 50 100 ISO Animl 57 100 200 150 200 Flip number 192 Chapter Nine - Swimming Endurance Fig. 2a-d. Distance travelled by individual tail flips, up to cessation of swimming in typical uninfcctcd (Stage 0) and Hematodinium-infected (Stages 1,2 and 34) lobsters The suggested point of transition to the 'second phase' of swimming (see text) is represented by a dashcd line. 35 30 20 10 3 35 30 25 20 13 I 35 30 23 20 13 10 35 30 35 4 10 20 is 0 I I I -, D- Stage I -0- Stage 2 --a- Stage 3-4 so 100 150 200 Fbp number 193 Chapter Nine - Swimming Endurance Fig. 3. Correlation plot of distance travelled against number of flips performed until cessation of swimming in uninfectcd (Stage 0) and Hematodinium-infected (Stages 1,2 and 34) N. norvegicus. 30 ýa Stage 1 I'(') I 0 Stage 0 y -0.182x. 0.2729 2 ýo R2 - 0.859 13 A Stage 2 25 10 Stage 3-4 ITN 1 Y-0.21ox- S 2n4 1.9773 R3 - 0.9527 0 20 40 60 80 100 120 140 160 180 200 Nwnber of ks Fig. 4. Mean percentage recovery in the total number of tail flips performed by uninfected (Stage 0) and Hemalodinium-infected (Stages 1,2 and 34) N. norvegicus in the 4h run. 100-1 90 ý 80 - M- 60- 50- 40- 30 - 20 IV o4 3&4 Infbdion staW 194 CMpter Nine - Swimming Endurance Fig. 5. Derived means of distance travelled and number of flips performed by uninfected (Stage 0) and Hematodinium-infectcd (Stages 1,2 and 34) N. norvegicus. Týe data for 0h (solid symbols) and 4h (open symbols) are plotted in relation to the lines of best fit for the 0h data set (see Fig. 2). 0 20 40 60 80 100 120 140 160 180 200 Nunibcr of flips 195 Chapter Nine - Swimming Endurance Fig. 6. Correlation plots of distance travelled against number of flips performed until cessation of swimming in uninfected (Stage 0) and Hematodinium-infected (Stages 1,2 and 34) N norvegicus. Lines of best fit are dashed (0 h run) or solid (4 h run). 35 , 30 ý 25 ý 20 Is 10 5 0 35 30 25 20 5ý 0 35 , 30 ý 23 ý 20 -j '51 30 25 20 ý is ý to 3. 00 hr c3 4 hr *0 hr a4 hr *0 hr 04 hr 0 a (%. - *, S tage 0 S tage 1 Stage 2 vo Stage 3-4 0 50 too 150 200 Number of flips a- ol, 9-0196ý' -0 196 Chapter Ten - Burrow-Rclatcd Behaviour Chapter 10 Alteration of burrow-related behaviour of the Norway lobster, Nephrops norvegicus (L. ) during infection by the parasitic dinoflagellate Hematodinium. Abstract The current study has used time-lapse video recordings to analyze the burrow-related behaviour of N. norvegicus under a 12 h light : dark regime in aquarium conditions, and compared this to the behaviour of lobsters infected by the parasitic dinoflagellate Hematodinium. Uninfected lobsters performed more burrow departures, of longer duration, during the hours of darkness while infected lobsters performed a relatively constant number of similar-duration departures in the light and dark periods. However, the absolute number of departures performed by infected lobsters (70 day") was more than double that of uninfected lobsters (30.1 day"), while the duration of burrow departures performed by infected lobsters (258.5 s. departure-1) was more than six times greater than in uninfected lobsters (38.7 s. departure-'). This led to a more than ten-times increase (from L7 to 19.4 %) in the percentage of the day spent out of the burrow by infected lobsters. The altered burrow-related behaviour could be due to the nutritional demands of the parasites on infected lobsters, causing an increased requirement to forage, or alternatively to a 'functional hypoxia', due to the additional respiratory demands of the parasite, causing the host to emerge from the burrow onto the sediment surface to seek oxygen. Implications for the increased time spent out of the burrow are discussed in relation to availability of infected lobsters to trawlers and predators, and to the use of infection prevalence data from trawl-caught samples in stock assessment models for X norvegicus. Submitted as: Stentiford, G. D., Neil, D. M. & Atkinson, RTA. (2001). Alteration of burrow-related behaviour of the Norway lobster, Nephrops norvegicus (L. ) during infection by the parasitic dinoflagellatc Hematodinium. Marine & Freshwater Behaviour & Physiology (submitted). 197 Chapter Ten - Burrow-Related Behaviour Introduction. Infection by parasites is known to have significant effects on the behaviour and life history of host organisms. Behavioural changes may bring the host to a more suitable site for the direct transmission of the parasite, or make intermediate hosts more susceptible to predation (Holmes & Bethel, 1972; Poulin, 1994). At the community level, parasites and pathogens are recognized as important factors in determining the density and long-term population dynamics of many animal populations (Dobson, 1988. Kuris & Lafferty, 1992). The discovery of a dinoflagellate parasitic infection in populations of the Norway lobster (Nephrops norvegicus) (Field et al., 1992) provides an opportunity to study the effects of a parasitic infection on some well-studied aspects of the burrow-related behaviour of a scavenging crustacean, and to consider the practical consequences of such changes for the large fishery which exists for this commercially important shellfish species. The Norway lobster (Nephrops norvegicus) is widely distributed on the continental shelf of the northeast Atlantic and in the Mediterranean sea, and inhabits burrows constructed in fine, cohesive mud substrata (Chapman, 1980). The general form of N. norvegicus burrows and the method of burrow construction have been well described (Rice & Chapman, 197 1, Fanner, 1974a). The burrows provide protection against predatory attacks (Rice & Chapman, 1971) and are important for the incubation of eggs by females (Farmer, 1974b). Studies on the burrow-emergence behaviour of N. norvegicus have shown that its pattern is dependent on depth, with shallow water animals most active during darkness, deep water animals during the light and animals from intermediate depth showing a crcpuscular activity pattern (Chapman, 1980). The basis for this emergence pattern may be associated with the reception of light by the eye and subsequent movement of the retinal shielding pigments (Ar6chiga & Atkinson. 1975. Shelton & Gaten, 1986). As such, the relatively higher light intensity experienced in the middle of the day may inhibit out-of-burrow activity (Ar6chiga & Atkinson, 1975). However, recent studies have shown that light- 198 Chapter Ten - Burrow-Related Behaviour induced eye damage has little effect on various aspects of N. norvegicus behaviour (including burrow-emergence) (see Chapman el al,. 2000), suggesting that activity rhythms may be entrained, at least in part, by light detection by other receptors, such as photosensitive neurones in the brain (Page & Larimer, 1976) and the caudal photoreceptor (Wilkens, 1988). In addition to the direct effects of light intensity, food availability and tidal strength may also be implicated in the observed burrow-emergence pattern (Atkinson & Naylor, 1976; Newland el aL, 1988), as may be some unknown endogenous determinant (Ardchiga and Rodriguez-Sosa, 1997). N. norvegicus supports a major fishery in the United Kingdom, with the main method of capture being otter trawling, which relies upon lobsters being out of their burrows (Atkinson & Naylor, 1976; Newland & Chapman, 1989). It follows that any factor that increases the amount of time spent out of the burrow may be expected to lead to larger catches by trawling, and that such changes in behaviour provide the most likely explanation for the observed short term variation in catches (Atkinson & Naylor. 1976. Fanner, 1974c). Stocks of N. norvegicus off the west coast of Scotland have been shown to harbour a seasonal infection by a parasitic dinoflagellate of the genus Hematodinium (Field et a/., 1992) which is responsible for a disease syndrome resembling the 'Bitter Crab disease' (BCD) reported in Alaskan tanner crabs (Chionoecetes bairdi and Copilio) (Meyers et a/., 1987. Taylor & Khan, 1995) and also Hematodinium infections in other decapod hosts (Newman & Johnson, 1975; Wilhelm & Boulo, 1988; Latrouite et al., 1988; Shields, 1992. Hudson et al., 1993). A number of studies have established the basic characteristics of Hematodinium infection in N. norvegicus in terms of its progression, diagnosis and pathology (Field and Appleton, 1995,1996), its effect on host physiology (Taylor et al., 1996). on haemolymph and tissue biochemistry (Stentiford et al.. 1999 - Chapter 4,2000a - Chapter 5) and on locomotion (Stentiford et al., 2000b - Chapter 8). 