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Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ research-enlighten@glasgow.ac.uk Sulphide metabolism in burrowing marine CrustaceaAntony Richard Johns B.Sc.(Hons ) WalesA thesis submitted for the degree of Doctor of Philosophy to the Faculty of Science at the University of Glasgow © September 1996Division of Environmental & Evolutionary Biology,Institute of Biomedical & Life Sciences,University of Glasgow, Glasgow G 12 8QQ&University Marine Biological Station Millport, Isle of Cumbrae, Scotland, KA28 OEG______________ProQuest Number: 10391287All rights reservedINFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.In the unlikely event that the author did not send a com p le te manuscript and there are missing pages, these will be noted. Also, if material had to be removed,a note will indicate the deletion.uestProQuest 10391287Published by ProQuest LLO (2017). Copyright of the Dissertation is held by the Author.All rights reserved.This work is protected against unauthorized copying under Title 17, United States C odeMicroform Edition © ProQuest LLO.ProQuest LLO.789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346THIS BOOK IS BOUND BY M. DOWNIE & SON UNITS F & G, PURDON ST PARTICK GLASGOW G11 6AF TEL: 0141-339-0333' l L x \>l o t nT Gl^ wGOW VNIVERSIT? USRAEYTo my wife and family, Bridget, Ellie and Peter and our parents. : _ :The conquerors of the shelf will have to get wet, (Cousteau & Dumas, 1953),Düsseldorf; Paul 'Parsley' Williams, Lynne Sneddon, Murray Roberts, Ruth Beaver, Francesca Marubini, June Freel, Jason Sheridan, Andy Hodds, Susanne Volkel, Steffi Wohlgemuth, Eva Stegen, Martin Till, Christian Ortmann and Daniel Haas.Acknowledgem entsThe work in this thesis was carried out in the Division of Environmental and Evolutionary Biology of Glasgow University funded by awards from the NERC and the British Council. I would like to thank Professor F. Huntingford for providing the research facilities and Dr. Alan Taylor and Dr. Jim Atkinson who supervised the project. I am also indebted to Professor Manfred K.Grieshaber and Professor Chris Bridges for invaluable discussions and allowing me to work in Institut fur Zoophysiologie at the Heinrich-Heine Universitat in Düsseldorf. I am also grateful to Dr Susanne Volkel for assistance with the | |'SiHPLC assays. Dr Jochen Koop and Christian Ortmann for help with Calo- respirometry. Professor K. Vickerman, Dr. Lawrence Tetley, Margaret Mullen,and Eoin Robertson for guidance, preparation and use of transmission electron 51microscopy, Phil Lonsdale, Ken Cameron and John Chamberlain of the UMBS diving team, the crews of research vessels Aora and Aplysia, Liz Denton for advice with graphics and June Freel for technical assistance. Many thanks also to my many friends and colleagues in the Universities o f Glasgow and |If:f— — : : .AbstractSulphide occurs widely in marine sediments and is highly toxic to most organisms. Its principal poisoning effect occurs at extremely low concentrations and is the result of inhibition of mitochondrial cytochrome c oxidase. Mud- shrimps, (Crustacea: Decapoda: Thalassinidea), construct burrows in sublittoral muddy sediments. The sediment in which they burrow may often be markedly reduced and conditions within the burrow are usually hypoxic and hypercapnie. Field measurements taken during this study also indicate significant exposure to sulphide in two species of mud-shrimp. Direct evidence has demonstrated that Callianassa subterranea may be exposed to potentially toxic levels of sulphide in the burrow water (37 ± 44 piM, range 0 - 206 piM). Sulphide, thiosulphate and sulphite were also found in the haemolymph and tissues of freshly caught animals {Calocaris macaudreae and Callianassa subterranea) indirectly indicating that they are exposed to ambient sulphide. The concentmtions of sulphide found in the burrow water and thiol concentrations within the mud-shrimps also showed seasonal variations which may be correlated with the input of organic material.Mud-shrimps appear to be physiologically adapted to tolerate elevated levels of sulphide that they may encounter in their natural habitat. Laboratory experiments carried out on three species, Calocaris macandreae, Callianassa subterranea and Jaxea nocturna, have shown that they have a high tolerance of sulphide. High micromolar concentrations of sulphide (360 ± 320 ptM) were also found not to affect the irrigation behaviour of the mud-shrimp Calocaris macandreae suggesting tolerance of sulphide exposure.The mud-shrimp Calocaris macandreae can tolerate substantial exposure to sulphide before showing signs of anaerobic metabolism. An oxygen-dependent 'oxidation* mechanism appears to exist which defends mitochondrial cytochrome c oxidase from sulphide poisoning. Sulphide diffusing into the body tissues is oxidized primarily to thiosulphate, in the hepatopancreas and muscle tissues, and____ ___ _________________________________________________accum ulates rapidly even during brief exposure to low m icrom olar concentrations of sulphide. Sulphite also appears as a secondary 'oxidation' product. No evidence of chemoautotrophic bacterial symbionts could be found and it is therefore proposed that the 'oxidation' of sulphide is facilitated by the animal's tissue.The 'oxidation' mechanism allows aerobic metabolism to be maintained despite the presence of sulphide although the mud-shrimp's sensitivity to sulphide is apparently greater at lower oxygen partial pressures. Sulphide also has a concentration-dependent capacity to either stimulate or inhibit aerobic metabolism in mud-shrimps. Aerobic metabolism appears to increase during exposure to sulphide, when oxygen is available, and can be maintained even under severe hypoxic and sulphidic conditions. The mud-shrimps resort to anaerobiosis (which may operate concurrently with aerobic metabolic pathways) when the 'oxidation' mechanism is saturated, when oxygen supply becomes limited and when intracellular sulphide concentrations become toxic. The possibility of metabolic depression to reduce energy expenditure may also occur in response to sulphide exposure and is discussed.Haemocyanin function was also found to be unaffected by sulphide and oxygen affinity increased in the presence of thiosulphate. This may be a physiological adaptation to increase the amount of oxygen available for sulphide 'oxidation' to produce the metabolites thiosulphate and sulphite during severely hypoxic and sulphidic conditions. Mudshrimps are therefore physiologically adapted to tolerate elevated levels of sulphide which they may encounter in their natural habitat. The 'oxidation' of sulphide in the mitochondria in a small number of other marine invertebrates has been established and it therefore seems likely that this may be the case in mud-shrimps. Indirect evidence suggests that the underlying biochemical mechanism of mitochondrial sulphide 'oxidation' is enzymatic ('sulphide oxidase'), although direct evidence of this, the isolation of such an enzyme and its characterization remain to be elucidated. : _________________ _______Table of contentsAcknowledgementsAbstractContentsChapter 1. Introduction1.1. Sulphide in the aquatic environment1.2. Sulphur and sulphide in the biosphere1.3. Sulphide chemistry and toxicity1.4. Animals in sulphide rich environments1.5. Thalassinidean biology and ecology1.6. Project aim and specific objectivesChapter 2. Sulphide in marine sediments2.1. Introduction2.1. 1. Sulphide chemistry in water2.1.2. Sulphide in marine sediments2.1.3. Sulphide exposure in the field2.2. Materials and methods2.2.1. Sulphide measurementPage Number4691319212325v i■a! _ _ _2.2.1a. Colorimetric determination of sulphide 252.2.1b. Chromatographic determination of sulphide 262.2.1c. Potentiometric determination of sulphide 312.2. Id. lodometric determination of sulphide 332 .2 .le. Comparison of the methods for the measurement of sulphide 342.2.2. Burrow water sampling at Loch Sween, the Clyde Sea and in laboratory mesocosms 352.2.3. Concentrations of sulphide in interstitial water in Loch Sweenand Clyde sediments 372.2.4. Sediment Eh 392.2.5. Thiol concentrations in freshly caught animals 392.2.6. Behaviour of mud-shrimps during sulphide exposure 402.2.7. Statistics 412.3. Results2.3.1. Sulphide measurement 432.3.2. Burrow water sampling at Loch Sween, the Clyde Sea andin laboratory mesocosms 432.3.3. Concentrations of sulphide in interstitial water in Loch Sween 462.3.4. Sediment Eh 462.3.5. Thiol concentrations in freshly caught animals 472.3.6. Behaviour of mud-shrimps during sulphide exposure 52 : :2.4. Discussion2.4.1. Sulphide measurement 552.4.2. Sulphide exposure in the field 562.4.3. The effect of sulphide on the behaviour and distribution of marineinvertebrates 612.4.4. Summary of Chapter 2 63C hap ter 3. Su lph ide to leran ce, ’o x id a tio n ’ a n d metabolism in mud-shrimps3.1. Introduction3.1.1. Sulphide tolerance 653.1.2. Sulphide 'oxidation' and metabolism 663.1.3. Symbiotic chemoautotrophic sulphur bacteria3.1.4. Sulphur amino acid metabolism67683.2. Materials and methods.3.2.1. Animal collection and maintenance 693.2.2. Sulphide tolerance of mud-shrimps3.2.3, Sulphide 'oxidation' and metabolism in mud-shrimps6972 '.a3.2.4. Symbiotic chemoautotrophic sulphur bacteria and mud-shrimps 733.2.5. Sulphur amino acid metabolism in mud-shrimps 743.2.6. Potentiometric oxygen sensors and sulphide interference 75.............................................3.3. Results3.3.1. Sulphide tolerance of mud-shrimps 783.3.2. Sulphide 'oxidation' and metabolism in mud-shrimps 783.3.3. Symbiotic chemoautotrophic sulphur bacteria and mud-shrimps 883.3.4. Sulphur amino acid metabolism in mud-shrimps 883.3.5. Potentiometric oxygen sensors and sulphide interference 883.4. Discussion3.4.1. Sulphide tolerance in mud-shrimps 913.4.2. Sulphide 'oxidation and metabolism in mud-shrimps 923.4.3. Potentiometric oxygen sensors and sulphide interference 963.4.4. Summary of Chapter 3 97Chapter 4. Aerobic and anaerobic metabolism of mud- shrimps during exposure to sulphide4.1. Introduction4.1.1. Anaerobic metabolism in marine invertebrates4.1.2. Anaerobic metabolism in Crustacea 1014.1.3. Sulphide-induced anaerobic metabolism in marine invertebrates 1024.1.4. Sulphide-induced anaerobic metabolism in mud-shrimps 1034.2. Materials and methods4.2.1. Collection and maintenance of animals 105___________________4.2.2. Whole animal oxygen consumption during sulphide exposure 1054.23. Anaerobic metabolism under sulphidic conditions4.2.3b. Method for the analysis of L-lactate4.3.1. Whole animal oxygen consumption4.3.2. Anaerobic metabolism under sulphidic conditions4.3.3. Accumulation of thiols during exposure to sulphide during normoxic and hypoxic conditions4.4. Discussion4.4.1. Whole animal oxygen consumption4.4.2. Sulphide-induced anaerobic metabolism4.4.3. Summary of chapter 41084.2.3a. Preparation of haemolymph and tissue samples using perchloricacid extraction 1081104.2.4. Accumulation of thiols during exposure to sulphide during bothaerobic and anaerobic metabolism 1114.3. Results112113114124127130Chapter 5. A preliminary calo-respirometric study of the sulphide metabolism in mud-shrimps5.1. Introduction.5.1.1. Metabolic adaptations to hypoxic, anoxic and sulphidic conditions 1325.2. Materials and methods5.2.1. Collection, transportation and maintenance of mud-shrimps 135I99I, |1■«'!?■: : : -----5.2.2. Direct and indirect calo-respirometry (the instrument)5.2.3, Preparation and calibration5.2.4. Normoxic, hypoxic and sulphidic exposures5.2.5. Calculation of metabolic rates5.3. Results5.3.1. Direct and indirect calorimetry5.4. Discussion5.4.1. Direct and indirect calorimetry5.4.2. Summary of chapter 5135137138 140141149154Chapter 6. Specific effects of sulphide and thiosulphate on the oxygen affinity of the haemocyanin of d e c a p o d crustaceans6.1. Introduction6.1.1. Haemocyanin oxygen transport properties6.1.2. The effect of sulphide on respiratory pigments6.1.3. The effect of thiosulphate on respiratory pigments6.2. Materials and methods6.2.1. Collection and maintenance of animals6.2.2. Collection of haemolymph samples6.2.3. The effect of sulphide on decapod crustacean haemocyanin155156 158160160160■ |IIIa■1Jam■iÎagÎ'• S :;;'a|ï■ ;|5 : :_____________________________________6.2.4. Oxygen transporting properties of the haemolymph from Calocaris macandreae and Nephrops norvégiens6.2.4a. Haemolymph ionic composition6.2.4b. Construction of oxygen binding curves6,2.4c. Calculation of oxygen affinity and cooperativity6.2.5. The effect of thiosulphate on the oxygen affinity and cooperativity of haemolymph from Calocaris macandreae and Nephrops norvégiens6.3. Results6.3.1. The effect of sulphide on decapod crustacean haemocyanin6.3.2. Oxygen transporting properties of the haemolymph from Calocaris macandreae and Nephrops norvégiens6.4.3. The effect of thiosulphate on the oxygen affinity and cooperativityof decapod crustacean haemocyanin6.4. Discussion6.4.1. Sulphide and haemocyanin6.4.2. Oxygen transporting properties of haemolymph6.4.3. Summary of Chapter 67. Summary, conclusions and direction of future work References----------------------------------------------------------------------- ___161162162163165166167168175177181182189!• Introduction.1.1. Sulphide in the aquatic environmentSulphide is a naturally occurring toxin which commonly occurs in aquatic environments. The role of sulphide as an environmental factor for aquatic animals, however, has received relatively little attention, although the risk to sediment-dwelling invertebrates of being exposed to sulphide was recognised during the late 1960s and early 1970s (Theede eta l., 1969; Fenchel & Reidl, 1970; Boaden & Plat, 1971). More recently, a rapidly growing interest in sulphide within marine and limnic environments has developed, stimulated by the discovery of complex communities of animals around geothermal (hydrothermal) vents on the ocean floor (Corless & Ballard, 1977; Pel beck, 1981; Grassle, 1986). Many of these animals may be exposed to high concentrations of sulphide and are apparently able to overcome its potentially toxic effects. Some of these animals form endo-symbiotic associations with chemoautotrophic bacteria to meet their energy requirements from geothermally produced hydrogen sulphide. Over 230 invertebrate species associated with hydrothermal vents have been described, which include molluscs, annelids, crustaceans and members of the new phylum Vestimentifera (Childress & Fisher, 1992). Biomass around these hydrothermal vent sites is highly localized and may be up to 300 times that of the surrounding abyssal regions.Extensive work has taken place over the last few years on hydrothermal vent organisms such as the vestimentiferan Riftiapachyptila (Felbeck, 1981), the bivalves Bathymodiolus thermophiliis and Calyptogena magnifica (Powell & Somero, 1986b) and the decapod crustacean Bythograea thermydron (which lacks symbionts) (Vetter e ta l , 1987). This work has also led to a renewed interest in animals which inhabit other sulphide-rich environments in shallower waters. Many infaunal marine organisms survive in habitats which contain potentially toxic concentrations of sulphide such as in marine sediments, and1may possess mechanisms to detoxify and derive energy from sulphide. A small and growing number of these animals have been investigated and include the polychaete Arenicola marina (Volkel & Grieshaber, 1992, 1995, 1996), the echiuran Urechis caupo (Arp et al., 1992, Eaton & Arp, 1993), the bivalve Solemya reidl (Anderson et ah, 1987) and the Baltic brackish water isopod Saduria entomon (Hagerman & Vismann, 1993). This project has investigated another group of animals, the thalassinideans or mud-shrimps, which form burrows in marine sediments and may be exposed to significant ambient concentrations of sulphide.1.2. Sulphur and sulphide in the biosphereSulphur (atomic number =16, relative atomic mass = 32.06) is an element in the oxygen group (VI) of the Period Table and exists in a wide range of oxidation states in the biosphere. These range from sulphide (2-) in its most reduced form through oxidation states 0, 2+ and 4+ to sulphate (6+) in its most oxidized form. It behaves principally as a non-metal forming covalent species, shows a great tendency to catenation and reacts combining with most other elements to form a wide range of inorganic and organic compounds (Atkins, 1989).The reduced and oxidized forms of sulphur can be inter-converted by various organisms in the biosphere during the sulphur cycle (Figure 1.1). The assimilatory reduction of sulphate by micro-organisms and plants leads to the formation of cysteine which may subsequently be incorporated into higher organisms as proteins via methionine (Schiff & Saida, 1987; Andreae, 1990). Cysteine and methionine may also undergo oxidation in micro-organisms, plants and animals to produce sulphate which may be used to form polysaccharide and steroidal sulphates. Chemosynthetic bacteria also mediate the inter-conversion of sulphide to form sulphate, whilst bacterial dissimilatory sulphate reduction may occur to form sulphide (Schiff & Saida, 1987; Andreae, 1990). - . '£1O)o nZ (5 CPI |ig E 2 :2 m "O04CLsz04«8 «In the aquatic environment sulphur commonly occurs in its reduced form as sulphide as a result of a variety of processes and mechanisms. These include geothermally produced sulphide at hydrothermal vent sites (Edmond e ta l., 1982), bacterial production of sulphide during periods of eutrophication and stratification of the water column (Kitching et al., 1976; Aure & Stigebrand, 1989; Mann & Lazier, 1991) and production of dimethylsulphide by marine plankton (Andreae, 1990). In marine sediments, sulphide is produced under anoxic conditions beneath the redox potential discontinuity layer due mainly to the activity of sulphate-reducing bacteria, and may reach concentrations in the micromolar to millimolar range. Sulphide is oxidized rapidly in the presence of oxygen (Theede et a l, 1969; Fenchel & Reidl, 1970; Theede, 1973; Jdrgensen & Fenchel, 1974; J0 rgensen, 1988). It can therefore accumulate to significant levels only in anoxic or severely hypoxic environments (Cline & Richards, 1969; Chen & Morris, 1972; Invorgensen & Jdrgensen, 1979; Jdrgensen, 1982). Thus, high sulphide levels are a correlate of ambient oxygen deficiency. Many infaunal marine organisms survive in habitats which contain potentially toxic levels of sulphide in interstitial water. Animals that inhabit marine sediments may therefore be subjected to sulphide and its potentially toxic effects.1.3. Sulphide chemistry and toxicityIn aqueous solution sulphide exists in three different chemical species; hydrogen sulphide (H^S), the hydrosulphide anion (HS~) and the bisulphide anion (S^-)* Sulphide therefore is conventionally expressed as total sulphide: [Sulphide] = [S^-] + [HS'] + [HjS] (Vismann, 1991a; Bagarinao, 1992). The proportion of each species is highly dependent on pH (see section 2,1.1.) (Goldhaber & Kaplin, 1975). At physiological and environmental pHs (7 - 8 ), however, hydrogen sulphide and the hydrosulphide anion predominate. This has a considerable effect on sulphide toxicity because H^S is the form which freely diffuses across membranes (see section 2.4.2.) (Bagarinao, 1992).I,Hydrogen sulphide is extremely toxic to aerobic organisms (Evans, 1967;Nicholls, 1975; National Research Council, 1979; Beauchamp e ta l., 1984;Vetter e ta l , 1991). Its principal poisoning effect, which is reversible, is the result of the inhibition of mitochondrial cytochrome c oxidase by binding to cytochrome aas. This results in the disruption of the electron transport chain and inhibition of aerobic metabolism (Nicholls, 1975). The isolated enzyme, taken from various organisms as homogenates, has been shown to be very sensitive to sulphide and is inhibited at nanomolar to low micromolar concentrations of sulphide (Hand & Somero, 1983; Bagarinao, 1992). Aerobic mitochondrial metabolism, however, is inhibited at higher sulphide concentrations (2 - 38 piM); approximately 1 - 2 orders of magnitude greater (Bagarinao, 1992). Since the enzyme is located at the inner mitochondrial membrane, sulphide may be oxidized by the mitochondria before cytochrome c oxidase is inhibited.To a lesser extent sulphide may also effect other enzymes (approximately 20), metabolites and cofactors. These include ATPase, carbonic anhydrase and succinate dehydrogenase (Bagarinao, 1992). Sulphide is a very strong nucleophile and may therefore reduce disulphide bridges and bind to haem-type enzymes which results in the inhibitory effects described above (National Research Council, 1979). Respiratory pigments may also be affected. In the presence of sulphide, haemoglobin may undergo a number of chemical reactions (Kraus etal., 1996) either binding to the iron-containing haem centre (Somero et al., 1989; Kraus et al., 1996) or to the tetrapyrrole ring of vertebrate1SÏ: mJ ihaemoglobins. Haemoglobins may react with sulphide to form haematins which may also catalyse the 'oxidation' of sulphide. These reactions may significantly reduce oxygen affinity causing the molecule to become dysfunctional. The effect of hydrogen sulphide on haemocyanin is unclear, although, in contrast to haemoglobin, it may not be poisoned