Louisiana State UniversityLSU Digital CommonsLSU Historical Dissertations and Theses Graduate School1993Physiological and Biochemical Responses toHypoxia in the Blue Crab, Callinectes SapidusRathbun, the Lesser Blue Crab, Callinectes SimilisWilliams, and the Southern Oyster Drill,Stramonita Haemastoma Linnaeus.Tapash DasLouisiana State University and Agricultural & Mechanical CollegeFollow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissthesesThis Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion inLSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contactgradetd@lsu.edu.Recommended CitationDas, Tapash, "Physiological and Biochemical Responses to Hypoxia in the Blue Crab, Callinectes Sapidus Rathbun, the Lesser BlueCrab, Callinectes Similis Williams, and the Southern Oyster Drill, Stramonita Haemastoma Linnaeus." (1993). LSU HistoricalDissertations and Theses. 5625.https://digitalcommons.lsu.edu/gradschool_disstheses/5625INFORMATION TO USERSThis manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer.The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction.In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book.Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order.U niversity M icrofilm s International A B ell & H ow ell Information C o m p a n y 3 0 0 North Z e e b R oad . A nn Arbor, Ml 4 8 1 0 6 -1 3 4 6 U SA 3 1 3 /7 6 1 -4 7 0 0 8 0 0 /5 2 1 -0 6 0 0Order Num ber 9419879Physiological and biochem ical responses to hypoxia in the blue crab, Callinectes sapidus R athbun, the lesser blue crab, Callinectes similis W illiam s, and the southern oyster drill, Stramonita haemastoma LinnaeusDas, Tapash, Ph.D.The Louisiana S tate University and Agricultural and Mechanical Col., 1993U M I300 N. Zeeb Rd.Ann Arbor, MI 48106PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES TO HYPOXIA IN THE BLUE CRAB, CALLINECTES SAPIDUS RATHBUN, THE LESSER BLUE CRAB, C. SIMIUS WILLIAMS, AND THE SOUTHERN OYSTER DRILL, STfRAMONITA HAEMASTOMA LINNAEUSA DissertationSubm itted to the G raduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirem ents for the degree of Doctor of PhilosophyinThe D epartm ent of Zoology and PhysiologybyTapash Das B.S., Burdwan University, India, 1981 M.S., Kalyani University, India, 1983 December 1993DEDICATIONI am dedicating th is d issertation to m y beloved p aren ts an d s is te r w hose sacrifice and inspiration is a co nstan t source of suppo rt in the eveiy w alk of m y life.ACKNOWLEDGMENTSThe com pletion of th is d issertation would not have been possible w ithout the help of m any others. I would like to extend my deepest gratitude and th a n k s to m y advisor. Dr. William B. Stickle jr . for h is guidance, counselling and co n stan t support over the last four and a half years. I am also indebted for all the help I have received from the m em bers of m y com mittee. Dr. J . F. Siebenaller, Dr. J . W. Fleeger, Dr. K. M. Brown and Dr. D. W. Foltz.Many th an k s go to Dr. M arieanne Hollay for her invaluable help w ith statistical analyses, Mr. JelTTam plinfor h is help in some of the illustrations and Mr. Ron B ouchard for h is con tinuous help with photography.Special th an k s are extended to Melanie Morgan, Phillip Corey Jackson . Jo h n L. G uerin, Dr. Shiao Wang and Dr. Jo h n McCall for the ir help and support.I wish to express my heartfelt g ratitude to my p aren ts and s is te r for their patience, love and understand ing during these trying years.TABLE OF CONTENTSDEDICATIONACKNOWLEDGEMENTSABSTRACTCHAPTER12345LITERATURE CITEDGeneral IntroductionR esponses of C rabs Callinectes sap idus and C. similis and the G astropod Stramonita haem astom a to Hypoxia and AnoxiaDetection and avoidance of Hypoxic W ater by Juvenile Callinectes sap idus and C. similisPhysiological and Biochemical R esponses of Juvenile Blue Crabs, Callinectes sapidus and C. similis, to D ium ally Varying Dissolved Oxygen TensionSum m ary and ConclusionsVITAABSTRACTThis s tudy m easured the physiological and biochem ical changes associated with exposure of the juvenile blue crab, Callinectes sap idus R ath b u n and th e lesser b lue crab, C. sim ilis Williams, to long term (28 d) hypoxia, sh o rt term (10 d) transfer from hypoxia to norm oxia and a d ium ally