199 Chaptcr Ten - Burrow-Rclated Bchaviour Animals showing symptoms of patent infection (moribund, cloudy haemolymph, bright shell coloration) are seen in catches taken during the spring and early summer (Field el al., 1992), and it was suggested by these authors that infected lobsters may be more prone to capture by trawlers than uninfected lobsters. Some evidence for a behavioural component to this susceptibility was obtained in a study which repeatedly sampled one fishing ground over a 24 h period (Field et al., 1998), and found diurnal changes in the percentage of infected lobsters making up the catch. However, no systematic study has yet been performed to identify the behavioural changes induced by Hematodinium infection. The present study was therefore carried out to characterise any changes that may occur in the burrow-related behaviour of aquarium-held N. norvegicus infected by Hematodinizim, and to consider these changes in relation to the catchability of infected lobsters. Methods. Collection and treatment ofanimals. Norway lobsters were collected with an otter trawl from depths of approximately 80 m at a location south of Little Cumbrae in the Clyde Sea area, Scotland, UK (55.410N, 4.56OW). Before being placed into experimental tanks, lobsters were maintained for approximately one week in a closed aquarium (90C, 33 ppt salinity) at the University Marine Biological Station Millport, Isle of Cumbrae, Scotland. Animals were fed ad libitum upon capture on squid (Loligo spp. ) and mussels (Mytilus sp. ) but were not fed thereafter. Only male N. norvegicus. which are known to have the same endogenous emergence rh5thms year round (Atkinson & Naylor, 1976) were used in experimental runs. Individuals with a carapace length of around 30 min were chosen, as Hematodinium infection is most common in animals of this size (Stentiford et al., 2000a - Chapter 5). All animals were in the intermoult state as defined by Aiken (1980). Since animals were to be held in the experimental tanks for five days, and patent infection would progress over this time, assessment of Hematodinium infection was performed at the end of 200 Chapter Ten - Burrow-Relatcd Behaviour each trial. For this, the pleopod staging method of Field & Appleton (1995) was used, according to which Stage 0 denotes an apparently uninfected animal, with patent infection progressing from Stage I through to Stage 4. All animals used were of Stage 2 or Stage 3 infection (medium to heavy parasite burden). Experimental set-up. Experiments designed to examine the effect of Hematodinium infection on the burrow-related activity of N. norvegicus were carried out in a circular tank (0 1.5 in, depth 0.75 in - see Fig. IA-F) containing well-settled sediment (0.35 m depth) obtained from the Clyde Sea area. The tank was placed in a light-controlled room and exposed to a 12 h: 12 h light-dark cycle (illumination from 0700 h to 1900 h). During the illuminated period, diffuse green lighting was used (c. 1.0 tiE. s-1 M-2), while red lamps provided sufficient light for video recording for the whole light-dark period. A video camera (Panasonic WV-CL350) fitted with a 6.5mm wide- angle lens was mounted above the tank, providing a view of the whole sediment surface. Tile signal from the camera was fed to a time lapse video recorder (Panasonic AG-6730) set to a framing rate of I per second. Animals were placed individually in the tank, and their behaviour was recorded for a period of 5 days. In total 10 uninfected and 10 Hematodinium-infected lobsters were monitored in this way. Video Analysis. The video recordings were analyzed using a Panasonic AG-5700 VCR linked to a Panasonic AG-570 single-frame editing facility controller and a Panasonic WV-CMIOOO colour monitor. The whole recording was reviewed, in order to assess the behaviour performed, but the first 24 h of the video recording was not used for the quantitative analysis of burrow-related behaviour. Thereafter, measurements were made over the next 3 201 Chapter Ten - Burrow-Rclated Behaviour consecutive days of the total time spent within and outside of the burrow, the number of burrow departures and the duration of individual excursions from the burrow. Thus data for a total of 30 animal-days were obtained for each of the two experimental groups. Lobsters were scored as 'in-burrow' when part or all of their bodies were within the confines of the burrow (Fig. IA, B and F) and 'out-of-burrow' when the whole body (including telson and uropods) were outwith the confines of the burrow (Fig. I C, D and E). Data obtained from the three days of filming was averaged for each hour and pooled with other animals from the group (uninfected or Hematodinium-infected) to allow assessment of the effect of parasitic infection on burrow-related behaviour. Data Analysis. Comparisons of behavioural data from uninfectcd and infected lobsters were performed either by one-way analysis of variance (ANOVA) or a Kruskal-Wallace test followed by between stage comparisons with a Tukey's pairwise analysis (for normal distributed data), or by a Mann-Whitney test (for non-normal distributions). Tests were considered significant if p<0.05. Results. Observations on burrow-related behaviour. When a lobster was placed into the experimental tank, it took up occupancy of an existing burrow, or in some cases excavated a new burrow. T'hereafter it extended and repaired the burrowl and evidence of this was obtained even when the animal was inside the burrow from the appearance of sediment plumes emerging from one or more openings. The lobster often occupied the main burrow opening (Fig. IB), with only its claws visible, and from this position it would periodically either retreat into the burrow (Fig. IA), or emerge onto the surface for an excursion (Fig. IC, D and E). Both uninfected and infected animals 202 Chapter Ten - Burrow-Related Behaviour showed these patterns of behaviour, although their duration and timing were different in the two groups (see below). Another observation was made that was unique to the Hematodinium-infected lobsters. In two cases, in the video recording beyond the 3 day period of measurement, the animals produced a series of relatively uncoordinated tail flexions ('spasms'), and between these spasms they lay in a quiescent state on the sediment surface. The frequency of these spasms increased over a 34 hour period, and after the final one of these the animals showed no further movement, and were found to be dead at the end of the trial. Quantitative analysis ofburrow-related behaviour. Burrow departures. Measures of the number of burrow departures, expressed in terms of the mean hourly values over a 24 hour period, are shown in Figure 2. Uninfected lobsters performed a greater number of burrow departures during the hours of darkness than during the hours of light, the mean value for the complete periods of dark (1.72 departures h") and light (0.79 departures W) being significantly different (p < 0.001) (Fig. 2). In contrast, Hematodinium-infected lobsters displayed a pattern of burrow departures that was relatively constant over the 24 h period (Fig. 2), and the number of departures performed in the complete dark period (3.08 departures W) and the complete light period (2.75 departures W) (Fig. 3) are not significantly different (p = 0.086). However, both these values for infected lobsters are significantly greater than those for the uninfected lobster in their more active period, the dark (p < 0.00 1 in each case). Over the whole 24 hour period, the mean total number of burrow departures performed by infected lobsters (70 departures day") was more than double the number performed by uninfected lobsters (30.1 departures day-) (p < 0.01) (Fig. 4). 203 Chapter Ten - Burrow-Related Behaviour Duration of excursions. Measures of the duration of individual excursions from the burrow onto the sediment surface, expressed in terms of the mean hourly values over a 24 hour period, are shown in Figure 5.717his shows that uninfected lobsters made longer excursions from the burrow during the hours of darkness than during the hours of light, and the mean values calculated for the complete periods of dark and light (49.84 s departure' and 27.65 s departure"' respectively) (Fig. 6) are significantly different (p < 0.001). The excursion duration's of Hematodinium-infected lobsters also showed some variation throughout the 24 hour period, though the difference in the mean excursion duration between the light (212.9 s departure-) and the dark (304.2 s departure-) was not significant (p = 0.06 1). This was probably due to the large variation in the dark data set (see Fig, 5). However, a more important feature of the excursions made by infected lobsters was their greatly increased duration, and values for both the dark and light periods are significantly greater than those for the uninfected lobster in their more active period, the dark (p < 0.00 1 in each case). Over the whole 24 hour period, therefore, the mean excursion duration for infected lobsters (258.5 s departure") was more than six times the duration of the excursions performed by uninfected lobsters (3 8.7 s departure") (p < 0.00 1) (Fig. 7). Total time spent out qf the burrow. By combining the data for the number of burrow departures and the duration of individual excursions, values were derived for the total time spent out of the burrow by uninfected and infected lobsters. Data expressed in terms of the mean hourly values over a 24 hour period are shown in Figure 8, and reflect the trends observed in the separate measures of burrow departures and excursion duration's. Thus uninfected lobsters spent significantly longer on the sediment surface during the period of darkness (mean for complete period 90.3 s h") than during the period of light (mean for complete period 30.4 s h") (p < 0.001) (Fig. 9). 