by sulphide (Vetter et a l, 1987; Hagerman & Vismann, 1993). In addition, sulphide may generate reactive radicals and altermembrane permeability (Bagarinao, 1992). Finally, it has also been established'■ithat the survival of animals which resort to anaerobiosis to overcome the poisoning of cytochrome c by sulphide may be reduced, due to the inhibition of anaerobic metabolic pathways (Theede, 1969; Vismann, 1990, Vismann, 1991b).1.4. Animals in sulphide rich environmentsAnimals inhabiting sulphide-rich environments, such as marine sediments, hydrothermal vents, methane seeps, and those exposed to sulphide during eutrophication events and the stratification of the water column, tend to have an increased tolerance of sulphide in comparison with other species which are less likely to be exposed to it. The sulphur present in most living organisms (sulphide tolerant and non-tolerant) occurs in the form of sulphur-containing amino acids (cysteine and methionine) and as sulphate (SOa^"). In sulphide- tolerant animals, however, additional forms of sulphur are often abundant in haemolymph and tissues: sulphide (S^-), thiosulphate (S2O32-), sulphite (SOj^-) and elemental sulphur (S®) (Childress & Fisher, 1992). These sulphur groups are 'oxidation' products of sulphide and their presence in sulphide-tolerant animals has been widely interpreted as sulphide 'detoxification' metabolites. A range of adaptive physiological and biochemical 'oxidation' mechanisms to counter the toxic effects of sulphide and in some cases derive energy have been proposed in a variety of animal groups (Vetter et al., 1991; Vismann, 1991b; Bagarinao, 1992; Volkel & Grieshaber, 1995). These adaptations are now believed to be fairly common in animals inhabiting the marine and limnic environments and include meiofauna (Fenchel & Reidl, 1970; Powell et a i , 1979, 1980; Ott e ta l , 1991, 1996) and various macroinvertebrates (Theede et al., 1969, 1973) such as annelids (Degn & Kristensen, 1981), polychaetes (Vismann, 1990; Llanso, 1991; Volkel & Grieshaber, 1994, 1995), oligochaetes (Felbeck et a l , 1983), sipunculan worms, (Volkel & Grieshaber, 1992), echiuran worms (Eaton & Arp, 1993), vestimentiferan and pognophoran worms : ■ -(Powell & Somero, 1983; Childress et aL, 1984), priapulidan worms (Oeschger & Vetter, 1992), bivalve molluscs (Felbeck, 1983, Powell & Somero, 1986a; Levitt & Arp, 1991; Herry eta l., 1989; O'Brien & Vetter, 1990), amphipods (Vargo & Sastry, 1977), isopods (Vismann, 1991a) and decapod crustaceans (Vetter et al., 1987; Astall 1993), as well as some shallow-water marine fish (Bagarinao & Vetter, 1989, 1990). These physiological and biochemical 'detoxification' strategies for protection against sulphide and perhaps for the derivation of energy are summarized below and include specific examples1. Evolution of an alternative sulphide insensitive cytochrome c oxidase, as has been proposed to exist within the mitochondria of the intertidal polychaete Arenicola marina (Volkel & Grieshaber, 1996).2. Exclusion of sulphide at the body wall because of impermeability (not described to date) or by the presence of a layer of sulphide oxidizing bacteria covering the cuticle as has been reported to occur in some marine nematodes (Ott etal., 1991)3. The use of anaerobic metabolism during exposure to sulphidic conditions when aerobic metabolism is inhibited. This has been established in a small number of marine invertebrates including the bivalve Solemya reidi (Anderson et al., 1990) and the Baltic brackish water isopod Saduria entomon (Hagerman & Vismann, 1993).4. The use of sulphide-binding proteins with subsequent unloading at sites of 'oxidation', or upon return to low external sulphide concentrations, as in the haemoglobin of the hydrothermal vent vestimentiferan Riftia pachyptila (Arp el a l, 1984, 1987).75. Symbiotic association with chemoautotrophic bacteria capable of sulphide ’oxidation'. The symbionts, using energy from sulphide 'oxidation', fix carbon dioxide into organic compounds for translocation to the host, e.g. the bivalve Solemya reidi (Pelbeck, 1983; Anderson e? a/., 1987) and the hydrothermal vent vestimentiferan pachyptila (Felbeck, 1981, 1983).6 . Eukaryotic sulphide 'oxidation' with the potential utilization of energy released in sulphide 'oxidation' to drive the generation of ATP, e.g. the fish F und iiliisparv ip inn is (Bagarinao & Vetter, 1990), the priapulid worm Halicryptus spinulosus (Oeschger & Vetter, 1992) and the polychaete worm Heteromastus flliformis (Oeschger & Vismann, 1994).7. Behavioural adaptations to actively avoid sulphide as described in Corophiimi volutator (Meadows et al^ 1981).Most studies of these mechanisms have been carried out in various 'worms' and some bivalves. Only very limited work, however, has been conducted on the sulphide metabolism of Crustacea (Vetter et al., 1987; Vismann, 1991b; A stall, 1993; Ha germ an & Vismann, 1993; Gorodezky & Childress, 1994). In comparison with other benthic invertebrates, it is generally believed that crustaceans have a low physiological tolerance of sulphide and may use behavioural adaptations to overcome its toxic effects (Theede et a l, 1969; Vargo & Sastry, 1978; Meadows et al., 1981; Vismann, 1991b). Much work has been conducted on the biology and ecology of the hydrothermal vent-crab Bythograea tliermydron (Vetter e ta l , 1987; Sanders & Childress, 1992; Gorodezky & Childress, 1994). Very little, however, has been carried out on other crustaceans from different sulphidic environm ents. M ud-shrim ps (Crustacea; Decapoda; Thalassinidea) construct burrows in sediments in eulittorai and sublittoral marine environments in which they may be exposed to sulphide.8They occur in a substantially different biotope than other animals previously reported from sulphidic environments. Given the abundance and dominance of mud-shrimps in many intertidal and sublittoral environments (Buchanan, 1963; Buchanan & Warwick, 1974; Calderon-Perez, 1981; Nash et aL, 1984; Atkinson, 1986; Anderson et a i, 1991), the ecophysiological effects of sulphide on these animals may be of considerable importance and interest.1.5, Thaïassinidean biology and ecologyMud-shrimps, (Crustacea: Decapoda: Thalassinidea), construct complex burrows of species-specific architecture, which usually penetrate deep into intertidal and sublittoral sediments which may often be anoxic and reduced (Dworschak, 1983; Nash e ta l., 1984; Atkinson & Taylor, 1988; Nickell & Atkinson, 1995; Ziebis et at., 1996). These animals have attracted increasing attention in many recent ecophysiological studies on marine soft-sediment benthos. This is because mud-shrimps are often abundant and may form a large component of the infaunal macrofauna (Buchanan, 1963; Buchanan & Warwick, 1974; Calderon-Perez, 1981; Rowden & Jones, 1994). In addition, their bioturbatoiy activities strongly influence the benthic community structure and the chemico-physical structure of the sediment water interface (Aller, 1983; Nickell, 1992, Ziebis et al., 1996).The Infra-order Thalassinidea (Superfamily Thalassinoidea) Latreille comprises of approximately 5(X) species (Saint-Laurent & Lelouef, 1979). There are eight thaïassinidean species in U.K. waters: Upogebia stellata (Montagu), Upogebia deltatira (Leach), Upogebia pusilla (Petagna), Calocaris tnacandreae Bell (Plate 1.1), Callianassa subterranea (Montagu) (Plate 1.2), Callianassa tyrrhena (Petagna), Jaxea nocturna Nardo and Axius stirhynchus Leach. A number of these infaunal species inhabit the muddy eulittorai and sublittoral sediments around the west coast of Scotland and may be locally abundant. During the course of this study the biology and ecology of two different specieswere investigated in relation to sulphide; C. tnacandreae (Plate 1.1) and C. subterranea. (Plate 1.2).Within the burrow environment, mud-shrimps lead a fairly sedentary and solitary lifestyle. Apart from a brief pelagic larval stage which may be abbreviated in some species, such as in Calocaris tnacandreae, they rarely if ever, leave their burrows, although brief excursions in the immediate vicinity of the openings for purposes of excavation and feeding may occur (Nickell & Atkinson, 1995). The burrows themselves are some of the deepest (> 50 cm; sometimes several metres deep) and most complex formed by decapod Crustacea (Dworschak, 1983; Atkinson & Taylor, 1988; Atkinson & Nash, 1990; Ziebis et al., 1996). Burrow architecture has been shown to be species-specific and varies considerably from simple 'Y' or 'U' morphs to more complex and intricate designs (Dworschak, 1983; Suchanek, 1985; de Vaugelas, 1990). The burrow of C. m acandreae (Plate 1.3) typically consists of groups of interconnecting tunnels (usually with three openings at the sediment surface), penetrating the sediment to around 10 cm, below which a secondary development of circular tunnels at around 20 cm typically occurs (Nash et al., 1984; Atkinson, 1986). In the case of Callianassa subterranea, the burrow (Plate 1.4) consists of a lattice of tunnels and chambers connected to the surface by one or more inhalant and exhalant shafts and may extend to depths of greater than 86 cm into the sediment (Atkinson & Nash, 1990; Nickell & Atkinson, 1995). Feeding behaviour may vary considerably amongst the thalassinideans but is believed to have a strong influence on burrow morphology (Nickell & Atkinson, 1995; Ziebis e ta l., 1996). Three major feeding types have been proposed; suspension feeders, deposit feeders and seagrass harvesters which are associated with different types of burrow architecture (Suchanek, 1985). The mud-shrimps C. tnacandreae and C. subterranea are primarily sub-surface deposit feeders (Buchanan, 1963; Calderon-Perez, 1981; Nickell & Atkinson, 1995). C. subterranea, however, may supplement its diet by suspension feeding (Nickell &10Atkinson, 1995). Due to these activities burrow shape may be continuously modified (Nickell & Atkinson, 1995). This plasticity in feeding and interaction of different trophic modes may be used to exploit the most beneficial food resource.In contrast to the burrowing activities of mud-shrimps, rather little is known about the reproductive behaviour of the animals. The timing, frequency and location of mating is virtually unknown, although it has been suggested that mating occurs in intersecting burrows (Rodriques, 1976; Felder & Lovett, 1989). The mud-shrimp Calocaris macandreae, however, is a protrandous hermaphrodite, the testes and ovaries develop simultaneously for approximately three years before the testes degenerate leaving the vas deferenia filled with spermatophores (Buchanan, 1963). Cross fertilization however, may not be precluded. The ovaries continue to develop before the first eggs are laid at a latter stage (during year 5 in Northumbrian specimens) (Buchanan, 1963). The breeding season in most species of mud-shrimp is known to be divided into two distinct peaks (minor and major episodes) which occur in the spring and summer (Hailstone & Stephensen, 1961; Buchanan, 1963; Forbes, 1979; Dworschak,1988). Single breeding seasons have, however, been reported in some thaïassinidean species (Tunberg, 1986; Felder & Lovett, 1989; Hanekom,1989). The life-spans of mud-shrimps, including callianassids, have been reported to range between 2- 5 years (Devine, 1961; Hailstone & Stephenson, 1961; Forbes, 1977; Dworschak, 1988) although the mud-shrimp Calocarisapparently survives for 5 - 1 0 years (Buchanan. 1963; Calderon- Perez, 1981). Moulting appears to occur annually in C. macandreae from 4 years old onwards, although several moults may take place throughout the year in younger individuals (Buchanan, 1963).The bioturbatory effects of mud-shrimps, which also include the recycling of organic material, nutrients and trace elements, may be considerable in terms of sediment and also in water movement through the burrow (Pemberton e ta l.,111976; Aller er a l, 1983; Suchanek, 1983; Waslenchuck et a l, 1983; Abu-Hilal, 1988; de Vaugelas, 1990; de Vaugelas & Buscail, 1990; Nickell, 1992; Ziebis et al., 1996). The composition and structure of the macro and microbiotic assemblages may also be strongly influenced by these activities (Suchanek, 1983; Posey, 1986; Branch & Pringle, 1987; Griffis & Chavez, 1988; Posey et a l, 1991).Conditions within the burrows are very different from those at the sediment surface. Irrigation may, however, be infrequent and large areas of the burrow lumen may not be in contact with oxygenated water (Forster & Graf, 1992; 1995). The burrow water is usually hypoxic and hypercapnie (Anderson, 1989; Anderson et a l , 1991; Nickell, 1992; Astall et a l , 1997; Ziebis et aL,1996). Oxygen availability within the burrows of Calocaris macandreae has been shown to be low, with minimum P02 values in the region of 15 Torr (Anderson et a l, 1991). As a consequence of permanent hypoxia and short periods of anoxia in mud-shrimp burrows, sulphide levels may be elevated to some degree in the burrow water. Additionally, during burrowing, these animals may be subjected to toxic levels of sulphide that are present in the interstitial water. Interestingly, mud-shrimps are among the few species to survive the low oxygen partial pressures and high sulphide levels in the vicinity of fish cages in sea lochs (Atkinson, 1987).Thaïassinidean mud-shrimps are known to be able to survive exposure to hypoxia (Anderson et a l , 1991) and show a number of adaptations to these conditions. These include behavioural responses to declining oxygen tensions such as irrigating the burrow to draw in oxygenated sea water (Anderson 1989; Astall, 1993; Astall et al., 1997). In addition, in an attempt to ensure oxygen delivery is not compromised, physiological adaptations have been shown, such as increasing ventilation, maintaining a constant heart rate, reducing metabolism to reduce energy expenditure and the possession of a high affinity respiratory pigment (Anderson, 1989; Astall, 1993; Nickell, 1992, Anderson e t a l , 1994).12v à ;.vSpecies such as Calocaris macandreae are also able to maintain their rates of oxygen consumption constant over a wide range of oxygen partial pressure down to a critical P02 (Pc) of approximately 20 Torr (Anderson et al., 1991). Similar or even lower Pc values have been recorded in several other species of mud-shrimp (Atkinson & Taylor, 1988; Paterson & Thorne, 1995; Astall et al.,1997). Mud-shrimps also appear to have a greater tolerance of anoxia than many other decapods. For example, Neotrypaea (as Callianassa) californiensis and Lepidophthalmus loiiisianensis (as Callianassa jarnaicense) can survive anoxia for 3 - 4 days (Thompson & Pritchard, 1969; Felder, 1979). Although their ability to survive long periods of reduced oxygen availability is now well established, there have been very few studies of their ability to cope with exposure to sulphide,1.6. Project aim and specific objectivesThe aim of the study was to investigate the possible ecophysiological mechanisms that allow thaïassinidean mud-shrimps to overcome the potentially toxic effects of sulphide that may be encountered in their burrows. The following specific objectives have been addressed during the course of the work. A field programme was established to estimate the concentrations of sulphide in burrow and sediment interstitial water to determine whether these animals are likely to be exposed to sulphide in the burrow environment. In addition, sediment characteristics (Eh & pH) were also determined. In the laboratory, the tolerance of sulphide of a range of thaïassinidean species was investigated and the distribution of sulphide and its 'oxidation' products in tissues and haemolymph following exposure to sulphide determined. In addition, the effects of sulphide on aerobic and anaerobic metabolism were also investigated and the effect of sulphide on whole animal respiration and metabolism was established. The effects of sulphide on haemocyanin function and the effect of thiosulphate (a sulphide 'oxidation' product) ort haemocyanin oxygen transport13 _ _ _properties from a small number of decapod crustaceans was also determined. Finally, the behavioural responses of mud-shrimps during exposure to sulphide were investigated..,iI14E(j(NIII0>1I1I15B0 (N II1 phenylenediamine in 5.5 N HCl and 125 pi of 11.5 mM FeClg in 0.6 N HCl to the sample tube. After thorough mixing, the Eppendorf tubes were then incubated in darkness for 15 minutes at 20° C before the absorbance (at 670 nm) of the solution was determined using a spectrophotometer (Philips PU 8700 or Shimazdu UV - 1201). The colour is stable for a number of hours (Gilboa- Garber, 1971). The concentration of sulphide in the solution was then determined using a previously-prepared calibration curve constructed using solutions containing 10 - 100 pM sulphide prepared by serial dilution of a lOOpM standard. This calibration conforms to Beer's law over the absorbance range of 0 - 0.8. To enable the assay to be used with a greater range of sulphide concentrations ( 0 - 1 mM sulphide), smaller volumes of the samples were added to the assay and the final volume of the assay mixture maintained by the addition of the appropriate quantity of distilled water. This allowed the absorbance values to be maintained within the range of the calibration curve.The 100 pM standard solution was prepared from Na2S.9H20 ciystals that had been pre-washed and then dried. The standard solutions were made up using distilled water through which nitrogen had been bubbled for at least 60 minutes to remove the oxygen from solution. The exact concentration of the 100 / 5 % departure from the original calibration values for the 100 pM standards solution the system was checked and recalibrated.The individual thiols were identified from their retention times on the column (Figure 2.2). In addition, to confirm that the peaks were thiols a small number of samples were pre-treated with 10 p\ 2,2'~dithiopyridine (PDS)28_ _______ _ _immediately prior to the derivatization process. This compound has a greater affinity for reduced thiols than the monobromobimane fluorescing agent. Any sample pre-treated in this manner, therefore, has a significantly reduced peak area on the chromatogram providing confirmation that the peaks were thiol compounds.Table 2.2 The HPLC mobile phase gradient 0.25 % acetic acid / methanol gradient in terms of % methanol.Time (min) % Methanol0 125 127 2015 3018 3022 5026.1 10029 10029.1 1235 12290.500Ê0.000ill!20Time (min)Figure 2.2 An HPLC chromatogram of a 100 jaM thiol calibration. Full details of the HPLC protocol are given in 2.2.2.I30:ïî*A.: _________ __2.2.1c. Potentioiîietric determination of sulphideThe direct determination of sulphide by an electrode has a number of distinct advantages over the two previously-described chemical techniques. An ion-selective electrode, such as a sulphide electrode, is an electrochemical half cell responding to a specific ion and obeying the Nemst equation. Rapid determinations of the concentration of sulphide can be achieved provided the pH, temperature and salinity of the sample are known. The conventional operation of sulphide ion-selective electrodes requires their calibration directly before use. This process is extremely time consuming, requires laboratory facilities and, as a result, is impractical for field studies. A technique has recently been described, however, whereby an electrode constant can be determined for any particular sulphide electrode (pers.comm. B. Vismann & L. Hagerman), This remains stable for a number of months and allows the calculation of total sulphide from the voltage generated by the electrode. This technique was adopted throughout the present study. A silver/sulphide ion selective electrode (Russell, 1SE94-4169/11, Aucterm uchty, Fife) in combination with a calomel reference electrode (Radiometer, Copenhagen, Denmark) was used. The principle and methodology of the technique is described below.The tip of the sulphide electrode is constructed of silver. This detects the activity of the S^- ion and measures a voltage dependent on the silver ion activity of the half cell reaction;A028 <=> AgS + Ag+ + e~This reaction can be expressed in terms of the Nernst equation, equation 1, (pers.comm. B. Vismann & L. Hagerman)1). E = k - 2.3 [RT / 2fg, log A^g+31where E = the Ag - A028 electrode potential in mV, k = an electrode constant specific to the Ag - Ag28 and reference electrodes, 2.3 [RT/2F] = Nernst factor where R is the gas constant (J.mol‘l.k ° -1), F is the Faraday constant (c.moh^), T is temperature (®K) and log[A^^+] is the activity coefficient of the silver cation. Defining A^g+ in terms of solubility product allows this equation to relate E to the 8 "^ activity (Ag2-):2). E = k - 2.3 [RT / 2F] log Ag2-The sulphide ion activity coefficient is directly related to the su lph ide concentration by the ionic strength of the solution, see equation 3.3). [8 -^] = Ag2- / Y§2-where Yg2-= ionic strength. It was therefore possible to calculate the electrode constant k (equation 4) by the method described below in a solution of known sulphide concentration.4). E = k - 2.3 [RT / 2F] log [S2-]The specific constant of the electrode was determined by titration. 