fluctuating oxygen regime for 28 days. The so u th ern oyster drill. Stram onita haem astom a L innaeus, w as also exposed to 28 days of co n stan t hypoxia to com pare the responses of a to lerant species with the two species of Callinectes. The 28 day LC50 estim ates for C. sapidus, C. similis and S. haem astom a were respectively 106, 43 and 11.5 Torr under co n stan t hypoxic exposure for 28 d. Feeding ra te s for the crabs of both species exposed to 50 and 25 Torr oxygen were significantly lower than for crabs exposed to higher levels of oxygen. Growth and m olting ra te s of crabs exposed to constan t hypoxia were always lower th an for crabs exposed to norm oxia. Feeding rate in S. haem astom a declined linearly with declining oxygen u n d er co n stan t exposure to hypoxia. Oxygen consum ption ra te s of the two crab species un d er various hypoxic levels were significantly different. M ean oxygen consum ption in C. similis exposed to hypoxia w as higher th a n for crabs exposed to normoxia. Rate of adap tation for b lue crabs transferred from hypoxia to norm oxia was faster th a n w hen transferred from norm oxia to hypoxia. Detection and avoidance of hypoxic w ater by the two species of crabs was also observed under laboratory conditions. Both species of crabs were able to detect and avoid hypoxic w ater and stay at an interm ediate oxygen tension. C rabs were found to be m ore active at higher oxygen tensions. Behavioral responses, w hen crabs were exposed to hypoxic treatm ents, were clearly due to hypoxic stress. The feeding ra te of C. sapidus exposed to dium ally varying oxygen tension w as significantly higher th a n for the crabs exposed to norm oxia while the feeding rate of C. similis exposed to d ium ally varying oxygen tension was significantly lower th an the ones exposed to norm oxia. RNA and DMAvconcentration decreased over time in both species of c rabs exposed to d iu rna l variation in oxygen tension. C oncentration of the individual nucleic acids were found to be a reliable m easu re of hypoxia s tre ss th a n the RNA:DNA ratio.viCHAPTER 1GENERAL INTRODUCTION1Seasonal depletion of dissolved oxygen in subpycnocline w aters is often observed in estuaries and nearshore w aters of continental shelves (Sanford el al. 1990). O ccurrence of hypoxic w ater m asses h a s been reported by a n um ber of researchers from a variety of locations worldwide, particularly in Europe an d America (Bedinger el al. 1981, T urner and Allen 1982, Boesch 1983, Officer el al. 1984, Stachow itsch 1984, R enaud 1985, 1986a, Rabalais e l a l 1986a, b, Rosenberg 1986, Parker and O’Reilly 1991, Portnoy 1991). The occurrence of hypoxic bottom w ater off the Louisiana coast w est of th e M ississippi Delta is a com mon and recu rren t phenom enon (Bedinger et al.1981, S tun tz e ta l. 1982, Boesch 1983, Leming and S tun tz 1984, R enaud 1985, 1986a, R abalais e t a l 1986a, b) locally know n as the occurrence of dead w ater (Turner and Allen1982, Boesch 1983, R enaud 1985, 1986a). Hypoxic w ater m asses m ay persist for weeks and are usually associated with stratification of the w ater colum n, phytoplankton bloom s, an d stable high p ressu re w eather system s (Ragan et al. 1978, Bedinger et al. 1981, H arper eta l. 1981, Boesch 1983, Renaud 1985, R abalais et a t 1991a, 1992b). On the Louisiana continental shelf, in p u t from the M ississippi and the A tchafalaya rivers are im portan t contribu tors of both fresh w ater and n u trien ts which can cause a strong halocline and stim ulate th e production of a large phytoplankton biom ass (Boesch and R abalais 1991). Decomposition of the sinking phytoplankton con tribu tes to oxygen depletion in the isolated bottom water. Diel variation in the dissolved oxygen tension of es tuarine w ater along the Louisiana Gulf coast occurs frequently, especially during the sum m er m onths, with w ater becom ing very hypoxic during the early m orning hou rs (Das an d Stickle, unpublished observation).Hypoxia can be tolerated by all organism s for a variable am ount of time although th is condition is ultim ately incom patible with survival of m ost organism s which use oxygen for tire production of energy. M ass m ortality of m arine and estuarine