204 Chapter Ten - Burrow-Rclatcd Behaviour Infected animals also spent a longer time out of the burrow during the period of darkness (mean for complete period 787.3 s h-) than during the period of light (mean for complete period 610.9 s h") (p < 0.05). In this case, therefore, the combined measure reveals a significant difference between dark and light activity, despite the separate measures of burrow departures and excursion times not being significantly different (see Figs. 3& 6). However, the most important feature of the data is again the large and significant difference between the measures for the uninfected and infected animals (Figs 8,9). Considered as the % time spent out of the burrow over the whole 24 hour period (Fig. 10), the value for infected lobsters (19.4 % of day out-of-the-burrow) is more than ten times greater than that for uninfected lobsters (1.7 % of day) (p < 0.00 1). Discussion. Burrow-related behaviour in uninfected lobsters. Data presented for the burrow-related behaviour of male N. norvegicus show that under the conditions provided, lobsters were most active during the hours of darkness, with reduced out-of-burrow activity during illuminated periods. A prominent feature of emergence behaviour in uninfected male N. norvegicus was the relatively small proportion of the whole day spent on the sediment surface (mean 1.7%). This degree of activity fits well with field observations of emergence (Chapman & Rice, 1971; Chapman, 1980) and catch profiles for lobsters living at depths of 75-90 m (Atkinson & Naylor, 1976). Uninfected male N. norvegicus make a greater number of burrow departures, of longer duration during the hours of darkness than during the hours of light. The burrows of N. norvegicus are principally for refuge from predators (Rice & Chapman, 1971) and emergence is probably governed by the availability of suitable food (Farmer, 1974c; Cristo, 1998) and the level of illumination (Atkinson & Naylor, 1976). The increased number and duration of departures made by uninfected lobsters during the hours of darkness probably 205 Chapter Ten - Burrow-Related Behaviour reflect a combination of a lowered perceived risk of predation and a physiological requirement to feed. Similar variations in diurnal activity are seen in crayfish: Pacifastacus leniusculis reduces the success of visual predators by remaining in shelter during the day (Blake & Hart, 1995), while Procambarus clarkii exhibits activity patterns which are strongly influenced by food availability (De Miguel & Ardchiga, 1994). As the lobsters in this study were not captured in light-controlled conditions, there is likely to have been some damage to the retinal shielding system of the eyes (Ar6chiga & Atkinson, 1975; Shelton & Gaten, 1986). However, the appearance of a light-dark rhythm, even in these visually compromised animals suggests that activity patterns in N. norvegicus from these depths may not be totally reliant on light reception by the eyes. Ilis reinforces data presented by Atkinson & Naylor (1976) which showed that an endogenous activity rhythm occurs even when lobsters are held in constant darkness, and that this activity may be entrained by photosensitivity other than through the eye, possibly via the caudal photoreceptor (Wilkens, 1988; Simon & Edwards, 1990). Furthermore, Richardson (in Chapman et al., 2000) has reported that light-induced damage has little effect on the burrow-emergence rhythm, feeding, agonistic behaviour or predator avoidance behaviour of N. norvegicus. The maximum absorbance of the retina of N. norvegicus is around 498 nm and the absorbency in the red end of the spectrum is known to be minimal (Loew, 1974). As constant red light was provided throughout the current experiments and reception of green light by the eyes during the illuminated period may have been compromised, the changes in burrow-cmergence activity recorded in uninfected N. norvegicus may be a remnant of an endogenous activity rhythm (Atkinson & Naylor, 1976; Ar6chiga and Rodriguez-Sosa, 1997). However, the current study was not designed to investigate the factors controlling burrow-related activity. 206 Chapter Ten - Burrow-Rclated Behaviour Burrow-related behaviour in Hematodinium-infected lobsters. Male Hematodinium-infected lobsters were found to spend significantly more time on the sediment surface (mean 19.4 %) than their uninfected counterparts. This increase in the total time spent out of the burrow was due to the significantly increased number and duration of burrow departures. The lack of any significant difference between the number and duration of burrow departures performed in the light and dark periods suggests that infected male lobsters do not show the same degree of rhythmicity seen in uninfected male lobsters. However, when the number and duration of burrow departures were combined to give total time out of the burrow, the difference in activity between the light and dark periods was significant. This suggests that although the absolute level of activity is greatly increased in infected male lobsters, they may still retain some degree of emergence rhythmicity. Infected male lobsters were also significantly more active during the illuminated period than uninfected male lobsters were during darkness. This suggests that the motivation of infected lobsters to leave the relatively safe confines of the burrow exceeds the perceived risk of predation associated with emergence during the illuminated period. Parasites are known to indirectly affect host behaviour through the depletion of the hosts energy reserves and by impinging upon their respiratory requirements (Barber et al., 2000), The increase in foraging time necessary to provide the nutritional requirements of the host (and the parasite) appear to be made at the expense of predator avoidance behaviour (Millinski, 1985). In such cases, altered behaviour may make parasitized hosts more prone to predation by non-specific and definitive hosts for the parasite (Brassard et al., 1982; Levri, 1999). The increased number and duration of burrow departures (especially during the illuminated period) observed in the current studv is consistent with an increase of 'risk- prone' behaviour in male Hematodinium-infected lobsters. The basis for this increase in risk prone behaviour could be purely nutritional, as Hematodinium-infected N. norvegicus 207 Chapter Tcn - Buffow-Relatcd Behaviour are known to have severely depleted plasma and tissue carbohydrate reserves (Stentiford et al., 2001, in manuscript - Chapter 6; Stentiford et al., 2000a - Chapter 5, respectively), reduced concentrations of plasma protein (Taylor et al., 1996) and a disrupted plasma and tissue amino acid profile (Stentiford et al., 1999 - Chapter 4,2000a - Chapter 5). It has therefore been suggested that Hematodinium-infected lobsters are undergoing 'physiological starvation' caused by the large burden of parasites in the haemolymph (Stentiford et al., 2000a - Chapter 5). Such nutritional stresses have been related to increased foraging activity in parasitically infected hosts (Cunningham et al., 1994). The parasitic utilization of oxygen in the host plasma has also been implicated in the alteration of host behaviour during infection (Lester, 1971). During Hematodinijim infection of N. norvegicus, a reduction in haemocyanin concentration leads to a deficit in the oxygen carrying capacity of the haemolymph. This coincides with an elevated oxygen demand from the developing parasites, leading to severe respiratory stress in the lobster (Taylor et al., 1996). During conditions of moderate environmental hypoxia, N. norvegicus is known to exit the burrow, which is relatively more hypoxic than the water, in favour of the sediment surface (Baden et al., 1990). 