20 ml of 30 mM sulphide solution in 1 M NaOH was placed in a beaker on a magnetic stirrer. The use of NaOH maintained the solution at pH 14 which results in all the sulphide being present as the 8 ^- species. The sulphide and reference electrodes were placed into the solution and the initial potential (E) and temperature were recorded. The solution was titrated with 0.2 M AgNo^ until the end point was reached. This was determined by a rapid increase in electrode voltage when all the sulphide was consumed to form AgS^-. The exact initial32concentration of sulphide in the beaker was calculated from the volume of silver nitrate consumed in the reaction and used to determine the electrode constant.The electrode was then used to determine the S^- ion activity coefficient from which the [S^i concentration in any given solution of known ionic strength can be calculated. This total sulphide concentration was determined by retrospectively calculating the concentrations of the HS" hydrosulphide anion and of hydrogen sulphide H2S. The activity of these species is dependent on pH (Figure 2.1). This calculation was considerably simplified using tables which describe the relationship between sulphide ion activity and total sulphide concentration (see Radiometer Instructions for F1212S & F1712S sulphide selectrodes) at any given pH and ionic strength. These tables were used in conjunction with a computer spreadsheet to calculate the total sulphide concentration from the electrode voltage, pH, temperature and salinity of the solution.2.2.1d. lodoinetric determination of sulphidelodometric titration was used to determine the exact concentration of sulphide in the solution prior to its use in the calibration of the three techniques described previously (2.2.1a,b & c). Throughout this study sulphide solutions were always made up in oxygen-free distilled water to minimize sulphide oxidation. The water was deoxygenated by bubbling a stream of nitrogen through the solution for at least 30 minutes to achieve this. In addition, the sodium sulphide crystals (Na2S. 9H2O) from which the solutions were made may be contaminated with thiosulphate (Vetter et al., 1989) and are difficult to weigh accurately. As a result, it is often difficult to prepare accurately a solution of known sulphide concentration. lodometric titration was therefore carried toI:determine the exact concentration of any prepared sulphide solution.:The technique is based on the principle that sulphide can be accurately determined by the titration of thiosulphate against iodine following the addition33of sulphide to the iodine solution (Parsons et al., 1984). Two separate stages were involved. Initially, the normality of thiosulphate (nominally 0.1 N) was accurately determined. This was achieved by titrating the thiosulphate against an accurately prepared solution of iodine (0.1 N) using starch as an indicator. This was repeated three times to confirm the precision of the standardization. The exact concentration of thiosulphate was then calculated. The reaction is described belowI2 + 2 Na2S20^ 4^ 21 + Na2S^OgIn the second stage, 25 ml of approximately 1 mM sulphide was added to 5 ml iodine. Iodine is consumed in this reaction (see below).S2-+l2 21-+ SiiThe remaining iodine was then titrated with the thiosulphate. Four replicates were carried out. The difference in the volume of thiosulphate used in the two titrations was used to calculate the exact initial concentration of the sulphide solution.2.2.1e. Comparison of the methods for the measurement of sulphide.iThe reproducibility of each technique was assessed and a direct comparison was conducted between the colorimetric, chromatographic and potentiometric techniques as described in 2.2.1. A solution of approximately 1 mM sulphide was prepared and its exact concentration determined by iodometric back titration. iThis was than then accurately diluted to 100 piM and assayed (10 replicates) by34the colorimetric and chromatographic methods as described above and the results compared. In addition, comparisons of the colorimetric and the sulphide electrode methods were carried out by using both procedures to determine the concentrations of sulphide in prepared solutions (sulphide range 0 - 200 piM).2.2.2. Burrow water sampling at Loch Sween, the Clyde Sea and in laboratory mesocosmsMeasurements of the concentration of sulphide in the water in the burrows of Callianassa subterranea and Jaxea nocturna were carried out at Caol Scotnish, Loch Sween (56® 02.6'N, 005® 35.5’W). This site was chosen since mud-shrimps (C. subterranea and J. nocturna) were common in moderately shallow water (< 10 m depth) enabling a large number of samples to be collected by diving and because the sediments at this site were the most reduced in the Loch (Nickel!, 1992). The majority of samples were taken from the burrows of C. subterranea which could be identified by their characteristic burrow openings, (Plate 2.1) (Nickell & Atkinson, 1995). W ater sam ples (approximately 10 ml) were collected by inserting a length of narrow bore catheter tubing supported by a piece of stiff wire (75 cm in length) into the burrow. With practice it was possible to insert the end of the catheter tube deep into the burrow (up to 50 cm) down the vertical inhalant shaft. Water was slowly extracted from the burrow lumen using a hypodermic syringe attached to the catheter tubing. The syringe was then sealed and quickly transferred to a support boat where a sub-sample (125 }i\) was pipetted into a solution of 500 pil of 0.12 M zinc acetate and 125 pi\ of 1.5 M NaOH. Any sediment particles drawn into the syringe during sampling were allowed to settle out for 1 - 2 minutes, in the sealed syringe, before fixation in the zinc acetate solution. Samples were subsequently analysed in the laboratory using the method of Cline (1969) as modified by Gilboa-Garber (1971), (see section 2.2.1.).35___36■SSûII3X)CLE(QX(Uc 40 cm with mud sediment taken from the Firth of Clyde or from Loch Sween. The tanks were placed in a temperature-controlled aquarium (10 ± 1 ®C) and the sediment allowed to settle for several weeks before individual animals were placed in each of the aquaria. Burrows were normally established within a few days but water samples were not taken until after the burrows had been established for more than 2 months to ensure that conditions within the burrows had stabilized.Water samples were subsequently taken from the burrows using a similar procedure to that used in the field (see section 2.2.1a.) except that an in-line filter (5 pm ) was used to prevent any suspended sediment entering the syringe. The transparent Perspex walls of the aquaria allowed the position of the sampling tube within the burrow to be seen. The concentrations of sulphide in the water samples were assayed using the colorimetric method described above (see section 2.2.1a.). The pH of the water samples were also determined using a Russell pH electrode (Auctermuchty, Fife) and pH meter (Jenway 3020).2.2.3. Concentrations of sulphide in interstitial water in L o c h Sween and Clyde sedimentsA number of attempts were made to use a suction interstitial water sampler to determine the concentration of sulphide in the interstitial water of the sediment in the vicinity of the mud-shrimp burrows throughout the Loch. The sampler37I,was constructed from a small plastic collector densely packed with glass wool, attached to a 10 cm length of 0.8 mm i.d. glass tubing which was inserted into the sediment (Volkel & Grieshaber, 1992). A 2 ml syringe was then used in an-attempt to withdraw the interstitial water by suction. The sampler, however, very quickly became blocked by the very fine sediment particles present; median size in the order of 4 pm (Anderson, 1989). An alternative approach was therefore adopted. A small number of sediment cores (9) were taken, by diving, at the Caol Scotnish site during May 1995 and July 1996. In addition, a small number of Craib cores (3) were also taken in different parts of the loch using theResearch Vessel A ora at Sailean Mhor (56® 01.4'N 005° 35.7’W) Srôn Bheith (56° 00.8' N 005° 36.5'W) and Achnamara (56° 00.7'N 005° 35.6'W) during May 1995. The corer (58 cm by 10 cm diameter) used during diving sampling was constructed from a length of brass tubing which could be split longitudinally. This was pushed into the sediment during a dive, sealed with two large bungs and brought to the surface. It was then placed in a nitrogen- filled glove bag which was continuously flushed from a plastic tube connected to 10 1 high pressure nitrogen cylinder (BOC). The How rate of the gas was controlled using a two-stage Nitrogen gas regulator (Apo Flame Equipment). The core was subsequently transferred to the shore and then split inside the bag. The effectiveness of this method for removing oxygen from the glove bag was tested in the laboratory prior to the field work by placing an oxygen electrode inside it (see section 4.2.1). Samples of sediment (approximately 5 g) were taken at 5 - 10 cm intervals along the core's length and placed in 10 ml plastic tubes that had been filled with nitrogen. The tubes were then centrifuged for 5 minutes at 6500g to separate the interstitial water from the sediment (Vetter ef al., 1989). A subsample of the water was then immediately fixed in alkaline zinc acetate (see section 2.2.1a.). The total time taken to process the sediment core was < 30 minutes and the samples were maintained under nitrogen for this period in an attempt to reduce the possibility of sulphide oxidation. The fixed38samples were subsequently returned to the laboratory where their sulphide content was determined using the colorimetric method as described above (see section 2.2. la). Craib cores (15 x 8 cm) taken using the RV Aora were handled and analysed as described above.2.2.4. Sediment EhAdditional sediment cores were also taken using the same technique from the Caol Scotnish site to enable determinations of the redox potential of the sediment to be carried out. Immediately after the core had been split lengthways the Eh of the sediment was determined by inserting a combination Eh electrode (CMPTRL, Russell, Auctermuchty, Fife) into the sediment at 5 - 10 cm intervals along the length of the exposed core. The electrode was inserted into the middle of the core and held in a retort stand to support it. The redox voltage normally became stable within 2 - 3 minutes although, occasionally, this took considerably longer. Box cores were also taken using the RV Aora from sites in the Firth of Clyde in the vicinity of the Calocaris macandreae trawl area (55° 46.7' N, 004°58.8' W). The Eh of these sediment cores was also determined as described above.2.2.5. Thiol concentrations in freshly caught animalsAttempts to obtain samples of water from the burrows of C. macandreae in the field were unsuccessful. This was due in part to the shape of the burrows which prevented the insertion of the catheter deep into the burrow (see Plate 1.3) and to the fact that, even in Loch Sween, most C. macandreae occur at depths >20 m making access by diving d ifficu lt. An alternative, indirect technique to determine whether C. macandreae is exposed to sulphide in its environment was therefore adopted (Gorodezky & Childress, 1994). Samples of haemolymph were taken from mud-shrimps immediately after capture by trawling and 'bait pump' (see section 3.2.1. Animal collection and maintenance). A sample of39 _________________________ L________________________________________________ :__________________________________________ :_______________ 'haemolymph (50 pï) was taken from each animal by inserting a narrow gauge hypodermic needle (25G) into the pericardium and withdrawing the haemolymph with a 1 ml syringe. The concentrations of accumulated thiol metabolites in the haemolymph were determined by high performance liquid chromatography following derivatization with monobromobimane as described above (2.2.1b.). The concentration of thiols in the haemolymph of a single specimen of Callianassa subterranea captured using the 'bait pump' at the Caol Scotnish site was also determined (see Chapter 3.2.1. Animal collection and maintenance).2.2.6. Behaviour of mud-shrimps during sulphide exposureThe effect of sulphide on the burrow irrigation behaviour of the mud- shrimp Calocaris macandreae was investigated. A glass burrow having three openings (approximately 200 ml in volume) was manufactured in the Department of Chemistry at the University of Glasgow. Its shape and size were similar to burrow casts of this species taken from the field (Atkinson, 1986; Atkinson & Taylor, 1988). Three small sampling apertures were positioned along the length of the burrow and sealed using a small plastic taps. A large glass tank (volume = 6 1) was placed above the glass burrow with the three openings sealed into the base of the tank. The tank and the burrow were then filled with previously UV- sterilized sea water (salinity = 35 ± 1 %o). Prior to any addition of sulphide an air stone and air line were used to oxygenate the overlying sea water reservoir in the tank. The experiment was conducted in a thermostatically controlled cool room at 10 ± 1 °C.Animals used during the experiment were collected from the Firth of Clyde and maintained in the sea water aquarium at in the University of Glasgow at 10 ± 1 °C and a salinity of 35 ± \%o (see Chapter 3 for details). An individual animal (0.9 - 2.0 g) was placed in the glass burrow and allowed to settle overnight. The overlying sea"water in the reservoir tank allowed the animal to draw water into its burrow. The mud-shrimp's general behaviour was then40observed and the frequency and duration of pleopod beating of the mud-shrimp were determined over 20 - 30 minute periods. During each observation period two stop-watches were used to cumulatively time the duration the animal remained quiescent and the durations of pleopod beating. The frequency of pleopod beating was estimated by counting during the irrigation activity. Initially, control measurements were determined for 8 individual animals in the glass burrow filled with sea water. The individual mud-shrimps were then exposed to two different concentrations of sulphide. This was achieved by slowly injecting of 2 ml of nominally 10 mM sulphide and 100 mM sulphide, for the low and high sulphide exposures respectively, into the glass burrow through the sampling apertures near the animal. The pH of both sulphide solutions was adjusted to 8.0 prior to the experiment. The concentration of sulphide in the burrow water throughout the period of the experiment was determined at 4 - 5 minute intervals (see section 2.2. la.). Control injections of distilled water and sea water were also carried out.2.2.7. StatisticsThe following statistical procedures were used throughout this study (Zar, 1984). Individual data sets were initially tested for normality (Anderson-Darling test) and homogeneity (Bartlett's test or Levene's test). The pairwise analysis of parametric data was conducted by a pooled /-test. Any non-parametric data were tested separately by individual pairwise comparison using the following tests: /- test not pooled (data normal but with heterogeneous variance), Mann-Whitney (data not normal and homogeneous variance) or Mood test (data not normal and heterogeneous variance). Multiple comparisons of data sets with homogenous variance were performed by one way analysis of variance and subsequent Tukey pairwise comparisons. In some cases these data sets were transformed by square root or log functions to increase homogeneity. Analysis of co-variancewas used to compare regression lines. The level of significant difference adopted,41was p < 0.05. The letters a/b/c/ were used to indicate significant differences graphically (i.e. a # b # c). Minitab version 10 was used to conduct these analyses. Data values cited in the text throughout this thesis are means ± standard deviation (unless otherwise stated) and also include sample sizes. The number of samples in any treatment occasionally varied. During the experiments a small number of animals occasionally died and were therefore ignored. In addition, it was not always possible to maintain large numbers mud-shrimps in the aquarium at the University of Glasgow due to collecting difficulties.42;|2.3. Results2.3.1. Sulphide measurementBoth the colorimetric and chromatographic techniques were highly reproducible. In a comparative experiment to determine the concentration of sulphide in a nominally prepared 100 pM solution values of sulphide were found to be 108.6 ± 8.5 pM (colorimetric) and 111.0 ± 6.1 pM (chromatographic) and were not significantly different (/ - test, p > 0.05). Only very small differences were observed between replicate samples and the accuracy and precision of the colorimetric and chromatographic methods were similar (Figure 2.3). Both techniques were also found to be in agreement with the iodometric titration (/ - test, p > 0.05) used to standardize the sulphide concentration. A significant correlation was also found between the determination of sulphide by the potentiometric and colorimetric techniques. (Figure 2.4), (ANOVA, p < 0.05, r^ = 0.969).2.3.2. Burrow water sampling at Loch Sween, the Clyde Sea and in laboratory mesocosmsThe concentrations of sulphide in the burrows of C. subterranea at the Caol Scotnish site (Loch Sween) were quite variable with an overall mean concentration of 36.6 ± 44.3 pM (range 0 to 206 pM , n = 37). A seasonal trend in the sulphide concentration of the burrow water was seen with the concentrations being significantly lower during the summer (Figure 2.5). No sulphide was detected, however, in the burrows of Upogebia deltaura at the two sites in the vicinity of the Isle of Cumbrae, although low concentrations of sulphide in the interstitial water were detected in cores (see section 2.2.3.) taken from the surrounding sediment (Farland Point 7.1 ± 4.0 pM , n = 16; White Bay 7.0 ± 5.1 / 0.05.44 ' _______ ___2001I 150Io" 100 Q)’USr3(A 0.05) from that of the control animals. During exposure to higher sulphide concentrations (6.25 ± 4.82 mM) a significant (ANOVA + Tukey, p < 0.05) increase in the frequency of pleopod beating and the duration of bouts of irrigation was observed (Figure 2.9 & 2.10). In addition, the mud-shrimps were observed to tail-flip in an escape-type response away from the sulphide source to a different part of the burrow and also to swim to one of the burrow openings using its pleopods. Even during the exposure to the high sulphide concentrations, however, the mud-shrimps did not abandon their burrows.5230 Jê :>"s(0oCL82 0 .10.M Control ü Distilled waterEl Sea water□ Sulphide (1)□ Sulphide (2)F igure 2.9 The effect of sulphide on the pleopod activity (% duration) of Calocaris macandreae maintained in a laboratory glass burrow Sulphide 1 = 0.36 ± 0.32 mM, Sulphide 2 = 6.25 ± 4.82 mM. Values are means ± SE, n = 7 - 8 . ANOVA + Tukey p < 0.05, a/b = significant difference.53_______________________ _____________________0 Control H Distilled waterEl Sea water□ Sulphide (1)□ Suiphide (2)Figure 2.10 The effect of sulphide on the pleopod beat frequency (beats, min- 1) of Calocaris macandreae maintained in a glass burrow in the laboratory. Sulphide 1 = 0.36 ± 0.32 mM, Sulphide 2 = 6.25 ± 4.82 mM. Values aremeans ± SE, n = 7 - 8 . ANOVA + Tukey p < 0.05, a/b = significant difference.I';542.4. Discussion2.4.1. Sulphide measurementThe colorimetric, chromatographic, potentiometric and iodometric methods used throughout this study to determine sulphide were found to give comparable and equally reproducible results. The colorimetric method offers practicality and simplicity which allows the determination of relatively large numbers of samples in the laboratory. In addition, it is particularly suitable for fieldwork since the samples can be easily fixed on site by the addition of a known sample volume to a pre-prepared tube containing alkaline zinc acetate prior to subsequent analysis in the laboratory.In contrast, the chromatographic technique was found to be a fairly time- consuming procedure. Samples must be derivatized for 30 minutes in the dai'k at room temperature and each takes 35 minutes to analyse using an HPLC system. Nevertheless, this technique has a number of distinct advantages the most important of which is that it is possible to measure simultaneously a range of reduced thiols including sulphide, sulphite, thiosulphate, glutathione and cysteine in both water and tissue samples. After the initial derivatization process the samples can be stored for over a year without deterioration. This method is also the most sensitive technique (high nanomolar concentrations can be detected).The electrode technique was found to be too cumbersome for field use due to the need for the accurate additional measurements such as pH, salinity and temperature. In the laboratory, however, the method proved to be a valuable technique for routine determinations.lodometric titration is not very suitable for the analysis of large numbers of samples because of the length of time taken for each titration. It is also impractical for use in the field because of the requirement for considerable quantities of glassware and access to laboratory facilities. However, because of its inherent accuracy and precision this technique was used to standardize the55suiphide solutions used for calibration of the other three methods.2.4.2. Sulphide exposure in the fieldThe results from the analyses