'Me increased out of burrow activity observed in male Hematodinium-infected lobsters may thus represent the expression of such a respiratory stress response. Is Hematodinium acliveýy manipulatin, 9 its host? A well-known paradigm in host-parasite interactions is the ability of parasites to actively manipulate the host, in order to facilitate their own transmission (Levri, 1999). TIlus alterations of host behaviour during parasitism may not simply be non-adaptive by- products of infection, but may represent a manipulation of host behavioral responses by the parasite (Poulin, 1994). However it is difficult to show unequivocally that manipulation of host behavioral responses by the parasite is occurring (Poulin, 2000), as this requires 208 Chapter Ten - Burrow-Rclated Bchaviour evidence for a fitness gain by either the parasite or the host (Poulin, 1995). In the case of Hematodinium infection of N. norvegicus, incomplete knowledge of the life cycle of the parasite and the mode of transmission between hosts (Appleton & Vickerman, 1998) preclude conclusions about the 'motivational' basis for the altered behaviour observed in the present study. The component of behaviour in infected lobsters that is the strongest candidate for active manipulation by the parasite is the spasmodic tail flipping that was observed in two infected lobsters in the last hours before death. It certainly conforms to the precept that an 'active manipulation' by a parasite is most likely to occur when the intensity of infection is maximal Poulin (1994). Previous aquarium observations of Hematodinium-infected N. norvegicus have identified a terminal stage of infection (parasite sporolation), where swarming stages of the parasite are extruded via the integumental membranes, gills and the mouth, leaving the host moribund (Field and Appleton, 1995. Appleton and Vickerman, 1998). It is possible that spasmodic tail flipping is a behavioral manifestation of this sporolation event, causing rupture of integumental membranes, and allowing the motile dinospores to emerge. This behaviour will also bring the infected lobster to the sediment surface where broadcasting of the spores would be more effective. Implications ofaltered behaviourfor the N. nonvgiatspopulation andfishery. Previous studies on Hematodinizim-infected N. norvegicus have shown that infected lobsters exhibit severely reduced tail-flip swimming performance, which is likely to increase their likelihood of capture by predators and trawl nets (Stentiford et al., 2000b - Chapter 8). Longer periods spent out of the burrow will exacerbate this effect by making the animals more susceptible to predation. However, increased predation is only likely to sustain infection in a population if infected hosts are consumed by other hosts for the parasite (Barber et al., 2000). Hematodinium infection has been discovered in the hermit crab (Tagurus bernhardus) captured from the X norvegicus fishing grounds in the Firth of 209 Chapter Ten - Burrow-Related Behaviour Clyde, Scotland, and it is possible that this species acts as an alternative host for this parasite on these grounds. Scavenging on moribund or recently dead Hematodinizim- infected N. norvegicus by P. bernhardus may perpetuate the infection from one season to another. Further studies on the epidemiology of Hematodinium infection in P. bernhardus are necessary to resolve this issue. Changes in the behaviour and locomotion of Hematodinium-infected N. norvegicus should also be considered when calculating infection prevalence from trawl-caught samples (see Stentiford et aL, 2000b - Chapter 8). Increased time spent out of the burrow when coupled with a reduced swimming performance, should increase the likelihood of infected lobsters being captured by trawl nets, thus leading to an overestimation of the true level of prevalence in the population sampled. Previous attempts to incorporate the observed high prevalence of Hematodinium infection into analytical stock assessments of the Clyde Sea area have modeled the effect of infection as an additional loading on the natural mortality rate (since animals showing symptoms of patent infection usually die) (ICES, 1997). However, this has led to unrealistic estimates of recruits entering the fishery, due in part to the fact that the infection may not necessarily add to overall natural mortality, but rather replace a proportion of it, since infected animals will also be more susceptible to predation through a reduced ability to escape. As such, it is suggested that tile incorporation of infection prevalence data into stock assessment models for populations of N. norvegicus exhibiting significant infection prevalence, should take into account the effect of behavioural and locomotory modifications in the alteration of catchability. Also, changes in behaviour have to be considered in relation to lobsters at different stages of infection, and to the more complex burrow-rclated behavioural pattern of female lobsters. 210 Chapter Ten - Burrow-Relatcd Behaviour References. Aiken, D. E. (1980). Moulting and growth. In: The Biology and Management of Lobsters. (eds. Cobb, J. S. & Phillips, B. F. ) Academic Press, New York, pp. 91-163. Appleton, P. L. & Vickerman, K. (1998). In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sp. ) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116,115-130. Ardchiga, H. & Atkinson, R. J. A. (1975). The eye and some effects of light on locomotor activity in Nephrops norvegicus. Marine Biology 32,63-76. Ardchiga, H. & Rodriguez-Sosa, L. (1997). Coupling of environmental and endogenous factors in the control of rhythmic behaviour in decapod crustaceans. Journal of the Marine Biological Association U. K. 77,17-29. Atkinson, R. J. A. & Naylor, E. (1976). An endogenous rhythm and rhythmicity of catches of Nephrops norvegicus. Journal of Experimental Marine Biology & Ecology 25,95-108. Baden, S. P., Loo, L. -O., Pihl, L. & Rosenburg, R. (1990). Effects of eutrophication on benthic communities including fish, Swedish west coast. Ambio 19,113-122. Barber, I., Hoare, D. & Krause, 1 (2000). Effects of parasites on ish behaviour: a revi wd fe an evolutionary perspective. Review in Fish Biology & Fisheries (in press). Blake, M. A. & Hart, P. J. B. (1995). Then vulnerability of juveni e signal cra ish to perch d I yf an eel predation. Freshwater Biology 33,233-244. Brassard, P., Rau, M. E. & Curtis, M. A. (1982). Parasite-induced susceptibility to predation in diplostornoiasis. Parasitology 85,495-501. Chapman, CT & Rice, A. L. (1971). Some direct observations on the ecology and behaviour of the Norway lobster, Nephrops norvegicus. Marine Biology 10 1,321-329. Chapman, C. J. (1980). Ecology of juvenile and adult Nephrops. In: The Biology and Management of Lobsters. (eds. Cobb, J. S. & Phillips, B. F. ) Academic Press, New York, pp. 143. 178. Chapman, CT, Shelton, P. M. J., Shanks, A. M. & Gaten, E. (2000). Survival and growth of the Norway lobster Nephrops norvegicus in relation to light-induced eye damage. Marine Biology 136,233-241. Cristo, M. (1998). Feeding ecology of Nephrops norvegicus (Decapoda: Nephropidae). Journal of Natural History 32,1493-1498. Cunningham, E. J., Tierney, J. F. & Huntingford, F. A. (1994). Effects of the cestode Schistocephahis solidus on food intake and foraging decisions in the three-spincd stickcback Gasterosteus aculeatus. Ethology 79,65-75. De Miguel, F. F. & Ardchiga, H. (1994). Circadian locomotor activity and its cntrainment by food in the crayfish Procambanis clarkii. Journal of Experimental Biology 190,9-2 1. 211 Chapter Ten - Burrow-Related Behaviour Dobson, A. P. (1988). The population biology of parasite-induced changes in host behaviour. Quarterly Reviews in Biology 63,139-165. Farmer, A. S. D. (1974a). Burrowing behaviour of the Norway lobster, Nephrops norvegicus (L. ) (Decapoda: Nephropidae). Estuarine & Coastal Marine Science 2,49-58. Farmer, A. S. D. (1974b). Reproduction in Nephrops norvegicus (Decapoda: Nephropidae). Journal of Zoology 174,161-183. Farmer, A. S. D. (1974c). Field assessments of diurnal activity in Irish sea populations of the Norway lobster, Nephrops norvegicus (L. ) (Decapoda: Nephropidae). Estuarine & Coastal Marine Science 2,37-47. Field, R. H. & Appleton, P. L. (1995). A Hematodinium-like dinoflagellate infection of the Norway lobster Nephrops norvegicus : observations on pathology and progression of infection. Diseases of Aquatic Organisms 22,115-128. Field, R. H. & Appleton, P. L. (1996). An indirect fluorescent antibody technique for the diagnosis of Hematodinium sp. infection of the Norway lobster, Nephrops norvegicus. Diseases of Aquatic Organisms 24,199-204. Field, R. H., Chapman, C. J., Taylor, A. C., Neil, D. M. & Vickerman, K. (1992). Infection of the Norway lobster Nephrops norvegicus by a Hematodinium-like species of dinoflagellate on the west coast of Scotland. Diseases of Aquatic Organisms 13,1-15. Field, R. H., Hills, J. M., Atkinson, R. J. A., Magill, S. & Shanks, A. M. (1998). Distribution and seasonal prevalence of Hematodinium sp. infection of the Norway lobster (7vephrops norvegicus) around the west coast of Scotland. ICES Journal of Marine Science 55,846-858. Holmes, J. C. & Bethel, W. M. (1972). Modification of intermediate host behaviour by parasites. In: Behavioral aspects of parasite transmission. (eds. Canning, E. U. & Wright, C. A. ), Academic Press, London, pp. 123-149. Hudson, D. A., Hudson, N. B. & Shields, J. D. (1993). Infection of Trapezia spp. (Decapoda: Xanthidae) by Hematodinium sp. (Duboscquodinida: Syndinidae): a ne%v family record. Journal of Fish Diseases 16,273-276. ICES (1997). Report of the working group on Nephrops stocks. ICES CM 1997/Assess: 9 Kuris, A. M. & Lafferty, K. D. (1992). Modelling crustacean fisheries: cffects of parasites on management strategies. Canadian Journal of Fisheries & Aquatic Science 49,327-336. Latrouite, D., Morizur, Y., Nodl, P., Chagot, D. & Wilhelm, G. (1988). Mortalite du tourtcau Cancer pagunis provoquee par le dinoflagelle parasite: Hematodinium sp. ICES Committee Meeting CM. 1988/K: 32. Lester, RTG. (1971). The influence of Schistocephahis plerocercoids on tile respiration of Gasterosteus and a possible resulting effect on the behaviour of the fish. Canadian Journal of Zoology 49,361-366. 212 Chapter Ten - Burrow-Rclated Behaviour Lcvri, E. P. (1999). Parasite-induccd change in host behaviour of a freshwater snail: parasitic manipulation or byproduct of infection? Behavioural Ecology 10,234-24 1. Loew, E. R. (1976). Light and photoreceptor degeneration in the Nonvay lobster, Nephrops norvegicus (L. ). Proceedings of the Roval Society of London (Series B) 193,31-44. Meyers, T. R., Koeneman, T. M., Botelho, C. & Short, S. (1987). Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan tanner crabs, Chionoecetes bairdi. Diseases of Aquatic Organisms 3,195-216. Millinski, M. (1985). Risk of predation of parasitized sticklebacks (Gasterosteus aculeatus L. ) under competition for food. Behaviour 93,203-216. Newland, P. L. & Chapman, C. J. (1989). Ile swimming and orientation behaviour of the Norway lobster, Nephrops norvegicus (L. ), in relation to trawling. Fisheries Research 8,63-80. Newland. P. L., Neil, D. M. & Chapman, C. J. (1988). The reactions of the Norway lobster, Nephrops norvegicus (L. ). to water currents. Marine Behaviour & Physiology 6.301-313. Newman, M. W. & Johnson, C. A. (1975). A disease of blue crabs (Callinectes sapidus) caused by a parasitic dinoflagellate, Hematodinium sp. Journal of Parasitology 64,158-160. Page, T. L. & Larimer, J1. (1972). Entrainment of the circadian locomotor activity rhythm in crayfish. Journal of Comparative Physiology 160,169-179. Poulin, R. (1994). The evolution of parasite manipulation of host behaviour: a theoretical analysis. Parasitology 109, S 109-S 118. Poulin, R. (1995). 'Adaptive' changes in the behaviour of parasitized animals: a critical review. International Journal of Parasitology 25,1371-1383. Poulin, R. (2000). Manipulation of host behaviour by parasites: a weakening paradigm? Proceedings of the Royal Society of London 267B, 787-792. Rice, A. L. & Chapman, C. J. (1971). Observations on the burrows and burrowing behaviour of two mud-dwelling decapod crustaceans, Nephrops norvegicus and Goneplax rhomboides. Marine Biology 10,330-342. Shelton, P. M. J. & Gaten, E. (1986). Accessory pigment distribution and migration in the compound eye of Nephrops norvegicus (L. ) (Crustacca: Decapoda). Journal of Experimental Marine Biology & Ecology 98.185-198. Shields, J. D. (1992). Parasites and symbionts of the crab Portunus pelagicus from Moreton bay, eastern Australia. Journal of Crustacean Biology 12,94-100. Simon, T. W. & Edwards, D. H. (1990). Light-evoked walking in crayfish: behavioural and neuronal responses triggered by the caudal photorcccptor. Journal of Comparative Physiology 166A, 745-755. 213 Chapter Ten - Burrow-Related Behaviour Stentiford, G. D., Chang, E. S., Chang, S. A. & Neil, D. M. (2001). Carbohydrate dynamics and the crustacean hyperglycaernic hormone (CHH): cffects of parasitic infection in lobsters. General & Comparative Endocrinology (submitted). Stentiford, G. D., Neil, D. M. & Coombs, G. H. (1999). Changes in the plasma free amino acid profile of the Norway lobster, Nephrops norvegicus at different stages of infection by the parasitic dinoflagellate (genus Hematodinium). Diseases of Aquatic Organisms 38,151-157. Stentiford, G. D., Neil, D. M. & Coombs, G. H. (2000a). Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the Norway lobster, Nephrops norvegicus (L. ) during infection by a parasitic dinoflagellate of the genus Hematodinium. Diseases of Aquatic Organisms 42,133-141. Stentiford, G. D., Neil, D. M., Atkinson, R. J. A. & Bailey, N. (2000b). An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infected by a parasitic dinoflagellate of the genus Hematodinium. Journal of Experimental Marine Biology & Ecology 247,169-181. Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The effects of Hemalodinium sp. - infection on aspects of the respiratory physiology of the Norway Lobster, Nephrops norvegicils (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228. Taylor, D. M. & Khan, R. A. (1995). Observations on the occurrence of Hematodinium sp. (Dinoflagellata: Syndinidae); the causative agent of Bitter crab disease in Newfoundland snow crab (Chionoecetes opilio). Journal of Invertebrate Pathology 65,283-288. Thompson, S. N. & Kavaliers, M. (1994). Physiological bases for parasite-induccd alterations of host behaviour. Parasitology 109, SI 19-S 13 8. Wilhelm, G. & Boulo, V. (1988). Infection de 1'etrille Docarcinus puber (L. ) par un dinoflagelle parasite de type Hematodinium. ICES Committee Meeting CM. 1988/K: 32. Wilkens, L. A. (1988). The crayfish caudal photoreceptor: advances and questions after the first half century. Comparative Biochemistry & Physiology 91C, 61-68. 214 Chapter Ten - Burrow-Related Behaviour Fig. 1A-F. Still images from video recordings of X norvegicus burrow-oriented activity; (A) completely within burrow (burrow entrances shown by arrows), (B) guarding main burrow entrance, (C) emerging for surface excursion, (1), E) on the surface of the sediment during an excursion and (F), guarding second burrow entrance. Scale bar = 50 cm. 215 Chapter Ten - Burrow-Related Behaviour Fig. 2. Number of burrow departures by hour made by uninfected and Hematodinium- infected lobsters. Header bar indicates the illuminated and dark periods. 88$ Eý $$$$88 (=; W; C4 m -W Lo to r- ad Cri 40 (D Cl C) C) CD 6 _; C. ý cli 4 Actual time (hr) 888888888 uri (6 r--: ad (7) Cý -: $ý 'i C14 " 216 Chapter Ten - Burrow-Related Behaviour Fig. 3. Mean number of burrow departures per hour made by uninfected and Hematodinium-infected lobsters in the light and dark periods. Unh*cted I Fig. 4. Mean number of burrow departures per day by uninfected and Hematodinhim- infected lobsters. 90 80 70 10 60 50 40 30 20 Io 0-' I Anfiiml swo Infected I Uninfected Anýmal status Infected 217 Chapter Ten - Buffow-Related Bchaviour Fig. 5. Burrow excursion duration by hour for uninfected and Hematodinium-infected lobsters. Header bar indicates the illuminated and dark periods. cA 7S 0 1000 - 900 - 800 - 700 - 600 - 500 - 400 300 200 100 0 88 C> (D Cý <ý CD C) 0 8 %0 rý o6 0 888 C5 -4 C-i [I Uniifected 13 Infcctcd I 0 0 rý 06 2ý Actual tim (1y) 8888888888 .......... 218 Chapter Ten - Burrow-Related Behaviour Fig. 6. Mean duration of burrow excursions made by uninfected and Hematodinium- infected lobsters in the light and dark periods. wnfected Atinml status Infected Fig. 7. Mean duration of burrow excursions over the whole day made by uninfected and Hematodinium-infected lobsters. 350 - 300- 250- , 1200 150 loo 50 minfected infected Anii-nal status 219 Chapter Ten - Burrow-Related Behaviour Fig. 8. Number of seconds per hour spent out of the burrow, by hour, for uninfected and Hematodinium-infected lobsters. Header bar indicates the illuminated and dark periods. 1400 1200 ý 13 Uninfected 13 Infected 888888888888888888888688 cý4 e-i "; tri s6 r--: o6 cý 6 cý ei -, j wi zi r- o6 c7, rý. Actual týir (fir) 220 Chapter Ten - Burrow-Related Behaviour Fig. 9. Mean time spent out of the burrow per hour by uninfected and Hematodinium- infected lobsters in the light and dark periods. I 900 - 800 -0 Light 700 - 1: 3 Dark 600- 500 - 400 - 300 - 200 - 100 0 Heahhy Anhm] status Infected Fig. 10. Mean percentage of the whole day spent out of the burrow by uninfected and Hematodinium-infected lobsters. 