of the concentrations of sulphide in the burrow water of Callianassa subterranea together with indirect evidence from the accumulation of thiosulphate in the haemolymph of trawled Calocaris m acandreae indicate that mud-shrimps can be exposed to significant concentrations of sulphide in their burrows. Sulphide was not detected, however, in the burrows of Upogebia deltaura sampled from around the Isle of Cumbrae. This may be because obtaining water samples from deep within the Y shaped burrow is very difficult; sampling is hindered by the small openings to the burrows and the convoluted shafts which prevent the insertion of the catheter sampler deep into the burrow where sulphide may be present. Upogebia spp. also tend to irrigate their burrows frequently as they are primarily suspension feeders (Nickell & Atkinson, 1995). This is in contrast to species of mud- shrimps, such as C. subterranea m d Jaxea nocturna, that are primarily deposit feeders. The ingress of oxygenated sea water drawn into a burrow by feeding currents would clearly prevent sulphide from accumulating in the burrow.There are very few recorded measurements of the concentrations of sulphide from the burrows of other crustaceans and marine invertebrates. Sulphide has been recorded, however, in the burrows of callianassids in Bermudan sediments (Waslenchuk et a i , 1983) and in the burrow water of the echiuran worm, Urechis caiipo, and of the polychaete worm, Arenicola marina (Table 2.3). These authors noted that the concentrations recorded were very variable and that in a large number of burrows sulphide could not be detected (Arp et al., 1992; Volkel et al., 1995). This was also found to be the case in the present study.56Table 2.3 Comparison of the sulphide concentrations in the burrows of four marine invertebrates and in the interstitial water of the sediment. Values are means ± SD and the range is also given. The values of n are given in brackets.Location Species BurrowsulphidePore water sulphideSourceLoch Sween (Scotland)Callianassasubterranea37 / 1 mM. Interestingly, the three non-vent crabs exhibited similar adaptations although they were significantly more sensitive to sulphide poisorimg.. Pachygrapsus crassipes, which burrows in sulphide rich sediments in salt marshes, was less affected by sulphide than Cancer antennarius, which lives on sand and rocks and Portunus xantusii, which is free swimming, was the most susceptible to sulphide. Vetter et al. (1987) suggested, therefore, that this mechanism of sulphide 'oxidation' also occurs in sulphide-tolerant crustaceans.Vismann (1991b), using similar techniques to Vetter et al. (1987), established that the isopod Saduria entomon was also unable to prevent sulphide from entering its body and also detoxified sulphide in an oxygen-dependant mechanism in the hepatopancreas and muscle tissues (Hagerman & Vismann, 1993). The end-products were found to be thiosulphate and sulphite. Vismann66(1991b) also demonstrated an oxygen independent binding of sulphide by iron in the hepatopancreas, accounting for 40% of the sulphide removed. The crustacean hepatopancreas accumulates iron, and that of Saduria entomon turns black with sulphide exposure (Gibson & Barker, 1979; Vismann, 1991b).Uncharacterized sulphide oxidases have been suggested as the biochemical system responsible for sulphide 'oxidation' in a range of marine invertebrates and fish (Powell & Somero, 1985; Vetter et aL, 1987; Bagarinao & Vetter, 1989; Powell & Arp, 1989; Vismann, 1990). The possibility of a sulphide 'oxidation' mechanism in the thalassinidean mud-shrimp Calocaris macandreae was investigated during the present study.3.1.3. Symbiotic chemoautotrophic sulphur bacteriaMany marine invertebrates that inhabit sulphidic environments have been found to harbour symbiotic sulphide-oxidizing bacteria (see Chapter 1). These bacteria have a dual function; they supply the host with chemosynthetically fixed carbon (Felbeck, 1985) and, in addition, 'detoxify' sulphide preventing the inhibition of the host's aerobic metabolism (Arp et aL, 1995). Whilst this form of association has been found to be relatively common among many animals from sulphidic environments it has not been observed in crustaceans. Since there have been relatively few studies of the sulphide metabolism of Crustacea, the occurrence of this symbiotic association with chemoautotrophic sulphur bacteria in some species may have gone undetected. The possible presence of symbiotic sulphur bacteria in the mud-shrimps, Callianassa subterranea and Calocaris macandreae was therefore investigated.67 :3,1.4. Sulphur amino acid metabolismSulphur amino acid metabolism is an essential aspect of protein construction. Only two amino acids contain sulphur, methionine and cysteine. These amino acids are required by eukaryotic organisms but only cysteine can be synthesized; methione has to be obtained from their food (Baker, 1987). Eukaryotic sulphur amino acid metabolism, however, may differ significantly between protozoans and metazoans (Thong & Coombs, 1985a; 1985b). In virtually all eukaryotic organisms, cysteine is manufactured from methionine- based precursors during the trans-sulphuration pathways. The direct incorporation of hydrogen sulphide into these metabolic pathways has until recently not been observed in eukaryotes. It has, however, now been described in eukaryotic parasitic protozoans, in some gastro-intestinal nematode worms and in mammalian cells during the little-studied reversible reaction between serine and hydrogen sulphide producing cysteine (Walker & Barrett, 1992; Thong & Coombs, 1985b). This reaction is catalysed enzymatically by L-serine sulphydrase (EC 4.2.1.22). In addition, homocysteine another important sulphur-containing compound in the trans-sulphuration pathways of parasitic protozoans, can also undergo enzymatic desulphuration with the production of hydrogen sulphide, ammonia and a ketobutyrate. This resaction is catalyzed by the enzyme homocysteine desulphurase (EC 4.4.1.1.2) which has been found to have very high activititles in some parasitic protozoans (Thong & Coombs, 1985a).The possibility exists, therefore, that the incorporation of sulphur into the amino acid cysteine, and subsequently into the sulphur amino acid trans sulphuration pathways, could act as a way of removing sulphide that has entered the body of an aquatic invertebrate. During this study the mud-shrimp Calocaris m acandreae was investigated for the presence of the enzymes L -serine sulphydrase and homocysteine desulphurase in its tissues.683.2. Materials and methods3.2.1. Animal collection and maintenanceCalocaris macandreae Bell were collected from depths of approximately 80 m in the vicinity of the Isle of Cumbrae, Firth of Clyde, Scotland (55*' 46.7' N, 004® 58.8' W) using an Agassiz trawl. Callianassa subterranea (Montagu) and Jaxea nocturna Nardo were collected from depths of 10 ~ 30 m in Loch Sween, west coast of Scotland (56® 02.6' N, 005® 35.5' W) using an anchor dredge and by using a mechanical diver-operated suction sampler (Alvey 'king size' bait pump), using a technique similar to that described in Manning (1975). Upogebia deltaura Leach were collected from depths of approximately 20 m in White Bay, Isle of Cumbrae (55® 47.8' N 004® 54.4' W) by anchor dredging. Immediately after capture, the animals were returned to the University of Glasgow where they were maintained under a 12:12 h light : dark regime in a recirculating sea water aquarium (salinity = 35 ± 1 %o; temperature 10 ± 1 ®C). Preliminary observations had indicated that these mud-shrimps frequently engage in agonistic behaviour which may occasionally result in the death of one of the animals. To prevent this, the mud-shrimps were kept individually in small perforated tubes in larger aquarium tanks through which sea water was continuously circulated. The mud-shrimps were used within one month o f capture and during this time they were not fed.3.2.2. Sulphide tolerance of mud-shrimpsSpecimens of Calocaris macandreae, Callianassa subterranea and Jaxea nocturna were exposed to a range of sulphide concentrations under both normoxic and hypoxic conditions to assess their tolerance to sulphide. The animals (fresh weight range = 1.0 - 2.5 g; n = 7 - 30) were placed in individual perforated tubes (to prevent aggression between individuals) in a glass aquarium (volume = 6 1) in a temperature-controlled room (10 ± 1 ®C). Sea water having the required sulphide concentration (0.6 - 1 mM) was produced using two69____________peristaltic pumps (Watson Marlow 10lU and 501U) to pump a concentrated sulphide stock solution and sea water (salinity = 3 5 + 1 %o) into a sm all, continuously-stirred mixing chamber (Figure 3.1). A flow-through technique (flow rate approximately 160 ml.min-1) was used to establish an equilibrium in the concentration of sulphide during each exposure. The sulphide-laden sea water then passed via a UV sterilizer (to suppress bacterial growth) into the exposure tank which was continuously stirred (Underwater stirrer, R ank Brothers). The tank was covered with a piece of polystyrene to reduce sulphide volatilization to the atmosphere. The sulphide-laden sea water, however, was allowed to overflow out of the tank into a sump before being drained away.The P02 of the water was normally reduced to below normoxic levels due to the chemical oxidation of sulphide. Since the reduction in P02 was generally < 10 %, no attempt was made to increase the P02 . Exposure to sulphide underhypoxic conditions was carried out by bubbling a gas mixture (supplied by a precision gas mixing system) having the required P02 through the water in thetank. The pH of the water was controlled by prior adjustment of the stock solution and by the addition of CO2 to the gas mixture, creating a naturallybuffered bicarbonate system, to ensure that any variability in pH was reduced(8.19 ± 0.08).The P02 of the water was monitored continuously using an oxygen electrode and meter (Strathkelvin 1302 oxygen electrode & oxygen meter model 781) connected to a pen recorder. The anode of the oxygen electrode was gold plated prior to use to protect it from sulphide poisoning (Revsbeck & Ward, 1983; O’Brien & Vetter, 1990) (see section 3.2.5.). The oxygen electrode was calibrated prior to use against aerated sea water at the experimental temperature of 10 ± 1 C and against a solution having a P02 of zero Torr (sodium sulphite in 0.01 M sodium tetraborate).70IIUJO c=a.a■os OD>CO01 •s5V) % .2IPII| iIII S- s cI “S) "CT3 Os :^ i l £ «I IC l‘tÜÛC1 1 1 11 1 i î 140 Torr) to examine the metabolic effects of sulphide exposure. The exposure apparatus is describedabove (see section 3.2.1.). The pH of the exposure medium, however, was reduced to 8.00 ± 0.05 to increase the relative proportion of the toxic H2S present. At this pH the relative amount of toxic H2S represents 8 % of the total sulphide present (Goldhaber & Kaplan, 1975). The concentrations o f accumulated thiol metabolites in the haemolymph of Calocaris macandreae (n = 9- 11) were determined using the technique described in Chapter 2, (see section 2.2.1b.). Blood samples were taken after 1, 6 , 12, 18 and 24 hours and then analysed by high performance liquid chromatography (HPLC) following derivatization with monobromobimane (see section 2 .1.1b.).72The concentrations of reduced thiols in hepatopancreas and abdominal muscle tissue were also determined at the same time intervals as the blood samples. Approximately 10 - 20 mg of hepatopancreas and abdominal muscle (taken from the first abdominal segment) were immediately dissected from an animal after removal from the sea water containing sulphide. The tissue samples were then placed in pre-weighed 1.5 ml Eppendorf tubes containing 10 pil of 46mM monobromobimane, IQOpil of 160 mM HEPES / EOT A 16 mM pH 8 and SOfil of acetonitrile. These were re-weighed to establish the accurate weight of the tissue and then the tissue was homogenized in the tube for 2 minutes using a plastic homogenizer. The sample was then placed in the dark at room temperature for 30 minutes to allow derivatization of the thiols to occur before 100 pt\ of 65 mM methansulphonic acid was added. The samples were centrifuged for 10 minutes at 11500 g and 200 pi\ of the subsequent supernatant were pipetted into another 1.5 ml tube. The samples were then stored at -70 in darkness before analysis by HPLC. Care was taken throughout the assays to reduce the exposure of the tissue to light which is known to degrade the fluorescing reagent monobromobimane..A preliminary experiment was also carried out to assess the reproducibility and accuracy of the HPLC thiol assay. Five replicate samples of hepatopancreas and abdominal muscle tissue were taken from the same animals which had previously been exposed to a sulphide concentration of 150 ± 11.5 jaM sulphide for 24 hours. The samples were then assayed simultaneously using the procedure described above and the results compared.3.2.4. Symbiotic chemoautotrophic sulphur bacteria and mud- shrimpsHepatopancreas and gill filaments from freshly caught C alocarismacandreae and Callianassa subterranea were fixed for electron microscopy toestablish if these species harbour symbiotic bacteria involved in sulphide'oxidation'. This involved immediately placing the dissected tissues from73 . . ; 'freshly-caught animals (< 30 min) in 1 % glutaraldehyde, 2 % paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 with 2 % sucrose and 1.5 % sodium chloride. On return to the laboratory the fixed tissues were rinsed in 0.1 M phosphate buffer containing 4 % sucrose, then post-fixed in 1 % osmium tetroxide in phosphate buffer for 1 h. Specimens were washed in several changes of distilled water and block stained with 0.5 % aqueous uranyl acetate for 1 h.Following dehydration through an ethanol series, the tissues were embedded in Spurr resin (Spurr, 1969), using propylene oxide as a transitional solvent. Thin sections of suitable areas were cut and mounted on uncoated 300 mesh copper/palladium grids and stained with uranyl acetate (methanolic) and lead citrate. Thin sections were examined with a Zeiss 902 transmission electron microscope operating at 80 kV.3.2.5. Sulphur amino acid metabolism in mud-shrimpsExperiments were carried out to try to establish whether any activity of the enzymes L-serine sulphydrase and homocysteine desulphurase could be detected in the hepatopancreas of Calocaris macandreae. The activity of both enzymes can be assessed spectrophotometrically by the formation of sulphide which then reacts with lead acetate to form a brown colloidal suspension. In the case of L- serine sulphydrase, a reversible reaction is catalysed by this enzyme whereby cysteine is desulphurated to form serine and hydrogen sulphide. The desulphuration of homocysteine to form hydrogen sulphide, ammonia and alpha ketobutyrate catalysed by homocysteine desulphurase, can be followed in the same manner as described above.A specimen of Calocaris macandreae was dissected and samples of hepatopancreas tissue resuspended in 0.1 M Tris pH 8.5 in combination with proteinase inhibitors at final concentrations of 1 E 64 trans-epoxysuccinyl-L- leu cy lam id o -(4 -g u an id in o ) butane, 4 piM. pepstatin A, 1 phenylmethylsulphonylfluoride and 2 /iM 1,10 phenanthroline in a final volume74. I y;S'if: #’■yof 0.5 ml. The solution was maintained on ice and homogenized in a 1.5 ml,Eppendorf tube with a plastic homogenizer for 5 minutes. Following centrifugation at 11500 g for 5 minutes, the resulting supernatant was used as a potential source of the enzymes. Homocysteine desulphurase and L-serine sulphydrase were assayed spectrophotometrically at 15 °C (Thong & Coombs, 1985a; 1985b). The reaction mixture for the L-serine sulphydrase assay consisted of 500 pti of 0.33 mM lead acetate, 300 ptl of 4 mM L- cysteine, 650 j a \ of 0.1 M Tris adjusted to pH 8.5 and 50 pil of homogenate to give a final assay volume of 1.5 ml. The reaction mixture for homocysteine desulphurase assay was identical except that it contained D, L-homocysteine at a final concentration of 3.3 mM instead of L-cysteine. The reactions w ere m onito red spectrophotometrically for 5 minutes at 15 ®C by determining the production of lead sulphide at a wavelength of 360 nm. Control assays were also run with both enzymes to confirm the viability of the technique. In addition, to control for any free sulphide that may have been present in the hepatopancreas sample the assay was also run in the absence of the two substrates (L- cysteine and D, L- homocysteine).3.2.6. Potentiometric oxygen sensors and sulphide interferencePolarographic oxygen sensors may be poisoned by sulphide (Hale, 1983). Since the P02 needed to be recorded in the experiments carried out during this study the affect of sulphide on potentiometric oxygen sensors was investigated. The platinum cathodes of Clarke type oxygen electrodes have been found to react with sulphide (Hale, 1983). The cathode of these electrodes can be protected from sulphide, however, by electroplating gold onto it (Revsbeck & Ward, 1983; O'Brien & Vetter, 1990). During the present study, this was achieved by first etching the cathode for 3 minutes in a solution of saturated KCN in a fume cupboard. This cleans the surface to enable efficient electroplating to occur. The cathode was then washed in 0.1 N HCl. Gold was electroplated onto the75■3cathode from a solution of 5 % KAu(CN)2 În 0.2 M ammonium citrate adjusted to pH 6.3. A simple electrolysis system was created by applying 1.5 V d.c., negative at the cathode, with a 680 Q resistor in series, and a graphite rod in the KAu(CN)2 This prevented the anode from being exposed to the plating solution. The electrodes were then placed in the solution for 5 minutes.O'Brien & Vetter (1990) state that electrodes modified in this way were largely insensitive to sulphide concentrations < 1 mM and had similar oxygen sensitivities to those of untreated platinum cathode electrodes. Throughout this study, the sulphide concentrations used in any experiment were generally in the low micromolar range. To confirm insensitivity to sulphide under these experimental conditions the gold cathode electrodes were always recalibrated, as described below, before and directly after use to establish whether any deterioration in the electrode's performance had occurred that may have been induced by sulphide exposure.A series of empirical sulphide exposure experiments were also carried out to investigate the effect of millimolar sulphide concentrations on the calibration parameters of the electrodes. Gold-plated cathode and standard oxygen electrodes were initially calibrated (see section 3.2.2) and placed in 200 ml beaker of distilled water maintained at 10 °C (Grant temperature controlled water bath combined with a Techne tempette TE-8D cooler, refrigerated bath RB-5) for a control period of 24 hours and then recalibrated to confirm the electrode's performance ( < 1% drift acceptable). Both electrodes were then placed in a range of sulphide solutions (nominally 1 mM, 5 mM and 50 mM, see section2.2. la.) also maintained at 10 ®C. The electrodes were then removed at 24 hour intervals, thoroughly rinsed with distilled water, and placed in a solution of oxygen-saturated distilled water to establish if any deterioration in the electrode's performance had occurred.In addition, an experiment was conducted to assess the extent to which sulphide and thiol compounds were able to diffuse across the membrane into the76.electrolyte. A standard, untreated oxygen electrode and an electrode in which the cathode had been gold plated were connected to two oxygen meters (Strathkelvin Instruments) and placed in a sulphide solution (final concentration = 543 ±113 piM). Prior to this the electrodes were calibrated against air-saturated sea water and in a stream of nitrogen in a small sealed container. The electrodes were left in the sulphide solution at 10 (as described above) for a total of 96 hours after which the electrodes were dis-assembled and samples (50 pi\) of electrolyte were taken and the concentrations of reduced thiols in the electrolyte determined by HPLC.Ii773.3. Results3.3.1. Sulphide tolerance of mud-shrimpsAll three species of mud-shrimp were tolerant of sulphide. The survival curves for animals exposed to high concentrations of sulphide were approximately sigmoidal (Figure 3.2 & 3.3). The LTgg values for Calocarismacandreae and Callianassa subterranea exposed to 1.0 ± 0.1 mM sulphide under near normoxic conditions (P02 > 140 Torr) were similar; 29 h and 24 hrespectively. Jaxea nocturna, however, showed an even greater tolerance of sulphide (LTgg= 62 h) even when exposed to sulphide (0.62 ± 0.14 mM) under hypoxic conditions (Po^ 38.5 ± 14.4 Torr). Control animals suffered little mortality (< 10 % over? days) throughout the experiments.Due to the difficulty in collecting C. subterranea and J. nocturna, insufficient mud-shrimps were available to allow the determination of their tolerance of anoxia. However, the LT^q for C. macandreae under anoxic conditions was 30 h (Figure 3.2). The survival curve of C. macandreae maintained under anoxia was very similar to that obtained when animals were exposed to 1 mM sulphide and normoxia.It is interesting to note that the pleopods, mouthparts and branchial chambers of mud-shrimps exposed to high concentrations of sulphide became blackened with what is believed to be metallic sulphides.3.3.2. Sulphide ’oxidation’ and metabolism in mud-shrimpsExposure to sulphide (87 ± 14 piM) under normoxic conditions for 24 h resulted in a significant (ANOVA + Tukey, Man-Whitney & Mood tests, p < 0.05) accumulation of thiols in the haemolymph and tissues of Calocaris macandreae (Figure 3.4,3.5, & 3.6). Although low concentrations of sulphide were found in the haemolymph and hepatopancreas these were well below the exposure concentration. The concentrations of sulphide in the hepatopancreas did increase significantly (Mood, p < 0.05) after 12 hours but did not increase78significantly (Mann-Whitney, p > 0.05) in the abdominal muscle tissue (Figure 3.6).Most of the sulphide entering the haemolymph and tissues was oxidized to thiosulphate (ANOVA + Tukey, P < 0.05). The concentrations of thiosiilphate in the haemolymph and the tissues increased rapidly during the first few hours of exposure to sulphide. In both the hepatopancreas and the haemolymph, the concentration of thiosulphate reached an equilibrium after 1 - 6 h. The concentration of thiosulphate in the abdominal muscle reached its highest value after 6 h exposure but then decreased significantly (Mood, p < 0.05). Sulphite occurred as a secondary 'oxidation' product in the haemolymph but did not accumulate significantly in the hepatopancreas and muscle tissue during the experiment (ANOVA, p > 0.05; Mann-Whitney, p > 0.05). The concentrations of cysteine and glutathione in the haemolymph, hepatopancreas and abdominal muscle of animals exposed to sulphide for 24 h, were found not to differ significantly (/ - test, Mann-Whitney, Mood, p > 0.05) from their respective controls (Figure 3.7 & 3.8). Sulphite and thiosulphate were detected in the water at the end of the 24 h exposure period due to the chemical oxidation of sulphide but only at very low concentrations (< 1 0 piM). Concentrations of cysteine and glutathione in the exposure water were below the limit of detection (< 1 piM).The preliminary experiment carried out to assess the reproducibility and accuracy of the HPLC thiol assay for muscle and hepatopancreas tissue found the repeatability of the assay to be good. When exposed to 150 ± 1 1 .5 piM sulphide under normoxic conditions for 24 h the concentrations of sulphite, thiosulphate and sulphide in the abdominal muscle tissue from a single specimen of Calocaris macandreae (6 replicates) were found to be 0.116 ± 0.019 jwmol. g“l, 2.119 ± 0.370 ptmol.g-^ and 0.178 ± 0.059 /

3O)1007 5 .5 0 .2 5 .0O\ \\ \I \V S0—r - 20—I—40 60Time (h)Figure 3,2 Cumulative survival (%) over time of Calocaris macandreae (n = 27 - 30) during exposure to normoxia (open circles), anoxia (closed circles) and 1.0 ± 0 .1 mM sulphide under near normoxic (P02 > 140 Torr) conditions (closed squares).81_______1007 5 .?E 5 0 .3 140 Torr) and Jaxea nocturna (closed squares, n = 19) exposed to 0.62 ± 0.14 mM sulphide under hypoxic conditions (P02 = 38 .5+14.4 Torr).821000 ’1wo£7 5 0 .IOEuX5 0 0 .2 5 0 .— Q - —12 24186Time (h)Figure 3.4 The accumulation of the thiols, thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in the haemolymph of Calocaris macandreae following 24 h exposure to 87 ± 14 piM sulphide under near normoxic conditions (P02 > 140 Torr), Values are means ± SE (n = 8 -11). a/b/c = significant difference (ANOVA + Tukey, Mood or Mann-Whitney, p < 0.05).831.00 NSTime (h)Figure 3.5 The accumulation of the thiols, thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in the hepatopancreas of Calocaris macandreae following 24 h exposure to 87 ± 14 piM sulphide under near normoxic conditions (P02 > 140 Torr). Values are means ± SE (n = 8 -11). a/b - significant difference (ANOVA + TUKEY, Mood or Mann- Whitney, p < 0.05).84/ ____ ___q>01 SUo£o(03Time (h)Figure 3.6 The accumulation of the thiols, thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in the abdominal muscle of Calocaris macandreae following 24 h exposure to 87 ± 14 piM sulphide under near normoxic conditions (P09 > 140 Torr). Values are means ± SE (n = 8 -11). a/b = significant difference (ANOVA + TUKEY, Mood or Mann- Whitney, p < 0.05).85T V ,- ' _______ iW': —10.0.24Time (h)GSH CysteineFigure 3.7 The concentrations of the organic thiols cysteine and glutathione (GSH) in the haemolymph of Calocaris macandreae following 24 h exposure to 87 ± 14 piM sulphide under near normoxic conditions (P02 > 140 Torr). Valuesare means ± SE (n = 8 - 11). There were no significant differences between the initial and final concentrations {t - test. Mood or Mann-Whitney, p > 0.05).862520 .T"1q>E 1 5 .3(0 •0 1 0 .is5 .0 24Time (h)GSH Hepatopancras GSH Muscle H Cysteine Hepatopancreas □ Cysteine MuscleFigure 3.8 The concentrations of the organic thiols cysteine and glutathione (GSH) in the hepatopancreas and abdominal muscle of Calocaris macandreae following 24 h exposure to 87 ± 14 j a M sulphide under near normoxic conditions (P02 > 140 Torr). Data point values are means ± SE (n = 8 - 11).There were no significant differences between the start and final concentrations (t - test. Mood or Mann-Whitney, p > 0.05).873.3.3. Symbiotic chemoautotrophic sulphur bacteria and mud- shrimpsCareful examination by transmission electron microscopy did not reveal any evidence of bacterial symbionts in either the hepatopancreas or gill tissue from freshly-captured Callianassa siibterranea (n = 2 for each tissue) and Calocaris macandreae (n = 2 for each tissue).3.3.4. Sulphur amino acid metabolism in mud-shrimpsThe enzymes L-serine sulphydrase and homocysteine desulphurase were not detected in the hepatopancreas (n = 1) of Calocaris macandreae. Control cuvettes were also run to establish the presence of any sulphide in the hepatopancreas, none was detected. To ensure this was a genuine negative result the enzymes were also added to the assay mixture to confirm its viability.3.3.5. Potentiometric oxygen sensors and sulphide interferenceComparisons of the performance of the two types of electrode showed that both the untreated and gold plated electrodes were unaffected by sulphide concentrations up to at least 6.0 ± 0.99 mM since, after prolonged exposure to sulphide (144 h), they could still be recalibrated to within 1 % of their original values. At higher sulphide concentrations (37.7 ± 2.7 mM) the standard POS could not be recalibrated after 24 hours, suggesting sulphide poisoning. The gold-plated electrode, however, could be calibrated within 1% of its original calibration after 72 hours after which it was also poisoned by the sulphide. In addition, in the anoxic sulphide solutions both electrodes consistently recorded a Po2of zero Torr in each of the three sulphide solutions.When exposed to concentrations of 543 ±113 pfM sulphide for 96 hours only very low micromolar concentrations of sulphite, thiosulphate and sulphide could be detected in the electrolyte of the untreated and gold plated electrodes (Fig. 3.9). Since the standard electrolyte does not contain any of these88■'compounds this may indicate that sulphide slowly diffuses across the membrane into the electrolyte and is oxidized to thiosulphate and sulphite.___________36 .1v>OPOS Gold POSElectrode TypeM Sulphite H Thiosulphate En SulphideFigure 3.9 The accumulation of sulphite, thiosulphate and sulphide in the electrolyte of an untreated Strathkelvin oxygen electrode and one in which the cathode had been gold plated following exposure to a solution of 543 ±113 sulphide for 96 hours (n = 3). Cysteine and glutathione were not detected in the electrolyte. The standard electrolyte was found not to contain any of the above reduced sulphur compounds. Values are means ± SD.90 ;3.4. Discussion3.4.1. Sulphide tolerance in mud-shrimpsThe considerable ability of the mud-shrimps studied here to survive sulphide exposure may be related to their burrowing lifestyle which frequently exposes them to significant concentrations of sulphide (see section 2.3.2.). A lthough there are very few comparative data on sulphide tolerance in crustaceans, the available evidence indicates that most species have a poor tolerance of sulphide. For example, the larval stages of the white shrimp Metapenaeus monoceros and the blue crab Portunus trituberculatiis have been shown to be very intolerant of low micromolar concentrations of sulphide (Kang et aL, 1993; Kang & Matsuda 1994). The survival rate of adult blue crabs P, trituberculatus was also found to decrease significantly when exposed to similar conditions (Kang et a l, 1995). Similarly, Hagerman & Vismann (1995) reported that the shrimp Crangon crangon suffered high mortalities when exposed to sulphide concentrations as low as 5 pM. In addition, when exposed to sulphide concentrations of 1 mM, the swimming crab Liocarciniis depurator suffered 50 % mortality within 11 hours (Astall, 1993). These species are benthic but are not burrow dwellers although they may periodically bury themselves in the surface sediment. Their non-burrowing lifestyle may explain their low tolerance of sulphide.Much greater tolerances of sulphide exposure have been demonstrated, however, in the hydrothermal vent crab Bythogmea thermydron (Vetter er at.,1987) and in the Baltic isopod Sadiiria entomon (Hagerman & Vismann, 1993) which may bury in sediment. There is also evidence, based on choice chamber experiments, that species such as the burrow-dwelling amphipod, Corophiurn volutator, may actively avoid high concentrations of sulphide by behavioural means (Meadows et aL, 1981). Similarly, burrow irrigation behaviour may reduce the concentration of sulphide in the burrow water (see section 2.4.3.) Thus, suspension feeding mud-shrimps such as Upogebia spp. might be91 ____________expected to experience lower sulphide concentrations in their burrows than species such as Calocaris macandreae and Callianassa subterranea that are primarily deposit feeders. The similar trends in mortality of C. macandreae between those animals exposed to anoxia and those exposed to sulphide suggest the total reliance on anaerobic metabolism whilst exposed to m illimolar concentrations of sulphide despite the presence of oxygen at near normoxic concentrations.3.4.2. Sulphide 'oxidation' and metabolism in mud-shrimpsThe presence of low concentrations of sulphide in the tissues of Calocaris macandreae exposed to sulphide in the laboratory indicates that they were unable to prevent the diffusion of sulphide into the body. This is in agreement with data from studies on some other crustacean species (Vetter et aL, 1987; Vismann, 1991b; Hagerman & Vismann, 1993). C. macandreae was able, however, to maintain the concentrations of sulphide within the body tissues much lower than those in the exposure medium. This was achieved in part by the 'oxidation' of sulphide to form thiosulphate which may be considered as a possible 'detoxification' mechanism. Internal pH may also be significant because during hypoxia a reduction in pH (acidosis) of body fluids and tissues may occur. This results in an increase in the internal concentration of undissociated hydrogen sulphide (Portner, 1987), In the event of an equilibrium between the internal and external hydrogen sulphide concentrations the total sulphide concentration will be lower within the animal (Groenendaal, 1981; Volkel & Grieshaber, 1992; Grieshaber et aL, 1996). The sulphide concentration in the haemolymph may also be reduced by an auxiliary 'oxidation' mechanism such as the immobilization of sulphide by proteins in the haemolymph acting as a sulphide buffer. This mechanism has been described in other marine invertebrates (Arp et aL, 1987; Doeller et aL, 1988; Powell & Arp, 1989; Vismann, 1990; Childress et aL, 1991; Oeschger & Vetter, 1992; Grieshaber gf aL, 1996) and is92 _' " minvestigated and discussed in Chapter 6 .Thiosulphate accumulated as the major 'oxidation' product in Calocaris macandreae with sulphite accumulating as a significant secondary metabolite and possible intermediate product (O'Brien & Vetter, 1990). The production of thiosulphate is energetically more efficient than sulphate synthesis since 1 mole of sulphide is removed for every 1.5 moles of oxygen whereas the 'oxidation' of one mole of sulphide to form sulphite and sulphate requires three and four moles of oxygen, respectively.It has been suggested that, in crustaceans, the hepatopancreas is the primary site of sulphide 'oxidation* (Vetter et aL, 1987). In the present study, both the muscle and hepatopancreas tissues accumulated similar concentrations of thiosulphate when the mud-shrimps were exposed to sulphide. It is unlikely, therefore, that in mud-shrimps sulphide 'oxidation' is confined to the hepatopancreas. It would appear that a possible alternative mechanism for the removal of sulphide via the direct incorporation of sulphide into sulphur amino acid metabolism does not exist in Calocaris macandreae since the enzymes L- serine sulphydrase and hom ocysteine could not be detected in the hepatopancreas.How millimolar concentrations of thiosulphate are removed by the animal remains to be addressed. Thiosulphate has a poor ability to pass through biological membranes and has consequently been used a blood marker (Holmes & Donaldson, 1969). In the lug worm, Arenicola marina, however, the body wall has been found to be permeable to thiosulphate (Hauschild & Grieshaber,1995). It has been suggested, therefore, that the metabolites of sulphide 'oxidation' such as thiosulphate which may accumulate to m illim olar concentrations in the tissues, may diffuse out of the animal into the burrow water where thiosulphate has been shown to be present only at low micromolar concentrations (Volkel et aL, 1995). It is possible that the cuticle of crustaceans is permeable to both sulphide and to 'oxidation' products such as thiosulphate. It93has yet to be established, however, whether thiosulphate can be lost from the body by passive diffusion or whether an active transport mechanism exists.Vetter et al. (1987), however, found no evidence that thiosulphate was rapidly, cleared from the blood of the hydrothermal vent crab Bythograea thermydron and also found that the gills were not freely permeable to thiosulphate. They proposed that thiosulphate may either be excreted after being converted to sulphate or by the direct disposal of thiosulphate via the antennule gland (Vetter cm /., 1987; Astall, 1993).It is has been established that, for some invertebrate species, chemoautotrophic bacteria have an important role in the 'oxidation' of sulphide entering the body (Somero et aL, 1989; Tunnicliffe, 1991; Vismann, 1991a; Southwood, 1994) and also in the provision of chemoautotrophically fixed CO2. In addition, recent studies on some marine nematodes have shown that sulphide- oxidizing bacteria on the cuticle may also restrict the amount of sulphide that diffuses into the body (Ott et aL, 1991; Polz et aL, 1992). The electron microscope studies on Calocaris macandreae and Callianassa subterranea, however, failed to establish the presence of such bacteria associated with either the gill or hepatopancreas tissues. This suggests that the only 'oxidation ' mechanism that exists in C. macandreae, and possibly in other mud-shrimps, is mediated solely by the animal's tissues.Electron microscopy studies of other upogebiid and callianassid mud- shrimps, however, have found the mid-gut to be extensively coated with filamentous bacteria which were absent in the hind-gut (Harris et aL, 1991, Finn, 1995). These bacteria appear to be very different from those ingested during feeding and it would be interesting to determine whether they may possibly be involved in sulphide 'oxidation'. It has been established that sulphur-oxidizing bacteria are present in the gut of the sediment-dwelling echinodenn, Echinocardiurn cordatum (Temara et aL, 1993). These symbiotic filamentous bacteria, morphologically similar to Thiothrix, a sulphide-oxidizing94species, thrive in the intestinal caecum of this deposit feeding animal and have been shown to oxidize sulphide and thiosulphate.Sulphide 'oxidation' by mitochondria linked to the electron transport chain has been demonstrated in the gill and foot tissue of the bivalve Solemya reidi (Powell & Somero, 1986a; O'Brien & Vetter, 1990), in the liver of the Californian killifish, Fundulus parvipinnis (Bagarinao & Vetter, 1990), in the body wall of the priapulid, Halicryptus spimdosus (Oeschger & Vetter, 1992) and in the polychaete, Heteromastus filiformis (Oeschger & Vismann, 1994). Sulphide 'oxidation' and the concurrent production of ATP has been found to be stimulated at low micromolar sulphide concentrations in these species but at higher sulphide concentrations (in excess of 50 pM) complete inhibition of the respiratory chain occurred. It is believed, therefore, that sulphide 'oxidation' is linked with the respiratory chain by cytochrome c oxidase, which could be the sulphide oxidase, transferring electrons to oxygen (Powell & Somero, 1986a).Recent studies of the intertidal worm Arenicola marina have indicated, however, that mitochondrial sulphide 'oxidation' is not inhibited by sulphide (Volkel & Grieshaber, 1994, 1995) and, recently, the possible existence of an alternative terminal oxidase appears to have been demonstrated (Volkel & Grieshaber, 1996). The biochemical mechanism and characterization of this proposed alternative cytochrome-c oxidase remains to be fully established. It seems highly unlikely that A. marina should be the only sulphide tolerant marine invertebrate to contain mitochondria with this alternative enzyme but further studies are required to establish its presence in other species.The concurrent synthesis of ATP during mitochondrial sulphide 'oxidation' has been established in a small number if marine invertebrates (Oeschger & Vetter, 1992; Oeschger & Vismann, 1994; Volkel & Grieshaber, 1994, 1995; Volkel e ta l., 1995; Volkel & Grieshaber, 1996). This energy resource may be very significant in providing the animal with a supplementary supply of ATP whilst under sulphide stress.95A small number of conceptual models have been designed to integrate the different features of sulphide 'detoxification'. They seem to indicate that the diffusion rate of sulphide into the cell and diffusion barriers may be important in preventing sulphide from reaching inhibitory concentrations at the site of cytochrome c oxidase on the inner membrane of mitochondria (Bagarinao & Vetter, 1993, Arp et aL, 1995). For this to be possible a balance between the rate at which sulphide diffuses towards the enzyme, dependent on pH and external sulphide concentration, and the rate at which sulphide is detoxified by external agents to the enzyme (such as the oxidising capacity of the respiratory pigment) must occur.It would appear, therefore, that mud-shrimps are adapted physiologically to tolerate exposure to sulphide in their natural habitat and that the mitochondria may be responsible for the 'oxidation' of sulphide to thiol metabolites during 'detoxification'. These animals may therefore be added to those marine invertebrates so far reported to 'detoxify' sulphide. It seems likely that the underlying sulphide 'oxidation' mechanism is mitochondrial and that the ATP produced at this time may be used as a supplementary energy source (Oeschger & Vetter, 1992; Oeschger & Vismann, 1994; Volkel & Grieshaber, 1994, 1995; Volkel etaL, 1995; Volkel & Grieshaber, 1996).3.4.3. Potentiometric oxygen sensors and sulphide interferenceThe experimental evidence from this study indicates that gold plated and standard potentiometric oxygen sensors are largely insensitive to micromolar and very low millimolar concentrations of sulphide and is in agreement with O'Brien & Vetter, (1990). The electrodes could be recalibrated after sulphide exposure for a considerable number of hours without any apparent loss of performance (6.0 ± 0.99 mM for 144 h). In contrast, at higher m illim olar sulphide concentrations (37.7 ± 2.7 mM) both types of electrode suffered considerable reductions in their performance. The gold plated electrode, however, appeared to%maintain its calibration for at least 72 hours whereas the standard electrode was apparently more susceptible to sulphide in that it could not be recalibrated after 24 hours exposure to sulphide.Low concentrations of thiols were also detected in the electrolyte even after prolonged exposure to sulphide (543 ± 113 pM sulphide for 96 hours). The membrane, which seals the cathode and anode within the electrode body, is therefore apparently largely impermeable to sulphide and prevents sulphide diffusion into electrode electrolyte. Sulphide poisoning may not therefore be a problem until mM concentrations of sulphide are reached.3.4.4. Summary of Chapter 3The findings of Chapter 3 in relation to mud-shrimps are summarized below:a. Mud-shrimps appear to be physiologically adapted to tolerate elevated levels of sulphide that they may encounter in their natural habitat.b. Sulphide diffusing into the body tissues is 'oxidized' prim arily to thiosulphate which may act as a possible 'detoxification' mechanism.c. Sulphite accumulates as a secondary and possible intermediate sulphide 'oxidation' metabolited. Symbiotic sulphide oxidizing bacteria were not detected in the gills or hepatopancreas in mud-shrimps, although their possible presence in the gut remains to be