25 -1 20 10 - 5 0 Uninfected Infected Arlýml status i 221 Cbapter Elcven - Conclusions and Prospccts Chapter 11 Conclusions and Prospects. This study has ftifthered our understanding of the complex relationship between the protozoan parasite Hematodinium and its host, the Norway lobster, Nephrops norvegicus. A diverse range of experimental approaches and analytical techniques have been used to investigate the epidemiology, etiology and pathology of the infection, and its effect on the biochemistry, physiology and behaviour of the host. In the previous chapters the significance of these various findings has been considered in relation to the literature relevant to the particular aspects. In this final chapter, therefore, consideration is restricted to the wider relationships between these findings, and emphasis is placed on unresolved issues, and on suggestions for fiinher studies. Description qf the parasite infecting N. non-egims. While still not described to species level, the dinoflagellate parasite found in N. norvegicus is now accepted to be a species of the genus Hematodinium (Appleton & Vickerman, 1998), and recent molecular studies have shown that the N. norvegicus isolate of Hematodinium is probably distinct from those infecting the other crustacean species that have been studied (Hudson & Adlard, 1996, Appleton & Vickcrman, 1998; Coats 1999), However, attainment of species status for the N. norvegi . cits isolate of Hematodinium is an important and urgent aim for future taxonomic studies. Field epidemiology. Data describing the diagnosis and epidemiology of Hematodinium infection in populations of N. norvegicus from the Clyde Sea area, Scotland (Chapters 2 and 3) have provided a new appreciation of the cycling of the parasite in the field in relation to the 222 Chapter Eleven - Conclusions and Prospects complex life history of the host, and add considerably to the work already reported by Field et al. (1992,1998). Chapter 2 described how a simple morphometric diagnostic method (the pleopod method) could be used to study the epidemiology of Hemalodinium infection in the field. It was shown that mean carapace length (the standard measure of animal size) of the population appears to be important in determining the absolute level of infection prevalence within a given year. Since an increased fishing effort has been implicated in the reduction of mean carapace length of N. norvegicus on certain fishing grounds (Sardi, 1998), studies are now required to determine whether increased fishing effort leads to the formation of a population that is particularly susceptible to disease epizootics. Chapter 2 also showed how the synchrony of the moulting seasons of male and fernale lobsters may play an important role in the pattern of infection seen within a particular year. In some years, a sharp peak of infection is seen, while in others a plateau of infection occurs (see below). As such, the length of the infection season may be as important as the absolute infection prevalence in determining the natural mortality attributable to Hematodinium infection in any given year. As a result of the finding that the pleopod method was ineffective in detecting sub-patent and latent Hematodinium infections (Field & Appleton, 1996), Chapter 3 described the further development and application of an diagnostic test for infection, based on detection of Hematodinium antigens in the haemolymph and tissues qf X norvegicus using immunoassay procedures. The data presented suggest that the p1copod method considerably under-diagnoses infection prevalence in trawl-caught samples, and that detection of parasite antigens by an immunoassay test may provide a more sensitive method for infection diagnosis. Further development of the immunoassay into a multi. sample, enzyme-linked immunosorbant assay (ELISA) is now being pursued. Such methods should allow for the routine assessment of Hematodinium infection prevalcricc in geographically-distinct N. norvegicus populations. In addition to antibody-antigen bascd 223 Chapter Eleven - Conclusions and Prospects detection methods, diagnosis of Hematodinium infection has also been achieved using molecular techniques based on the polymerase chain reaction (PCR) (Hudson & Adlard, 1994,1996). This technique utilizes nucleotide sequences that are specific to the parasite genome in order to amplify extremely small quantities of parasite DNA present in host tissue. The method is highly sensitive and is likely to be invaluable in future studies of Hematodinium parasite latency in N. norvegicus. Development of nucleotide primers specific to the N. norvegicus isolate of Hematodinium is being pursued. Linking patholoýy to ecoloSy. Due to the severe pathological effects associated with advanced Hematodinium infections (Meyers et al., 1987; Hudson & Shields, 1994; Field & Appleton, 1995; Wilhelm & Mialhe, 1996 and Chapters 4,5 and 6 of this thesis), the survival of infected lobsters under aquarium conditions (Field et al., 1992, Field & Appleton, 1995) and the sporolation response which leads to the death of the host lobster (Appleton & Vickerman, 1998), it is unlikely that recovery from patent infection is possible. TIlis would explain why seasons of high infection prevalence have been associated with lower burrow densities on the fishing grounds, and with reductions in landings per unit effort from trawling (Field el al,, 1998). However, infection with Hematodinjum may also contribute to determining the availability of N. norvegicus for capture, and an important aim of the present study was to identify the extent to which changes in the physiology, biochemistry and behaviour of the host upon infection determine the catchability of the infected lobsters. Chapter 6 reported the reduction in hepatopancreatic glycogen and plasma glucose reserves during patent infection. This reinforced the data presented in Chapter 5 which showed that muscle glycogen was also greatly depicted during infection, Taken together with changes measured in the concentration of the crustacean hypcrglycacmic hormone (CHH) in the plasma, these findings suggest that Hemalodinium parasites act as a 224 Chapter Eleven - Conclusions and Prospects 'carbohydrate sink' in the haernolymph of N. norvegicus, by consuming free glucose and preventing repletion of tissue glycogen. It is thus envisaged that infected lobsters are in a state of 'physiological starvation' -a condition that may be compounded by 'functional hypoxia', due to the parasites consuming oxygen in the blood (Taylor et al., 1996). Schmidt and Platzer (1980) note that the condition of the host may determine the degree to which a parasitic infection develops, with hosts in the best physiological condition harbouring the largest parasite burdens. The prevalence of Hematodinium infection was seen to be highest in N. norvegicus during the spring, and in females (Chapter 2 and 3). Female crustaceans have relatively larger amounts of hepatopancreatic tissue than males as a means of supplying the nutritional requirements for egg rearing and spawning (Farmer, 1974). The higher incidence of Hematodinium infection in female lobsters may reflect some relative advantage of the female host to the parasite. Coincidence of peak infection with the onset of the main moulting period of N. norvegicus (Chapter 2) may reflect the fact that crustaceans are in their best physiological condition during the late intermoult and the premoult (Johnson, 1980), allowing Hematodinhim parasites to develop and multiply under optimal conditions in the haemolymph. Data presented in Chapters 4,5 and 6 have suggested that simple carbohydrate reserves may provide the initial energy source for developing parasites. This is followed by progressive depletion of more complex carbohydrates (such as glycogen) and then consumption of amino acids and proteins (see Taylor et al., 1996). Ile sporolation phase of the parasite, in which motile parasite forms emerge from the host lobster, may occur when all available host resources have bccn depleted. This may explain why sporolation of the parasite can even occur in lobsters displaying only Stage I or 2 infection (Appleton & Vickcrman, 1998). Further studies on the nutritional requirements of the parasite during different stages of development may reveal the nature of these more complex nutritional relationships between Hemalodinium and its host. 225 Chapter Eleven - Conclusions and Prospects The concepts of physiological starvation and functional hypoxia are also relevant to the interpretation of data presented in Chapters 8 and 9 on the swimming performance of Hematodinium-infected lobsters. These chapters described the progressive reduction in swimming performance during patent infection, and associated this mainly with the reduced glycogen reserves reported in infected muscle (Chapter 5). However, it was also suggested that the increased haemolymph and tissue lactate concentrations previously reported to occur in Hematodinium-infected lobsters (Taylor et al., 1996), when coupled with alterations in water content and damage to the sarcolemmal membranes of infected muscle (Chapter 5), may also play a role in reducing swimming performance. Furthermore, the two-phase swimming pattern reported in Chapter 9 has highlighted how different metabolic substrates may be utilized throughout the normal swimming sequence of N. norvegicus. Further studies of the disruptions to metabolic processes in muscle