investigated.e. Sulphur amino acid metabolism seems not be directly implicated in sulphide 'oxidation'.97f. Cathode gold plated oxygen electrodes are insensitive to micromolar concentrations of sulphide.984. Aerobic and anaerobic metabolism of mud-shrimps during exposure to sulphide.4.1. Introduction4.1.1. Anaerobic metabolism in marine invertebratesMany marine invertebrates which inhabit environments that can become hypoxic or anoxic may rely on anaerobic metabolism to survive these conditions. Anaerobic mechanisms during exposure to environmental anoxia have been described in a variety of marine invertebrates (Zebe, 1982; de Zwaan & Putzer, 1985; Hochachka, 1986; Gade & Grieshaber 1986, Bryant, 1991; de Zwaan, 1991; Grieshaber et ah, 1992, 1994). The biochemical adaptations seen in these animals which enable them to survive the effects of hypoxia or anoxia depend primarily on the duration of the period of exposure and the energy requirements of the animal.As soon as severely hypoxic or anoxic conditions prevail, adenosine triphosphate (ATP) may be provided by the exploitation of phosphagens, such as creatine phosphate and phospho-L-arginine (Grieshaber et aL, 1994). In addition, the synthesis of ATP also occurs in the cytosol during anaerobic glycolysis. To maintain the glycolytic flux and cytosol y tic redox balance, NADH, which is produced during the reaction of glyceraldehyde 3 phosphate to form 3 phosphoglycerate, must be reoxidized to NAD+. This involves the reduction of pyruvate to form D, L-lactate or opines by the pyruvate oxydoreductases (Grieshaber & Gade, 1986) and results in the accumulation of metabolic end-products such as L-lactate and opines which are functionally analogous (Fields, 1983). The rate of ATP production in these reactions can be very high because the glycolytic flux may increase, although biochemical efficiency (ATP yield per mol glucose or glycogen) is low since they involve the incomplete oxidation of glycogen (Shick, 1991; Grieshaber et a i, 1992, 1994,1996). Anaerobic glycolysis results in the production of only 3 moles of ATP99per mole of glycogen, whereas during aerobic metabolism the yield of ATP is considerably higher at 38 moles of ATP per mole of glycogen (Eckert et aL,1988). Anaerobic glycolysis, however, may be more important during exercise or functional anaerobiosis, requiring a higher rate of ATP production, than during environmental anaerobiosis which, in contrast, involves low energy expenditure. Anaerobic glycolysis may therefore have been selected for periods of high power output during the course of evolution. For example, the escape responses of many marine invertebrates to avoid predation, such as the tail- flipping escape response of many decapod crustaceans and the strong muscular contractions of the polychaete worm Arenicola marina during burrowing, and the fast white muscle of fish, which require a high rate of ATP production (Grieshaber et aL, 1992, 1994).Over long periods of anoxia or severe hypoxia, however, ATP can be produced more efficiently by anaerobically-functioning mitochondria than during anaerobic glycolysis in the cytosol (Grieshaber et aL, 1994). The glucose- succinate pathway ferments glycogen and produces succinate as a transient end- product (which cannot be excreted) in the mitochondria. Succinate is further metabolised to propionate and acetate which may then be excreted (Grieshaber et el., 1994). These end-products have been found in a range of marine and limnic invertebrates, particularly in molluscs and annelids. The succinate-propionate pathway is energetically more efficient than anaerobic glycolysis because approximately 5 moles of ATP are produced in comparison to the 3 moles of ATP produced during anaerobic glycolysis. Less substrate is, therefore, used for a given amount of energy production. Depletion of glycogen, during long periods of ambient oxygen reduction, may be even further reduced by the attenuation of the metabolic rates, to approximately 5 - 20% of normoxic values.1004.1.2. Anaerobic metabolism in CrustaceaThe Crustacea tend to have low tolerances of anoxia (Zebe, 1982, 1991). This is strongly correlated with their exposure to reductions in oxygen concentrations in their environment which for many decapod crustaceans is unlikely. In general they are also highly mobile, which allows them considerable scope to avoid anoxic or hypoxic conditions. Almost without exception, therefore, the crustaceans rely entirely on anaerobic glycolysis (see section 4.1.1) if they encounter short periods of anoxia and hypoxia (Zebe, 1982; de Zwaan & Putzer, 1985, Gade et aL, 1986). The production of only glycolytic end-products (D, L-lactate and D, L alanine) during crustacean anaerobic metabolism rather than any other metabolic end-products contrasts strongly with other groups of marine invertebrates which tend to use a greater variety of anaerobic pathways (Zebe, 1982; Fields, 1983; Grieshaber et aL, 1992, 1994). As has been described above, this may in part be an evolutionary reflection of the possibly more important role of anaerobic glycolysis in crustaceans. The supply of oxygen or the aerobic capacity of muscle tissues during functional anaerobiosis are limited for short periods during which escape responses which involve intense muscular activities occur (see above). The increased demand for ATP can be quickly met by anaerobic glycolysis with the potential of a 100 fold increase in flux rates (de Zwaan, 1977).A small number of crustaceans however, may be able to survive anaerobically for longer periods than most species by producing succinate in addition to the anaerobic end-products D, L-lactate and Alanine (de Zwaan & Skjoldal 1979; Zebe, 1982; Carlson & Gade, 1986; Hagerman & Szaniawska, 1990). These include the isopods Natatolana borealis, Sadiiria entomon and the thalassinideans Upogebia pugettensis and Neotrypaea (as C allianassa) californiensis. In the two thalassinidean mud-shrimps, however, both alanine and succinate were found to be minor end-products and were produced at only very low concentrations during exposure to anoxia over 24 hours (Zebe, 1982).101Phosphagen transphosphorylation and anaerobic glycolysis leading to the formation of ATP and L-lactate in these two invertebrates is therefore the major mode of anaerobic metabolism.4,1.3. Sulphide-induced anaerobic m etabolism in m arine invertebratesThe presence of sulphide in the water column or in interstitial water is strongly correlated with anoxic or hypoxic conditions (Theede et al., 1969; Fenchel & RiedI, 1970). Sulphide is highly toxic by mainly inhibiting cytochrome c oxidase (Nicholls, 1975; National Research Council, 1979) and may therefore prevent aerobic respiration in many marine invertebrates (Bagarinao, 1992). Sulphide-tolerant animals, however, may be able to sustain their aerobic metabolic pathways, defending the cytochrome c oxidase in the electron transport chain within their mitochondria, through a range of mechanisms (Vetter et aL, 1991; Vismann, 1991a; Bagarinao, 1992). In addition, these mechanisms may also be involved in the synthesis of ATP which could act as a supplementary energy source during sulphide stress (Powell & Somero, 1986a; Volkel & Grieshaber, 1996).The use of anaerobic metabolism may allow an animal to survive exposure to sulphide (Vetter et a i, 1991). Sulphide-induced anaerobiosis in the presence of oxygen has been reported in a number of marine invertebrates and is believed to be relatively common (Anderson et al.,\990; Oeschger & Vetter, 1992; Volkel & Grieshaber, 1992, 1994; Hagerman & Vismann, 1993, 1995; Grieshaber etaL, 1996). The maintenance of aerobic metabolism during exposure to sulphide before resorting to anaerobic metabolic pathways has, however, been shown to be very variable and dependent on oxygen provision as well as ambient sulphide concentrations (Hagerman & Vismann, 1993, 1995; Volkel etaL, 1994).1024.1.4. Sulphide-induced anaerobic metabolism in mud-shrimpsMud-shrimps show a number of adaptations to declining oxygen tensions which include behavioural responses such as irrigating the burrow to draw in oxygenated sea water (Anderson 1989; Astall, 1993; Astall e ta L , 1997). In addition, in an attempt to ensure oxygen delivery is not compromised, physiological adaptations have been shown, such as increasing ventilation, maintaining a constant heart rate, reducing metabolism to reduce energy expenditure and the possession of a high affinity respiratory pigment (Anderson, 1989; Astall, 1993; Nickell, 1992, Anderson et aL, 1994). The mud-shrimp Calocaris macandreae is also able to maintain its oxygen consumption constant over a wide range of oxygen partial pressures down to a critical P02 (Pc) of approximately 20 Torr (Anderson et aL, 1991). Similar Pc values have been recorded in several other species of mud-shrimp (Atkinson & Taylor, 1988; Paterson & Thome, 1995; Astall et aL, 1997). C. macandreae has been shown to maintain aerobic metabolism during moderate hypoxia above the Pc (P02 > 20 Torr) and survive periods of severe hypoxia (below the Pc) and anoxia for many hours (Anderson et aL, 1994). Similarly, other species of mud-shrimp appear to have an even greater tolerance of anoxia than many other species of decapod crustaceans. For example, Neotrypaea (as Callianassa) californiensis and Lepidophthalmus louisianensis (as Callianassa jamaicense), can survive anoxia for 3 - 4 days (Thompson & Pritchard, 1969; Felder, 1979).In addition to low partial pressures of ambient O2 mud-shrimps may also be exposed to sulphide in their burrows and in the surrounding reduced sediments. They tolerate sulphide oxidizing it to thiosulphate and sulphite (see section 3.3.2 and 3.4.2). Since they rarely, if ever, leave their burrows (Nickell & Atkinson, 1995) it would be interesting to establish their oxygen consumption requirements when exposed to sulphide. Of additional importance is under what sulphidic conditions do the animals resort to anaerobic metabolism? The oxygen consumption and onset of anaerobiosis at various sulphide concentrations and103different ambient oxygen partial pressures were therefore investigated in the mud-shrimp Calocaris macandreae.1044.2. Materials and methods4.2.1. Collection and maintenance of animals.Calocaris macandreae were collected by Agassiz trawling in the Clyde and returned to the University of Glasgow where they were maintained under a 12:12 h light : dark regime in a recirculating natural sea water aquarium (salinity = 35 ± 1 %o; temperature 10 ± 1 °C). Further details are given in section 3.2.1. Theanimals were not fed and allowed at least 48 h to acclimate to these conditionsbefore any physiological measurements were made.4.2.2. Whole animal oxygen consumption during sulphide exposureThe rates of oxygen consumption of Calocaris macandreae under varying oxygen partial pressures (P02) and sulphide concentrations were determined by flowthrough respirometry (Figure 4 .1). Eight mud-shrimps of similar weight (fresh weight range = 1.5 - 2.0 g) were placed in individual 40 ml clear Perspex tubes through which natural sea water (salinity = 35 ± 1 %o, pH 7.99 ± 0.08, temperature 10 ± 1® C) flowed at a constant rate of 75 ml.h'l^. An additional two tubes without animals acted as controls to allow the rates of microbial oxygen consumption and chemical oxidation of sulphide to be calculated. In addition, to reduce the effects of bacterial respiration during the experiment, the sea water was sterilized using ultra-violet light (Tropical Marine). Exposure to different sulphide concentrations (0 - 300 /^M) and normoxic and hypoxic, 47 ± 7 Torr conditions was controlled as described in 3.2.2. The animals were left undisturbed overnight to acclimatize and to allow the experimental conditions to reach equilibrium.105XIJOO)a .SB3CMD ) 90 % exchange of water within the tubes (Steffensen, 1989). Water samples (0.75 ml) were withdrawn from the outflow of each tube using a 1 ml syringe and immediately injected into an oxygen cell (EH 100, Strathkelvin Instruments, Glasgow) thermostatted at 10 ± 1 ®C, containing a sulphide-insensitive oxygen electrode (1302, Strathkelvin Instruments, Glasgow) connected to an oxygen meter (Model 781, Strathkelvin Instruments, Glasgow) and pen recorder. The anode of the electrode was gold plated prior to use to protect it from sulphide poisoning (see section 3.2.2). The concentration of sulphide in the water from the control tubes was determined as described below. Prior to each experiment the respirometers and associated tubing were sterilized with a solution of sodium hypochlorite to reduce bacterial respiration and then thoroughly rinsed with distilled water.Weight specific rates of oxygen consumption (M02) were calculated after subtraction of the rates of microbial respiration recorded in the control tubes. The mass of the mud-shrimps used was intentionally limited to a narrow range (1.5 - 2.0 g) to reduce size-related effects which affect oxygen consumption estimates (Bridges & Brand, 1980). It was noted that a reduction of the P02 in the control tube (by approximately 6 %, depending on the concentration of sulphide used) occurred and was attributable to both microbial respiration and thechemical oxidation of sulphide..The mean oxygen consumption rate of each individual animal (/2 “ ([O2] control lube " [O2] animal tube) ^ flow rate / masswhere 1^02 = oxygen consumption (/ 0.05). Exposure to sulphide concentrations greater than 100 under normoxia resulted in a pronounced decrease in Ivlo^ This decrease did not become significant (ANOVA + Tukey, p< 0.05), however, until the mud-shrimps were exposed to sulphide concentrations greater than 180 piM. A similar reduction in M02 occurred whenthe mud-shrimps were exposed to sulphide under hypoxic conditions although this reduction occurred at significantly lower sulphide concentrations (ANOVA + Tukey, p < 0.05). In addition, even at concentrations between 200 - 300 piM, M02 did not approach zero indicating that some animals continued to consumeoxygen .4.3.2. Anaerobic metabolism under sulphidic conditionsExposure of Calocaris macandreae to increasing sulphide concentrations for 24 h under near normoxic conditions, resulted in a gradual but significant accumulation of L-lactate in the abdominal muscle (Figure 4.3). When exposed to the same range of sulphide concentrations under hypoxic conditions (P02 =20.1 ± 1.3 Torr), however, there was a pronounced and significant (ANOVA +112Tukey, p < 0.05) increase in the L-lactate content in the muscle when sulphide concentrations exceeded 25 piM (Figure 4.3). A further increase in L-lactate muscle content at higher sulphide concentrations (25 - 160 ^M ) was also seen, although this was not significant (ANOVA 4- Tukey, p > 0.05).Similarly, the concentration of L-lactate in the haemolymph of mud- shrimps exposed to sulphide under near normoxic conditions increased significantly (ANOVA 4- Tukey, p < 0.05) only at sulphide concentrations greater than 150 ptM (Fig 4.4). Under hypoxic conditions, however, L-lactate accumulated significantly (ANOVA 4- Tukey, p < 0.05) at much lower sulphide concentrations (35 / 0.05) at higher sulphide concentrations (Figure 4.6). Under both sets of conditions, however, low but significant (ANOVA 4- Tukey, p < 0.05) concentrations of both sulphide and sulphite were recorded in the haemolymph.Similar trends in the accumulation of thiols during sulphide exposure under113normoxia and hypoxia were also observed in the hepatopancreas of Calocaris macandreae. When exposed to hypoxia, thiosulphate accumulated significantly (ANOVA + Tukey, p < 0.05) in the hepatopancreas at an exposure concentration of 7 yM. sulphide (Figure 4.7). Under normoxic conditions, however, the increase in thiosulphate occurred only at higher sulphide concentrations. This trend did not become significant (ANOVA -f Tukey, p < 0.05) until an exposure sulphide concentration of 63 yM was reached (Figure 4.8). Sulphite did not accumulate significantly (ANOVA, p > 0.55) in the hepatopancreas under hypoxic conditions but did increase significantly (ANOVA + Tukey, p < 0.05) under normoxia at sulphide exposure concentrations in excess of 150 y M sulphide. Sulphide was seen to increase significantly (ANOVA + Tukey, p < 0.05) to low micromolar concentrations under both normoxic and hypoxic conditions.The concentrations of sulphite and sulphide in the abdominal muscle tissue of Calocaris macandreae were not found to increase significantly (ANOVA + Tukey, p > 0.115) when exposed to sulphide under both normoxic and hypoxic conditions (Figure 4.9 & Figure 4.10). In contrast, the concentration of thiosulphate was found to increase significantly (ANOVA + Tukey Set ~ tests, p < 0.05) during exposure to sulphide. A significant (ANOVA + Tukey, p < 0.05) decrease in the concentration of thiosulphate in the muscle tissue was found to be coincident with the decrease of thiosulphate in the haemolymph (see above) at sulphide concentrations in excess of 35 yM under hypoxic conditions. This trend, however, was not found to be significant (pooled t - test, p > 0.12) during exposure to sulphide under normoxic conditions.114_______________________^ 0 .6 .u>C4o 0.4 V01 o*100 200 Sulphide (pM)300Figure 4.2 The effect of sulphide on the rate of oxygen consumption (M02 ) of Calocaris macandreae under near normoxic (Po^ > 140 Torr) (open circles) and under moderate hypoxic conditions (Po^ = 46.9 ± 7.2 Torr) (closed circles). Values are means ± SE (n = 6 - 16). a/b = significant difference (ANOVA + T ukey, p < 0.05)11540-— ^3 30-20 -10 -0 50 100 150 200Sulphide (pM)Figure 4.3 The concentration of L-lactate in the abdominal muscle of Calocaris m acandreae following 24 hour exposure to differing sulphide concentrations under near normoxia (P02 > 140 Torr) (closed circles) and severe hypoxia, (P02 = 2 0 + 1 Torr) (open circles). Values are means ± SE (n = 6 - 10). a/b = significant difference (ANOVA + Tukey, p < 0.05)1162015-£ï(0101Eo £% 51XQp=jQBO — r -50 100 150 200Sulphide (pM)Figure 4.4 The concentration of L-lactate in the haemolymph of Calocaris macandreae following 24 hour exposure to differing sulphide concentrations under near normoxia (Po^ > 140 Torr) (closed circles) and severe hypoxia, (P02 = 20 ± 1 Torr) (open circles). Values are means ± SE (n = 6 - 10). a/b/c = significant difference (ANOVA + Tukey or Mood te st, p < 0.05)1173.0.jOOZS 2 .0 .E>*01X, b bL — - - p — — I b25 50 75 100 125Sulphide (pM)Figure 4.5 The concentrations of thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in the haemolymph of C a locaris macandreae following 24 h exposure to differing sulphide concentrations under severe hypoxia (P02 = 20 ± 1 Torr). Values are means ± SE (n = 6 - 10). a/b/c = significant difference (ANOVA + Tukey, p < 0.05).118_______sEjOo£JC,Q.E>%01X1 .5 .1.0 .0 .5 .50 100 150 2000.00Sulphide (pM)Figure 4.6 The concentrations of thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in the haemolymph of C a locaris macandreae following 24 h exposure to differing sulphide concentrations under normoxia (P02 > 140 Torr). Values are means ± SE (n = 6 - 10). a/b/c = significant difference (ANOVA + Tukey, p < 0.05)119à>Im.2£IüE&§I2.5 _40Sulphide (pM)dû NSFigure 4.7 The concentrations of thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in the hepatopancreas of Ca locaris macandreae following 24 h exposure to differing sulphide concentrations under severe hypoxia (P02 = 20 ± 1 Torr). Values are means ± SE (n = 6 - 10). a/b/c = significant difference (ANOVA + Tukey, t , Mann-Whitney or Mood Tests, NS = not significant, p < 0.05).■33;9:1: i !I3iI;1120: '3%-' /- '...^ 2.0 .50 100 150Sulphide (^M)200Figure 4.8 The concentrations of thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in the hepatopancreas of Ca locaris macandreae following 24 h exposure to differing sulphide concentrations under normoxia (Po2 > 140 Torr). Values are means ± SE (n = 6 - 10). a/b/c = significant difference (ANOVA + Tukey, p < 0.05).121_____ ______cp01 oHoN3s120Sulphide (pM)Figure 4.9 The concentrations of thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in abdominal muscle tissue of Calocaris macandreae following 24 h exposure to differing sulphide concentrations under severe hypoxia (P02 = 20 ± i Torr). Values are means ± SE (n = 6 - 10). a/b = significant difference, NS = not significant, ANOVA + Tukey, p < 0.05.