that may affect the swimming capabilities of infected lobsters are required. Alterations in tissue (Chdpter 5) and haemolymph (Chapter 4) biochemistry provide further evidence for the severe pathological disruption that occurs in Hematodinium infection of N. norvegicus. While FAAs in the plasma of crustaceans are generally present at very low concentrations, during Hematodinium infection the plasma concentration of several FAAs increased considerably. The increase of taurine was the most marked, increasing 12-fold. Taurine is known to mimic the inhibitory effects of X-aminobutyric acid (GABA) in crustaceans (Zatta, 1987) and glutamate, which also increases considerably during infection, is known to have pharmacological activity at low concentrations (Lin & Cohen, 1973). During infection, the concentration of free glutamate in the plasma increased to values that exceed the threshold for stimulating crayfish muscle. Due to open nature of the crustacean circulatory system, these elevated concentrations of ncuro-activc compounds could be at least partly responsible for the reduced swimming ability of Hematodinium-infected lobsters reported in Chapters 8 and 9. Additionally, high plasma 226 Cbapter Eleven - Conclusions and Prospects concentrations of these compounds may be involved in the unusual tail flipping spasms seen in heavily-infected lobsters shortly before death (Chapter 10). Taurine and other FAAs (such as glutamate) have been identified as potent chemo- attractants in the marine environment. Altered excretion of chemo-attractive compounds from the antennal glands of N. norvegicus, which are known to be damaged during Hematodinium infection (Field & Appleton, 1995), and the subsequent predation of infected lobsters by other hosts, may provide an interesting link to parasite transmission in the field. This interaction between Hematodinium-infected lobsters and predators may be compounded by the increased amounts of time spent out of the burrow (Chapter 10) and the reduced swimming performance (Chapters 8 and 9) noted in Hematodinium-infected N. norvegicus. The changes in FAA concentrations in the deep abdominal flexor muscle of patently infected lobsters suggest that the normal biochemistry of the muscle cells may also be considerably disrupted. An up-regulation in the production of taurine in the deep abdominal flexor muscle, as shown in Chapter 5, may explain the increased plasma concentration of this compound reported in Chapter 4. Additionally, as this muscle provides the majority of the commercially useful meat of N. norvegicus, changes to its composition may impact upon the marketability or edibility of infected lobsters (see Shirai et al. 1996). Alterations in the taste of Hematodinium-infectcd meat have already been described in the aptly-named 'Bitter Crab disease' of Alaskan tanner crabs Chionoccetes opilio and C bairdi, though in this case it was assumed to be due to some exudate from the parasite itself (Meyers et al., 1987; Taylor & Khan, 1996). As FAAs and other low molecular weight compounds are important components of the 'taste profile' of crustacean meat, future studies on the biochemical composition of Hemalodinium-infectcd lobster meat are recommended. 227 Chapter Eleven - Conclusions and Prospects Hematobdum infection as a stressor. Evidence presented in the current study has suggested that Hematodinium infection may cause biochemical and physiological changes in host lobsters which are similar to those occurring under a range of other stresses (see Paterson & Spanoghe, 1997). In particular, a reduction in tissue glycogen and an elevation of plasma CHH are both common responses to hypoxia (see Baden et al., 1990 and Santos & Keller, 1993, respectively). Additionally, changes in the concentration of plasma and tissue taurine (especially in respect to its ratiometric association with the amino acid glycine) are also known to be sensitive indicators of acute and chronic stress in marine invertebrates (Livingstone, 1982). An interesting link was made in Chapter 6 between the onset of infection patency and the moult (itself a period of considerable physiological stress). Here, it was shown that CHH, the plasma concentration of which changes when animals are exposed to a number of different stressors (see Chung et al., 1999), is significantly increased in sub-patently infected lobsters. Other recent studies have shown that the concentration of CHH is also increased significantly in the hours preceding ecdysis. This presumably leads to mobilization of the host's carbohydrate reserves, thereby providing the necessary energy for moulting (Chung et al., 1999). The results of Chapter 6 suggest that the increased CIIH titre in sub-patently infected lobsters may cause a similar response, producing conditions in the haemolymph that are suitable for the development of patent infection (i. e. colonization of the haemolymph by latent tissue forms). Elevated plasma CHH concentrations have also been implicated in the uptake of water by crustaceans immediately before the moult (Chung et al. 1999). The high concentration of CHH observed during the later stages of Hematodinium infection may therefore be responsible for the bloated appearance of heavily infectcd lobsters. This may eventually cause integumental membranes to break, allowing extrusion of the motile dinospores. Such swarming stages have been noted on several occasions under aquarium conditions (Appleton & Vickerman, 1998). As such, Chapter 6 228 Chapter Eleven - Conclusions and Prospects provides the first description of parasite-induced hormonal disruption in a crustacean host. Furthermore, it suggests that Hematodinium infection in N. norvegicus may provide an excellent model for studying general stress responses in marine invertebrates, especially those involving hormonal and immunological mechanisms. Effects ofother stressors. Although not an initial objective of the current study, in the course of routine examination of catches, an unusual pathology was noted in the abdominal muscle of trawler and creel-caught N. norvegicus. Chapter 7 provides the first description of this pathology, and showed that the condition is similar to the idiopathic or spontaneous muscle necrosis previously reported to occur in several other crustacean species (see Evans et al. 1999). The pathology described in N. norvegicus involves a rapid degeneration of the muscles of the abdomen, which renders the lobster immobile and then dead within 34 days. The economic impact of this condition has already been felt by wholesalers on the west coast of Scotland. This resulted from the mass mortality of X norvegicus during vivier transport to the continent in the summer and autumn of 1999. The data presented in Chapter 7 for the prevalence of muscle necrosis immediately after trawl capture, and 4 hours later, reinforce the theory that the holding conditions of the animals in the period immediately following capture are crucial in determining whether the necrotic condition develops or regresses. This has important consequences both for the transport of live N. norvegicus, and for the quality of the meat of lobsters 'tailed' at sea. As sorting of undersized lobsters may take up to 4h (see Chapman et al., 2000), the pathology may also contribute to the high mortality of discarded N. norvegicus which are returned to the sea after trawl capture and several hours of cmersion (Ulmcstrand el al.. 1998). The pathology of muscle necrosis described in Chapter 7 was shown to be different from that occurring during Hematodinium infection (Chapter 5). As the protein composition of the deep 229 Chapter Eleven - Conclusions and Prospects abdominal flexor muscles of the abdomen were largely unchanged during Hematodinium infection, it could be suggested that anecdotal reports of 'watery' and unstructured meat from the tails of apparently Hematodinium-infected lobsters may have in fact been describing the tail pathology noted in Chapter 7. Further studies on the conditions which lead to expression of this pathology and to the structure, biochemical composition and taste of cooked tail muscle from lobsters showing symptoms of necrosis would allow such issues to be addressed. Hematodinium and the catchability of infected lobsters. In terms of burrow-related behaviour, infected male N. norvegicus were shown to spend significantly longer periods out of the burrow, and appeared not to show the diel emergence rhythm seen in uninfected lobsters (Chapter 10). These changes in behaviour may be driven by the increased nutritional and/or respiratory requirements of Hematodinium-infected lobsters as mentioned above. As such, changes in burrow-related behaviour are likely to be implicated in an increased catchability of infected lobsters by trawl nets, especially when coupled with the reduced swimming performance noted in Chapters 8 and 9. Increased catchability may lead to overestimation of infection prevalence in the field, and may impact upon the accurate assessment of natural mortality attributable to Hematodinium infection (Anon, 1997). These findings suggest why previous attempts to incorporate the high observed prevalence of Hematodinium infection into anahlical stock assessments of the Clyde Sea area, which modelled the effect as an additional loading on the natural mortality rate (since animals showing symptoms of patent infection usually die), led to unrealistic estimates of the number of recruits entering the fishery (Anon, 1997). Infected animals, while being more prone to capture by trawlers, may also be more susceptible to predation through both a reduced ability to escape and an increase in 'risk prone' behaviour (Chapter 10), 230 Chapter Eleven - Conclusions and Prospects Current fisheries independent methods for N. norvegicus stock assessment are based on the estimation of larval production, the use of underwater television for making burrow density counts, and the use of trawl surveys. Comparisons between the number of lobsters present on the sediment surface and the burrow density suggest that only around 10 % of lobsters are out of their burrows at any given time (Tuck et al., 1997). At higher burrow densities, proportional emergence may be greater than this, as individuals in such populations may have to spend longer foraging for food. However, while analytical models assume equal capture availability throughout the distribution of the stock (Tuck et al., 1997), stocks exhibiting significant Hematodinium infection prevalence may also be showing greater proportional emergence than stocks with lower infection prevalence. As such, estimates of N. norvegicus abundance from trawler capture may overestimate true infection prevalence on these grounds. Further studies on the infection prevalence of trawl- caught populations in relation to the density of burrows and the proportional emergence of lobsters on the same grounds would represent a field test of the theories of increased burrow emergence presented in Chapter 10. 231 Chapter Eleven - Conclusions and Prospects References. Anon 1997. Report of the working group on Nephrops stocks. ICES CM 1997/Assess: 9 Appleton, P. L. & Vickerman, K. V. (1998). In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sp. ) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116,115-136. Baden, S. P., Depledge, M. H. & Hagerman, L. (1990). Glycogen depletion and altered copper and manganese handling in Nephrops norvegicus following starvation and exposure to hypoxia. Marine Ecology Progress Series 103,65-72. Chang, E. S., Chang, S. A., Keller, R., Reddy, P. S., Snyder, M. J. & Spees, J. L. (1999). Quantification of stress in lobsters: crustacean hyperglycemic hormone, stress proteins and gene expression. American Zoologist 39,487495. Chapman, C. J., Shelton, P. M. J., Shanks, A. M. & Gaten, E. (2000). Survival and growth of the Norway lobster Nephrops norvegicus in relation to light-induced eye damage. Marine Biology 136,233-241. Chung, J. S., Dirckscn, H. & Webster, S. G. (1999). A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas. Proceedings of the National Academy of Science USA, 96: 13103- 13107. Coats, D. W. (1999). Parasitic life styles of marine dinoflagellates. Journal of Eukaryotic Microbiology 46,402409. Evans, L. H., Fotedar, S., Fan, A. & Jones, B. (1999). Investigation of idiopathic muscle necrosis and circulating hemocytes in the freshwater crayfish Cherax tenuimanus exposed to acute and chronic stressors. Freshwater Crayfish 12 (in press). Farmer, A. S. D. (1974). Reproduction in Nephrops norvegicus (Decapoda: Ncphropidac). Journal of Zoology, London 174,161-183. Field, R. H. & Appleton, P. L. (1995). A Hematodinium-like dinoflagellate infection of the Nom, ay lobster Nephrops norvegicus: observations on pathology and progression of infection. Diseases of Aquatic Organisms 22,115-128. Field, R. H. & Appleton, P. L. (1996). An indirect fluorescent antibody technique for the diagnosis of Hematodinium sp. infection of the Nonvay lobster, Nephrops norvegicus. Diseases of Aquatic Organisms 24,199-204. Field, R. H., Chapman, C. J., Taylor, A. C., Neil, D. M. & Vickerman, K. (1992). Infection of the Norway lobster Nephrops norvegicus by a Hematodinium-likc specics of dinonagellatc on the west coast of Scotland. Diseases of Aquatic Organisms 13,1-15. Field, R. H., Hills, J. M., Atkinson, R. J. A., Magill, S. & Shanks, A. M. (1998). Distribution and seasonal prevalence of Hematodinizim sp. infection of the Norway lobster (Nephrops norvegicus) around the west coast of Scotland. ICES Journal of Marine Science 55,846- 858. 232 Chapter Eleven - Conclusions and Prospects Hudson, D. A. & Adlard, R. D. (1994). PCR-techniques applied to Hematodinium spp. and Hematodinium-like dinoflagellates in decapod crustaceans. Diseases of Aquatic Organisms 20,203-206. Hudson, D. A. & Adlard, R. D. (1996). Nucleotide sequence determination of the partial SSU rDNA gene and ITSI region of Hematodinium cf. perezi and Hematodinium-like dinoflagellates. Diseases of Aquatic Organisms 24,55-60. Hudson, D. A. & Shields, J. D. (1994). Hematodinium australis n. sp., a parasitic dinoflagellate of the sand crab Portunus pelagicus from Moreton bay, Australia. Diseases of Aquatic Organisms 19,109-119. Johnson, P. T. 1980. The gut. In: Histology of the blue crab, Callinectes sapidus. A model for the Decapoda. Praeger Scientific, New York. pp. 149-247. Lin, S. & Cohen, H. P. (1973). Crayfish ventral nerve chord and hemolymph : content of free amino acid and other metabolites. Comparative Biochemistry & Physiology 45B, 249- 263. Livingstone, DR (1982). General biochemical indicators of sublethal stress. Marine Pollution Bulletin 13,261-263. Meyers, T. R., Koeneman, T. M., Botelho, C. & Short, S. (1987). Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan tanner crabs, Chionoeceles bairdi. Diseases of Aquatic Organisms 3,195-216. Paterson, B. D. & Spanoghe, P. T. (1997). Stress indicators in marine decapod crustaceans, with particular reference to the grading of western rock lobsters (Panuhrus cygnus) during commercial handling. Marine & Freshwater Research 48,829-834. Santos, E. A. & Keller, R. (1993). Regulation of circulating levels of crustacean hyperglycaemic hormone: evidence for a dual feedback control system. Journal of Comparative Physiology 163B, 374-379. SardA, F. (1998), Symptoms of overexploitation in the stock of the Norway lobster (Nephrops norvegicus) on the "Serola bank" (western Mediterranean sea off Barcelona). Scientia Marina 62,295-299. Schmidt, S. P. & Platzer, E. G. (1980). Changes in body tissues and hcmolymph composition of Culex pipiens in response to infection by Romanomermis culicivorax, Journal of Invertebrate Pathology 36,240-254. Shirai, T., Hirakawa, Y., Koshikawa, Y., Toraishi, H., Terayama, M., Suzuki, T. & Hirano, T. (1996). Taste components of Japanese spiny and shovel-noscd lobsters. Fisheries Science 62,283-287. Taylor, A. C., Field, R. H. & Parslow-Williams, P. J. (1996). The cffccts of Hematodinium sp. -infection on aspects of the respiratory physiology of the Nonvay lobster, Nephrops norvegicus (L. ). Journal of Experimental Marine Biology & Ecology 207,217-228. 233 Chapter Eleven - Conclusions and Prospects Taylor, D. M. & Khan, R. A. (1995). Observations on the occurrence of Hematodinium sp. (Dinoflagellata: Syndinidae); the causative agent of Bitter crab disease in Newfoundland snow crab (Chionoeceles opilio). Journal of Invertebrate Pathology 65,283-288. Tuck, I. D., Chapman, C. J., Atkinson, R. J. A., Bailey, N. & Smith, R. S. M. (1997). A comparison of methods for stock assessment of the Norway lobster, Nephrops norvegicus, in the Firth of Clyde. Fisheries Research 32,89-100. Ulmestrand M., Valentinsson D., Sangster G. I., Bova D., Kynoch R. J., Breen M., Graham G. N., Soldal A. V., Cruikshank 0., Moth-Poulson T. & Lowry N. (1998). Nephrops survival after escape from commercial fishing gear or discarded from deck. ICES Nephrops life history study group meeting. La Coruila, Spain. 1998. Wilhelm, G. & Mialhe, E. (1996). Dinoflagellate infection associated with the decline of Necora puber crab populations in France. Diseases of Aquatic Organisms 26,213-219. Zatta, P. (1987). Calcium and taurine interaction in the heart of the crab Carcinus maenas. Canadian Journal of Fisheries & Aquatic Science 44,1765-1768. I.. I cilm. ýýz 'ýC! I umt=t-7 I Lmmk", 234 'rhe Cumbracs. Firth of Clyde (G. D. Stcntiford) it is not stonn or cahn, butyesterdav The wild winds leapt in sudden thunder down; Shook the dark waters into a starry sprqv, And thrilled the soul ofmany a seaside town. 'The Sorrow of the Sea', John Hogben. ==, Ualily UZMLMI-7 Ln2=

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