____________122 '2.5 _2.0 .T-IO)01i2.2£3S 0 .5 . O ”0.050 100 200150Sulphide (j j M )Figure 4.10 The concentrations of thiosulphate (closed circles), sulphite (open circles) and sulphide (closed squares) in abdominal muscle tissue of Calocaris macandreae following 24 h exposure to differing sulphide concentrations under normoxia (P02 > 140 Torr). Values are means ± SE (n = 6 - 10). a/b = significant difference (ANOVA + Tukey or r - tests, NS = not significant, p < 0.05).1234.4. Discussion4,4.1. Whole animal oxygen consumptionThe effect of sulphide on whole animal oxygen consumption has been investigated in relatively few marine invertebrates (Childress & Mickel, 1982; Chen et a l 1987; Anderson et a l 1987, 1990; Eaton & Arp, 1993). Animals such as the bivalves Solemya reidi and S. velum that inhabit reduced sediments, and which contain endosymbiotic sulphide-oxidizing bacteria, appear capable of maintaining their rates of oxygen consumption over a range of sulphide concentrations, before resorting to anaerobic metabolism at higher sulphide concentrations (Chen et a l , 1987; Anderson et a l , 1987, 1990). This has also been shown to occur in the deep sea hydrothermal vent clam, Calyptogena pqcifica (Childress & Mickel, 1982). Similarly, the intertidal echiuran worm, Urechis caupo and the polychaete worm Arenicola marina have also been shown to maintain their oxygen consumption when exposed to low micromolar concentrations of sulphide, before resorting to anaerobic metabolism at higher sulphide concentrations (Eaton & Arp, 1993; Grieshaber et al., 1996). The rate of oxygen consumption of coelomic fluid from U. caupo, however, increased significantly when exposed to low micromolar concentrations of sulphide. This trend, however, was not established during the determination of the whole animal oxygen consumption of U. caupo.There are very few published reports on the direct effects of sulphide on oxygen consumption in Crustacea. Vetter et al. (1987) observed that both the heart and scaphognathite rates of the hydrothermal vent crab B ythograea tliermydron were maintained during exposure to high sulphide concentrations (> 700 pM), This has been interpreted as indicating continued oxygen consumption under these conditions (Eaton & Arp, 1993). In another study, Gorodezky & Childress (1994), showed that the oxygen consumption of B. tliermydron increased both during and after exposure to sulphide but the reasons for this remain unclear.124 _ _Kochevar & Childress (pers. comm, in Eaton & Arp, 1993), suggest that the callianassid mud-shrimp (as Callianassa) californiens is couldnot regulate its oxygen consumption during sulphide exposure. In the present study, however, the calocaridid mud-shrimp Calocaris macandreae was apparently able to maintain its oxygen consumption when exposed to sulphide concentrations in excess of 150 pM under normoxic conditions and in excess of 100 p M sulphide under hypoxia. This suggests that the sulphide 'detoxification' mechanism is oxygen-dependent. Further evidence to support this hypothesis is discussed below and in Chapter 7.The increase in oxygen consumption at low sulphide concentrations, although not significant, may indicate a trend that is masked by variation in the responses of individual animals, since additional oxygen will be required to oxidize sulphide to thiosulphate. This trend has apparently been observed in other experimental studies of benthic marine invertebrates (Eaton & Arp, 1993, Grieshaber et al., 1996). It has not however, been clearly demonstrated (significantly). This may be due to the high degree of variation which is typically observed in this type of whole animal experiment. Despite the statistical non-significant inference seen in these data, sulphide entering the tissues is apparently oxidized to thiosulphate. Therefore additional oxygen may be required if aerobic respiration is maintained.The oxygen consumption of the mud-shrimp Calocaris macandreae was seen to increase during exposure to sulphide concentrations of less than 100 pM sulphide. This may be interpreted as the oxygen required to produce the 'oxidation' metabolites thiosulphate and sulphite, in addition to maintaining normal aerobic metabolism. At higher sulphide concentrations, however, oxygen consumption declined and may reflect oxygen provision within the tissues become limiting as the amount of oxygen required to oxidize sulphide to thiosulphate and sulphite increases. When the 'oxidation' mechanism becomes saturated in this manner as the oxygen supply becomes limited and intracellular125sulphide concentrations become toxic the mud-shrimps resort to anaerobiosis (which may operate concurrently with aerobic metabolic pathways, see Chapter 5).The 'oxidation' mechanism may therefore allow aerobic metabolism to be maintained despite the presence of sulphide, although the mud-shrimp's sensitivity to sulphide is apparently greater at lower oxygen concentrations. Sulphide therefore appears to have a concentration-dependent capacity to either stimulate or inhibit aerobic metabolism in mud-shrimps which is also related to the oxygen availability. Aerobic metabolism appears to increase during exposure to sulphide, when oxygen is available, and can be maintained even under severe hypoxic and sulphidic conditions.An increase in oxygen consumption following exposure to sulphide has also been observed in the priapulid worm Halicryptus spinulosis (Oeschger et ah, 1992), the hydrothermal vent crab Bythograea tliermydron (Gorodezky & Childress, 1994) and in the isopod Saduria entomon (pers. comm. Hagerman). This may in part be due to the re-oxidation of anaerobic metabolites such as L- lactate (Oeschgerera/., 1992) which can accumulate during exposure to sulphide through the initiation of anaerobic metabolic metabolism (see below).The reduction in oxygen consumption during exposure to sulphide concentrations greater than 150 pM , under both oxygen saturation treatments, suggests the use of anaerobic metabolism under these conditions. The mean rates of oxygen consumption, however, did not approach zero as might be expected if all mud-shrimps were respiring anaerobically during exposure to sulphide. This may be explained by examining the data for individual mud- shrimps and not just the mean values. Many animals were clearly not consuming oxygen indicating that the 'oxidation' mechanism was saturated (reached its maximum rate) and anaerobic metabolism was initiated. A small but significant number of mud-shrimps, however, continued to consume measurable quantities of oxygen which indicates that there may be variation between individuals in126their ability to cope with exposure to such high sulphide concentrations.Interestingly, if the concentrations of sulphide in the burrows of Calocaris macandreae are similar to or lower than those found in the burrows of Callianassa subterranea, (low micromolar, see section 2.3.2.) these oxygen consumption data indicate that aerobic metabolism can be maintained and regulated in the burrow environment. An am bient su lph ide-oxygen environmental threshold, however, seems to exist which dictates the bias between aerobic and anaerobic metabolism. It may also be possible for aerobic and anaerobic metabolisms to function concurrently. This question is addressed in Chapter 5.4.4.2. Sulphide-induced anaerobic metabolismAt some critical ambient sulphide concentration and oxygen tension when either the availability of oxygen to 'oxidize' sulphide is limited or the maximum rate of 'oxidation' mechanism is reached, a switch to anaerobic metabolism seems to occur. Anaerobic metabolism has been observed in a small number of marine invertebrates during sulphide-induced anaerobiosis (Anderson et aL, 1990; Oeschger & Vetter, 1992; Volkel & Grieshaber, 1992, 1994; Hagerman & Vismann, 1993, 1995; Grieshaber et al., 1996). These include the priapulid worm Halicryptus spinulosus which has been found to accumulate the anaerobic metabolite succinate in the haemolymph and body wall tissue after 4 days exposure to 200 pM sulphide during normoxic incubation (Oeschger & Vetter, 1992). This suggests that the worms could not maintain their aerobic metabolism during long-term exposure to sulphide. The intertidal polychaete worm Arenicola marina has been observed to maintain aerobic metabolism during exposure at to least 100 pM sulphide before initiating anaerobic metabolic pathways that produce succinate as the major end-product (Volkel & Grieshaber, 1992, 1994). In the absence of sulphide the accumulation of succinate commenced at 10 kPa (equivalent to 75 Torr), although when exposed to 200127___p M sulphide for 8 hours under normoxia, succinate was found to accumulate. A similar trend in the increase of accumulation of succinate has also been seen in the peanut worm Sipunculus nudus. When exposed to 200 pM. sulphide under normoxia the animal resorted to anaerobic metabolism (Volkel & Grieshaber,1992). Similarly, the brackish water isopod Saduria entomon resorted to anaerobic metabolism when exposed to a concentration of 150 pts/[ sulphide under hypoxic conditions between 4 - 6.7 kPa (equivalent to 30 - 50.3 Torr) during a 48 hour exposure (Hagerman & Vismann, 1993). L-lactate and alanine both accumulated significantly under these conditions. At these hypoxic oxygen tensions in the absence of sulphide the concentrations of these anaerobic metabolites did not differ significantly from the normoxic controls indicating anaerobiosis induced by sulphide during these sulphidic conditions. Exposure to only low micromolar concentrations of sulphide in another crustacean Crangon crangon, however, induced total anaerobic metabolism (indicated by the accumulation of L-lactate). This occurred at an oxygen saturation which would permit aerobic metabolism in the absence of sulphide (Hagerman & Vismann,1995).Direct comparisons between these studies is complicated by the duration of exposure to the various conditions since the initiation of anaerobic metabolism is also time dependent. Despite this, a clear trend in the initiation of anaerobic metabolism seems to exist and occurs at a critical sulphide-oxygen threshold when conditions become too severe for the animal. Clearly, differences in sulphide tolerance will affect the position of the critical point. This difference Ican be illustrated by two of the species of marine invertebrate described above. The brown shrimp C. crangon does not tolerate sulphide and must rely on its limited anaerobic metabolism at far lower sulphide concentrations than the polychaete worm Arenicola marina which does tolerate sulphide and can maintain its aerobic metabolism.a128A critical transition point between anaerobic and aerobic metabolism (indicated by an accumulation of lactate in the abdominal muscle tissue and in the haemolymph) in the mud-shrimp Calocaris macandreae occurs when ambient oxygen is severely limited. When exposed to sulphide concentrations of up to 25 pM under severe hypoxia (P02 = 20 ± 1 Torr) aerobic metabolism can, however, be maintained. Under normoxic conditions a similar critical transition between aerobic and anaerobic metabolism is also apparent. The change to anaerobic metabolism, however, occurred at greater sulphide concentrations than under hypoxia. Interestingly, the transition under normoxia occurred over a far less defined range of sulphide concentrations. As was suggested above during the discussion of the whole animal oxygen consumption data, some variability in the tolerance of individual animals to sulphide seems to exist. This may account for the less well-defined initiation of anaerobic metabolic pathways when ambient oxygen is not limiting.The earlier onset of anaerobiosis under hypoxic and sulphidic conditions is also seen in the accumulation of thiols in mud-shrimps. Lower concentrations of thiosulphate were produced under hypoxic conditions than under normoxia (Figure 4.5 - 4.10). In addition, when exposed to hypoxia, a significant decrease in the concentration of thiosulphate in the haemolymph occurred at low micromolar ambient sulphide concentrations, whereas under normoxia, thiosulphate in the haemolymph did not decrease significantly even during the highest sulphide exposures. The concentration of thiosulphate was also seen to increase significantly in the hepatopancreas and abdominal muscle tissues of Calocaris macandreae under both normoxic and hypoxic conditions. Low but significant concentrations of sulphite also accumulated in the haemolymph of C. macandreae under both oxygen treatments but sulphite was not observed to accumulate significantly in abdominal muscle tissues. The concentration of intracellular sulphide in the abdominal muscle tissue of the mud-shrimp C.Si129macandreae did not differ from control values despite exposure to severe sulphidic and hypoxic conditions. This suggests that a considerable degree of protection is afforded to this body compartment by the 'oxidation' mechanism since thiosulphate was found to be the only metabolite present. Differences in the concentrations of thiols were observed in the hepatopancreas. Although sulphite accumulated significantly under normoxia, no change was seen under hypoxic conditions. Interestingly, sulphide began to accumulate at a lower ambient sulphide concentration when the mud-shrimps were exposed to hypoxia than during normoxic conditions. This also appears to indicate the existence of an oxygen-dependent 'detoxification' mechanism. The reduced availability of oxygen required for the 'oxidation' of sulphide in the mitochondria during low oxygen tensions may prevent effective sulphide 'oxidation' (Volkel & Grieshaber, 1996). As has been discussed here and in previous chapters, it is clear that a number of factors, acting in a complex manner at different biological hierarchies, influence the sulphide 'oxidation' capacity of mud-shrimps. These mechanisms and in particular the requirement of oxygen to produce thiosulphate from sulphide in addition to aerobic metabolic processes will be discussed further in chapter 74.4.3. Summary of Chapter 4The main points of Chapter 4 in relation to sulphide metabolism in mud- shrimps are summarized below:-a. A sulphide 'oxidation' mechanism apparently allows aerobic metabolism to be maintained to a certain extent despite the potential inhibitory effects of sulphide.b. The 'oxidation' mechanism is apparently oxygen-dependent and appears to operate 'oxidizing sulphide' below a critical transition point.130c. At sulphide concentrations in excess of! the critical transition point the 'oxidation' mechanism becomes saturated (reaches its maximum rate). Mud- shrimps therefore rely on anaerobic metabolism to survive these conditions.1315. A preliminary calo-respirometric study of the sulphide metabolism in mud-shrimps.5.1. Introduction5 .1 .1 . Metabolic adaptations to hypoxic, anoxic and su lp h id ic conditionsSulphide has been shown to induce anaerobiosis in a variety of marine invertebrates (Anderson, 1990; Oeschger & Vetter, 1992; Volkel & Grieshaber, 1992; 1994; Hagerman & Vismann, 1993, 1995; Grieshaber et a i, 1996). An ambient sulphide-oxygen threshold seems to be apparent which dictates the bias between aerobic and anaerobic metabolism in these animals (Grieshaber et aL,1996). There also appears to be considerable inter-specific differences in the ability of many marine invertebrates to maintain aerobic metabolic pathways during exposure to sulphide before resorting to anaerobic metabolism (see Chapter 4). During exposure to sulphide it may be possible for both aerobic and anaerobic metabolisms to run concurrently and also for specific tissues to function anaerobically while others remain aerobic.For many years calorimetric techniques (the estimation of heat dissipation) have been widely used to determine the metabolic rates of many animals. This technique, however, has been restricted to large animals because of the precision required (Eckert e taL , 1988), More recently, the availability of micro-calo- respirometric techniques has enabled the metabolism of small invertebrates, giving out very little heat and having low oxygen consumption rates, to be investigated. Direct calorimetry (the rate of heat dissipation) allows the continuous determination of total metabolism, while simultaneous measurements of oxygen uptake (indirect calorimetry) monitor the changing rates of oxygen consumption (Gnaiger 1983b). When the rate of heat dissipation and oxygen consumption are compared, in equivalent units of energy (m J.h 'kg '^), the relative proportions of aerobic and anaerobic metabolism can be calculated during132different experimental conditions (Pamatmat, 1983a; 1983b). The simultaneous determination of heat dissipation (direct) and oxygen consumption (indirect) therefore provides a value insight into aerobic and anaerobic metabolic rates (Gnaiger, 1983b, Gnaiger et aL, 1989). Using this technique, a small number of marine invertebrates have been shown to rely on both aerobic and anaerobic metabolism simultaneously. These include the bivalve mollusc Mytilus edulis (Pamatmat, 1983b; Shick e ta l., 1986) and the sea anemone Actina equina (Shick, 1981) during exposure to air. In addition, a small number of marine invertebrates have also been described as having the ability to depress their metabolism to assist in their survival during a reduction of ambient oxygen tension. These include the sipunculid worm Sipunculus nudus, the priapulid Halicryptus spinulosus (Oeschger, 1992) the polychaetes Marenzelleria viridis and Hediste diversicolor (Fritzsche & von Oertzen, 1995) and the bivalve molluscs M ytilus edulis. M odio lus dem issus, P olym esdoa caro lin ia n a (Pamatmat, 1979, 1980), Arctica islandica and Astarte borealis (Oeschger, 1990). Although the mechanisms which underlie these metabolic adaptations are poorly understood, the ability of a variety of intertidal and benthic marine invertebrates to rely partially on anaerobic metabolism during periods of hypoxia, and to depress their metabolism during hypoxic and anoxic conditions, has been widely interpreted as ecophysiological adaptations to survive adverse environmental conditions (Oeschger, 1990, 1992; Guppy e ta l, 1994). Only one study to date has investigated the relationship between aerobic metabolism and anaerobic metabolism during exposure to sulphide using calo-respirometric techniques (Schneider, 1996). Schneider has recently reported that, in contrast to the species described above, the metabolic rate of both the larvae and adults of the brackish water polychaete Marenzelleria viridis increased during exposure to sulphidic and hypoxic conditions.Calo-respirometric analytical equipment, however, is rarely available for physiological work. During the course of this, project an opportunity arose to133conduct a small calo-respirometric study at the Institut fur Zoophysiologie in the Heinrich Heine Universitat of Düsseldorf. Some preliminary calo-respirometric investigations into sulphide metabolism of mud-shrimps under normoxic, hypoxia and sulphidic conditions were therefore conducted and are presented here. The metabolic rates in the mud-shrimp Calocaris macandreae were measured respirometrically as oxygen consumption and calorimetrically as heat dissipation during exposure to different ambient oxygen concentrations and sulphidic conditions. A comparison of these aspects of metabolism was conducted to establish the effect of hypoxia and sulphide on the metabolic requirements of mud-shrimps and to determine the balance between aerobic and anaerobic mechanisms.134------135I■.;m :. §5.2. Materials and methods5.2.1. Collection, transportation and maintenance of mud-shrimpsApproximately 30 mud-shrimps {Calocaris macandreae) were collected byAgassiz trawling in the Clyde Sea (see section 3,2.1) approximately one week'before being transported by air to the Institut flir Zoophysiologie, Heinrich Heine Universitat in Düsseldorf. Prior to departure, the mud-shrimps were placed individually into small mesh containers (to prevent fighting) in a sea water filled cool box. On arrival in Düsseldorf, the animals were placed in a re-circulating sea water aquarium (salinity 35 ± l%o; temperature 15 ± 1 ®C ) and allowed 48 hours to recover from any stress incurred during transit.5.2.2. Direct and indirect calo-respirometry (the instrument)Metabolic activity was determined simultaneously by a micro-calorimeter(Thermal activity monitor, Thermetric) and a twin flow respirometer (Cyclobios)■containing two Orbisphere potentiometric oxygen sensors (set up Figure 5.1). The difference in heat detected between the animal and reference chamber represents the heat dissipation of the animal (direct metabolic rate). Sea water was drawn into the calorimeter through the animal chamber and reference chamber by a multi-channel peristaltic pump (Ismatec IPC) at a flow rate of 38 m l.h'l. The sea water was supplied from a 500 ml reservoir immersed in a 20 1 water bath which was maintained at 10 ± 0.1 (RC6 Lauda Cooler connected to a copper cold-finger and external thermistor). The 500 ml flask wasM :;continually refilled by a peristaltic pump (Ismatec IPC) from an external 5 1 sea'water reservoir.Oxygen consumption was estimated using the twin flow respirometer (Cyclobios) containing two sulphide-insensitive gold cathode potentiometric oxygen sensors (Orbisphere) each connected to an oxygen monitor (Cyclobios).Is iSeawaterSulphide stock SolutionPeristalticPump Mixing Chamber-X Z H —I h-PeristalticPumpCalorimeterWater bathRespirometerExternalCoolerSea water ReservoirAnimalChamber ReferenceChamberExternalCoolerPeristaltic PumpSulphide & sea water reservoirFigure 5.1 Diagram of the calorimetry and respirometry apparatus used to estimate simultaneously the metabolic heat dissipation (tQ = piW) and oxygen consumption (ÎVloo = /^mol.Oo.g'hh"^) of the mud-shrimp Calocaris macandreae when exposed to normoxic, hypoxic, anoxic and sulphidic conditions.136This allowed the continuous measurement of oxygen tension in the water immediately before and directly after the animal chamber. The water in oxygen the electrode chambers within the respirometer was continuously mixed by two magnetic stirrers. The respirometer was immersed in the temperature controlled water bath, as described above, and connected to the 500 ml flask containing sea water. A valve, changing the flow route of the water from before the animal chamber to behind the animal chamber, was set to operate automatically every 6 hours, in order to eliminate any differences in the individual electrodes performance. The whole system was operated inside a small air-conditioned laboratory.5.2.3. Preparation and CalibrationPrior to conducting the experiment the whole system was flushed through with ethanol for 6 hours to reduce bacterial respiration. The Wheaton tubing used throughout the system, to prevent diffusion, was also sterilized in a solution of sodium hypochlorite for 1 hour and then thoroughly rinsed in filtered Millipore water (Sartorius 0.45 ptm). The calorimeter was dried with air passed through a column of silicate gel and cobalt chloride indicator and then calibrated internally over a range of 0 - 300 piW. The control rate of bacterial heat dissipation (blank) was also determined before and after the experiment. Calibration of the oxygen electrodes was carried out by passing water with a P02 of zero, supplied from the sea water reservoir which was bubbled with nitrogen, through the respirometer (Figure 5.1), The electrodes were then equilibrated in oxygen-saturated sea water supplied from a reservoir through which air was bubbled. Control runs were conducted to establish the residual bacterial rate of oxygen consumption in the absence of an animal immediately prior to the experiment and again on completion.After the calibration and initial preparation of the calo-respirometer system, a single mud-shrimp (fresh weight approximately 2.0 g) was placed in a 20 ml137gold-plated stainless steel animal chamber containing sea water and connected to the calorimeter. The external surfaces of the animal and reference chambers were then thoroughly dried with a low pressure air line and slowly inserted into the calorimeter. This was done in 4 stages (approximately 15 minutes for each step). The operating temperature of the calorimeter was set at a temperature of 10 ± 0.01 and maintained by an external cooler (5510 Therm ostat, Thermetric).Artificial sea water was used throughout the experiments and prepared from Millipore water and Seamarine marine mix salts (Wiegandt GmbH). The salinity of the solution (salinity = 35 %o) was carefully adjusted by adding very small amounts of the marine mix salts or Millipore water and confirmed using a refractometer (ATAGO S/MILL, Japan). The pH of the sea water was adjusted to 8.00 using 1 M HCl and pH meter. Prior to use the sea water was filtered through a 0.45 pim cellulose acetate filter (Sartorius). Finally, the pH and salinity of the sea water was confirmed and re-adjusted if necessary to pH 8.00 and 35 %o.5.2.4. Normoxic, hypoxic and sulphidic exposuresTwo experiments were conducted on separate mud-shrimps. In the first, an animal (mass = 2.16 g) placed inside the chamber and exposed to normoxic conditions for 24 hours before the ambient oxygen tension was sequentially reduced to 20, 15, 10, 5 % saturation and, finally, to near anoxic conditions. The manipulation of ambient oxygen tension was achieved using a gas mixing pump (H. Wosthoff 2M301) supplied with nitrogen and oxygen from high pressure cylinders. The output from the pump was fed via an airline to an air- stone in the sea water reservoir. In the second experiment, a different animal (1.93 g) was initially exposed to normoxia for 24 hours and then to sulphidic conditions (71.9 ± 11.6 piM) under hypoxia (31.2 ± 6.4 Torr). The sulphide exposure was achieved by preparing 500 ml of a 5 mM sulphide solution (pH138adjusted to 8.00) using oxygen-free water (see section 2.2.1a. ). 50 ml of the sulphide solution was then added to 450 ml of a sea water solution having a salinity of 39 %o. The greater salinity of this solution was used to allow for the dilution effect during the addition of the sulphide solution. The sulphide-laden sea water was then used to fill a second 500 ml flask in the water bath (Figure5.1). This solution was allowed to react in the flask and to equilibrate to 10 °C for an hour. A valve was then operated to allow the sulphide-laden sea water to supply the system instead of the normoxic sea water. In an attempt to maintain the concentration of sulphide in the sea water reservoir as constant as possible it was continuously refilled with the sulphide laden sea water. This was achieved using a magnetically-stirred mixing chamber supplied with sea water (39 %o) and the sulphide stock (5 mM) solution by two peristaltic pumps (Ismatec IPC & Pharmacia LkB-Pump PI) at a combined flow rate of approximately 38 ml. h 'l and a dilution ratio of 10 sea water ; 1 sulphide. The concentration of sulphide was determined every 2 hours, using the method described in section 2.2.1a. Samples were taken from the reservoir immediately before the system and at both the animal chamber and reference chamber outlets. The pH and salinity of the sea water was also determined at two-hourly intervals. Since it was not possible to measure the concentration of sulphide in the animal chamber during the experiment an indirect estimate was made. The concentrations of sulphide entering and leaving the system and when the animal was removed at the end of the experiment were determined. These values were used to determine a ratio which was subsequently used to obtain an estimate of the concentration of sulphide in the animal chamber throughout the experiment.1395.2.5. Calculation of metabolic ratesThe output from the total heat dissipation rates (tQ = /) and cooperativity (nso).:1656.3. Results6.3.1. The effect of sulphide on decapod crustacean haemocyaninThe addition of 20 mM sulphide (final concentration nominally 1 mM) to haemocyanin appeared to have no effect on the respiratory pigment of Calocaris macandreae, (Figure 6.2). Despite the presence of sulphide the haemocyanin remained functional since it was possible to form deoxyhaemocyanin using nitrogen and subsequently oxyhaemocyanin by bubbling air through the sample. In contrast, the use of KCN induced the formation of deoxyhaemocyanin which could not be subsequently oxygenated (Figure 6.2). Very similar effects were seen with the haemolymph of both Nephrops norvégiens and Carcinus maenas.In addition, a rapid and significant reduction (ANOVA + Tukey test, p < 0.05) in the concentration of sulphide was seen in the sample cuvettes indicating a large sulphide oxidizing capacity of the haemolymph from C alocaris macandreae, Nephrops norvégiens and Carcinus maenas (Table 6.1). Much lower sulphide oxidation rates were seen in the control cuvette containing distilled water.Control samples of Calocaris macandreae haemolymph obtained during January (when the pooled haemolymph samples described above were also prepared) and fixed in the laboratory of the RV Aora were used to determine the concentrations of thiols in similar freshly caught specimens (see section 2.3.5). The concentration of thiosulphate and sulphide were found to be very low (2.3 ± 1.15 pM & 0.44 ± 0.57 pM) respectively (n = 15). Sulphite was not detected in the control haemolymph samples. The concentration of L-lactate was also determined (see section 4.2.2b.) in similar control animals (0.44 ± 0.18 mM, n = 12) although 48 hours was allowed after capture to enable the mud-shrimps to recover from the stress induced during trawling.166_Table 6.1 The concentration of sulphide, thiosulphate and sulphite (mean ± SD mM, n = 3) in 1.5 ml cuvettes (see methods). Details of the associated sulphide oxidation rate (pmoL min-^) and statistics used (ANOVA + Tukey test, aJb/c = significantly different, p < 0.05) are also included.Species Sulphide (; was found to be larger in the haemolymph of Nephrops norvégiens.168; « : :__________________________6.3.3. The effect of thiosulphate on the oxygen affinity and cooperativity of haemocyanin from Calocaris m acan dreae and N ephrops norvégiensThe addition of control salines (containing no thiosulphate) to the haemolymph of Calocaris macandreae and Nephrops norvégiens (Figure 6.3 & 6.5) did not significantly affect the size of the Bohr shift (ANCOVA, p > 0.999 and p > 0.800, respectively) or the oxygen affinity of the haemocyanin (ANCOVA, p > 0.147 and p > 0.658, respectively). The addition of thiosulphate, using the prepared salines to the haemolymph of Calocaris m acandreae and N ephrops norvégiens, resulted in significant increases (ANCOVA, p < 0.001) in the oxygen affinity of the haemocyanin of both species (Figure 6.3 & Figure 6.5), although the size of the Bohr shifts were not significantly affected (ANCOVA, p > 0.1). The addition of the control salines had no significant effect (ANOVA, p > 0.1) on the cooperativity of the haemocyanin of Calocaris macandreae and Nephrops norvégiens, at 10°C (Figure 6.4 & 6.6). The saline containing thiosulphate was also found to have no significant affect (ANOVA, p > 0.05) on the cooperativity of the haemocyanin of Calocaris macandreae, although a significant decrease (ANOVA + Tukey, p < 0.001) in the cooperativity of the haemocyanin of Nephrops norvégiens, however, was seen (Figure 6.6).169_________________________0.60.50)0 C10.4O^ 0.3<0.20.1400380300 340 360320Wavelength (nm)Figure 6.2 Characteristic absorbance spectra for the haemocyanin of the mud- shrimp Calocarii: macandreae, the Norway lobster Nephrops norvegicus and the shore crab Carcinus maenas: a) oxyhaemocyanin oxygenated with air and after the addition of sulphide, b) haemocyanin deoxygenated with nitrogen, deoxygenation with KCN and also after the addition of KCN and oxygenation with air.170____: :-------------------------olOQ.g0.45\0 .3 0 .0 .1 5 . O \0.007.77.3 7.57.1PHFigure 6.3 The effect, at 10 °C, of thiosulphate on the haemocyaninoxygen affinity of Calocaris macandreae, The gradients of the regression lines fitted to the data of untreated haemocyanin (closed circles) and haemocyanin with saline (open circles) and haemocyanin with saline and 1.5 mM thiosulphate (closed squares) were not significantly different from each other (ANCOVA, p > 0.623); the elevations between the untreated haemolymph and haemolymph with the added saline control were also not significantly different (ANCOVA, p > 0.0.146); however, the elevation of the thiosulphate-treated haemolymph was significantly different (ANCOVA, p < 0.001) from the untreated and the saline- treated haemolymph.171se ooeo7.1 7.3 7.5 7.7pHFigure 6.4 The effect, at 10 °C, of thiosulphate (S2O32-) on the cooperativity of the haemocyanin of Calocaris macandreae: Untreated haemocyanin (closed circles), haemocyanin + saline (open circles), haemocyanin + saline containing thiosulphate (closed squares).172a■■iî :'iIiaIi_________ __________s7.25 7.50 7.75 8.00pHFigure 6.5 The effect, at 10 °C, of thiosulphate ou the haemocyaninoxygen affinity of Nephrops norvegicus. The gradients of the regression lines fitted to the data of untreated haemocyanin (closed circles) and haemocyanin with saline (open circles) and haemocyanin with saline and 1.5 mM thiosulphate (closed squares) were not significantly different from each other (ANCOVA, p > 0.531); the elevations between the untreated haemolymph and the saline and haemolymph were also not significantly different (ANCOVA, p > 0.657); however, the elevation of the thiosulphate-treated haemolymph was significantly different (ANCOVA, p < 0.001) from the untreated and the saline treated haemolymph.173______________________4_SO o°2 .»01s>•ro oo ■ m eo7.25 7.50 7.75 8.00pHFigure 6.6 The effect, at 10 °C, of thiosulphate on the cooperativityof the haemocyanin of Nephrops norvegicus. Untreated haemocyanin (closed circles), haemocyanin + saline (open circles), haemocyanin + saline containing thiosulphate (closed squares).174____ _ ___________ ___________________________ _____6.4. Discussion6.4.1, Sulphide and haemocyaninSulphide has been shown to be present in the haemolymph of freshly- caught specimens of the mud-shrimps Calocaris macandreae and Callianassa subterranea at low micromolar concentrations (see section 2.2.5.). In addition, during experimental exposure to sulphide, it has also been shown to accumulate in the haemolymph of Calocaris m acandreae (see section 3.3.2.), in the haemolymph of the hydrothermal vent crab Bythograea thermydron (Vetter et a i, 1987), and in the haemolymph of the Baltic brackish water isopod Saduria entomon (Vismann, 1991). Accumulation of sulphide could potentially affect the haemocyanin oxygen transport in these animals. The in vitro study of the haemocyanin from the three benthic crustaceans investigated, C alocaris macandreae, Nephrops norvegicus and Carcinus maenas, showed that the haemocyanin function was apparently not affected by sulphide because oxygen remained able to bind reversibly to the haemocyanins. Hagertnan & Vismann (1993) investigating the effect of sulphide on the haemocyanin of the Baltic brackish water isopod Saduria entomon, established that haemocyanin also remained functional when exposed to sulphide. The synthesis of haemocyanin, however, was apparently inhibited by sulphide. This is in contrast to the result of an earlier study on Saduria entomon and other benthic crustaceans which, in response to hypoxia (without the presence of sulphide), may synthesize haemocyanin to facilitate oxygen transport (Hagerman & Oksama, 1985).The 'oxidation' of sulphide to form metabolites, such as thiosulphate and sulphite, requires oxygen and apparently occurs within mitochondria (Volkel & Grieshaber, 1996). In the case of crustaceans, the transport of oxygen to sites of 'oxidation' must therefore be primarily mediated by the haemolymph, either in solution or bound to the haemocyanin or respiratory pigment. The haemolymph of Bythograea thermydron may be insensitive to sulphide and does not apparently contain a sulphide-binding protein involved in active transport175(Childress e ta l., 1987). The transport of sulphide in physical solution in the haemolymph has therefore been proposed in the hydrothermal vent crab Bythograea thermydron (Childress eta l., 1987). This is in contrast to other hydrothermal vent species such as the haemoglobin-containing vestimentiferan worm R iftla pa ch yp tila . (Arp et a l., 1987) and other members of the Vestimentifera (Somero et a l, 1989) where sulphide may be reversibly bound to a non-haem site on the haemoglobin molecule. Interestingly, the cytoplasmic haemoglobin within the gills of the clam Solem ya reidi, which contains intracellular sulphide-oxidizing bacteria remained functional in the presence of sulphide. Despite this, haemoglobin deoxygenation was significantly reduced and incomplete when sulphide was present. This suggests that sulphide may directly influence the rate of haemoglobin deoxygenation and may therefore prolong the intracellular oxygen store when sulphide levels increase and/or oxygen becomes limited (Kraus et a l, 1996). Although the haemocyanins of the three decapod species studied here were found to be insensitive in the presence of sulphide, there may be sulphide-mediated changes in the reaction kinetics of these haemocyanins which may affect oxygen transport in the haemolymph.In this study, the haemocyanin from Calocaris macandreae, Nephrops norvegicus and Carcinus maenas was also seen to oxidize sulphide strongly in vitro. This may be important in terms of sulphide 'oxidation'. The details of this mechanism and of any possible 'oxidation' metabolites produced remain to be established. In contrast, the haemolymph of Saduria entomon apparently does not oxidize sulphide (Vismann, 1991). A number of haemoglobin- containing animals from sulphide-rich marine habitats have, however, been shown to possess non-haemoglobin haem compounds which can oxidize sulphide. These include haematin (a brown oxidized or ferric compound not associated with protein) found in the coelomic fluid of the echiuran worm Urechis caupo (Powell & Arp, 1989) and the poiychaete worms Hediste (as Nereis) diversicolor BXid Neanthes {as Nereis.) virens (Vismann, 1990). In176 .............................................................................addition, similar haematin compounds, capable of sulphide oxidation have been observed in the haemolymph from the gill tissues of some haemoglobin- containing bivalves. These bivalve species include Calyptogena magnifica, Lucinom a annulata (Powell & Arp, 1989) and Scapharca inaequivalvis (Vismann, 1993). Importantly, the haematin was found to be present in vivo in these species and it has therefore been suggested that these pigments catalyse the oxidation of sulphide and may therefore protect the animals from sulphide poisoning. Although similar mechanisms have been suggested in the poiychaete worm Arenicola marina and other annelids (Patel & Spencer, 1969; Mangum, 1976; Wells & Parkhurst, 1980) their presence in vivo has been questioned (Powell & Arp, 1989). Haematins result from the breakdown of haemoglobin during stress induced by handling, or poor condition, and may not be naturally present. No evidence of the formation of additional pigments was seen in the haemolymph of the decapod crustaceans investigated in this study. The in vitro oxidation and removal of sulphide in the haemolymph of these species has been established, although this aspect clearly requires further work.6.4.2. Oxygen transporting properties of haemolymphComparison of data between species is notoriously difficult due to the need to make such comparisons under similar experimental conditions and pH values. Despite this, the haemocyanin oxygen affinities of crustaceans that may regularly be exposed to hypoxia, such as burrow-dwellers and hydrothermal vent crabs, appear to be consistently higher than those of decapods from normoxic environments (Childress & Fisher, 1992; Astall, 1993). The values for the Bohr shift ((j) = -0.639), oxygen affinity (P50 = 5.22 at pH 7.8) and cooperativity (n^o = 2.4 ± 0.27) for Calocaris macandreae and (f = -0.991, P50 = 8,75 at pH 7.8, cooperativity = 2.8 ft 0.78) for Nephrops norvegicus determined in this study were found to be very similar to those estimated by other workers (Bridges 1986; Anderson, 1989). These values indicate that the haemocyanin of these177species has a high oxygen affinity. In the case of Calocaris macandreae these may be adaptations to the often severely hypoxic burrow environment. Haemocyanin having a high oxygen may also be significant during exposure to hypoxic and sulphidic conditions in that oxygen is required, in addition to normal aerobic metabolic processes, to 'oxidize' sulphide (see Chapter 4).A few haemocyanins from crustaceans from the sulphidic and hypoxic environment of hydrothermal vents have also been extensively studied. Arp and Childress (1981) showed that the haemocyanin from the hydrothermal vent crab Bythograea thermydron had a moderate oxygen affinity and Bohr shift (P50 = 5.8 Torr at pH 7.55 and 5 ®C, = -0.34, respectively), although there is some suggestion that there may be genetic differences in the haemocyanin of this species of crab from different vent sites (Sanders, 1989). The haemocyanin from another vent crab Cyanograea praedator (Childress & Fisher, 1992) has also been found to show a large Bohr shift ((j> = -0.94 to -1.30). In addition, the haemocyanin of the hydrothermal vent shrimp Alvinocaris liisca has a high oxygen affinity (P50 = 1.3 to 2.8 Torr at pH 7.95) and a moderate Bohr effect (-0.77 at 5 ®C). The haemocyanin oxygen affinities of Bythograea thermydron, Cyanograea praedator and Alvinocaris lusca also show some degree of insensitivity to temperature (Childress & Fisher, 1992). High oxygen affinities, substantial Bohr effects and thermal insensitivity appear to be typical features of hydrothermal vent crustacean haemocyanins. It has therefore been suggested that these properties may be adaptations to the widely varying conditions, of P02 , temperature and sulphide, that may be encountered at hydrothermal vents. As similar trends in oxygen affinity and Bohr shift have been established in mud- shrimps in this study and by others (Anderson, 1989; Astall, 1993) these adaptations may be also be significant in the potentially sulphidic burrow environment of mud-shrimps.178 ■ ■ : : _6.4.3. The effect of thiosulphate on the oxygen affinity and cooperativity of decapod crustacean haemocyaninThiosulphate has been detected at micromolar concentrations in the haemolymph of mud-shrimps experimentally exposed to sulphide and also in freshly caught animals (see Chapter 2). Interestingly, in this study, thiosulphate has been shown to have a significant effect (increase) on the haemocyanin oxygen-binding affinity of both Calocaris macandreae and N ephrops norvegicus. In many studies, however, haemolymph samples are often dialysed against a physiological saline to reduce the concentrations of small molecular weight modulators of oxygen affinity, such as L-lactate and urate, prior to the determination of oxygen affinity. Since only a very small volume of blood Was available from each mud-shrimp {Calocaris macandreae) the pooled samples were not dialysed. The concentrations of thiosulphate and sulphide in the control haemolymph pools, taken from freshly caught animals, were found to be less than 3 /

Sulphide metabolism in burrowing marine Crustacea

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Sulphide metabolism in burrowing marine Crustacea

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