organism s h a s been widely reported from areas affected by low dissolved oxygen in the w ater(Santos and Sim on 1980a, b, H arper e t a l 1981, R abalais e t a l 1985, Rabalais and H arper 1991b, 1992a). Commercial crab-pot fisherm en have frequently reported dead blue crabs in po ts (Carpenter and Cargo 1957, T atum 1979, V an Engel 1982). Hypoxia is a m ajor environm ental variable affecting the perform ance and ultim ately survival of m arine organism s w hich respire aerobically.The blue crab Callinectes sap idus R athbun occurs in the lower reaches of rivers, estuaries, and coastal w aters along the Atlantic seaboard and Gulf of Mexico (Churchill 1919, G un ter 1938, Odum 1953, Cam eron 1978). Juveniles and ad u lts occupy hab ita ts ranging from freshw ater to hypersaline w aters of as high as 117°/00 S (Williams 1984) b u t larvae are relatively stenohaline (Costlow and Bookhout 1959). Tolerances m ay vary with life stages and hypoxia m ay also be stressfu l to these species. Their high abundance, diverse feeding hab its and im portance as prey species for a variety of organism s m ake them an integral p a rt of the coastal ecosystem. C. similis Williams is an offshore congener of the C. sap idus and occurs in the oceanic littoral zone in salinities above 15°/00 and a t a depth of alm ost 100 m eters (Williams 1984). The distributional p a tte rn s and niche characteristics of these two species overlap quite frequently (Engel 1977). They prim arily utilize the sam e prey groups (Hsueh et a l 1992). The high overlap in diet and niche characteristics of these two crab species suggests a com petition for com m on resources (Engel 1977). Norse and Fox-Norse (1979) suggested a common p a tte rn of occurrence am ong various species w ithin the genus Callinectes along all three ocean borders (eastern Pacific, w estern A tlantic and easte rn Atlantic) where blue crabs occur. Based on the evolutionary relationship of these species there are large inshore species which belong to bocourti group and sm all offshore species which belong to danae and m arginatus group. Based on the ir ecological and geographical patterns, Norse (1977) suggested different abilities to w ithstand physical and chem ical conditions for these species in the spectrum of biotopes they inhabit. In the no rthern Gulf of Mexico, C.sapidus is th e large estuarine species and C. similis is th e sm all offshore species (Perry 1975). Therefore, studying th e effect of hypoxia on these two species w ould provide u s with valuable inform ation abou t the ir susceptibility to th is environm ental variable (hypoxia) and th e ir differential responses to cope up w ith the changes in the dissolved oxygen tension in the am bient water.The sou th ern oyster drill, Stramonita(=Thais) haem astom a, is a n im portant p redato r on oyster reefs and o ther h ard su b stra te s in the n o rthern G ulf of Mexico. It is responsible for losses of up to half the yearly oyster crop in Louisiana (Cake 1983). S. haem astom a is exposed to d iu rnal fluxes of dissolved oxygen, ranging from 16 to 120% of sa tu ra tio n over a 24-h period and is very to lerant of chronic hypoxia (Kapper and Stickle 1987, Stickle e t a l 1989). Use of th is species provided baseline d a ta required to com pare the responses of the two crab species with a very hypoxia to leran t species from the sam e habitat.The overall aim of th is study is to m easure and com pare the responses of the two crab species and the gastropod, Stram onita haem astom a, to hypoxia an d anoxia. My null hypothesis is, responses to hypoxia in th e two species of crabs, Callinectes sapidus and C. similis, are sim ilar as are the responses of S. haem astom a. The alternative hypothesis is, responses to hypoxia differs in the two species of crabs a s well as in the oyster drill. The specific objectives of th is study are to: (1) com pare the hypoxia tolerance of juvenile C, sap idus and C. similis and adu lt S. haem astom a, (2) quantify the effects of long-term exposure (28 d) to hypoxia on the feeding rate, growth, molting and respiration of the juvenile C. sapidus and C. similis a s well as on the feeding ra te s of adu lt S. haem astom a, (3) detem iine the effects of ab ru p t changes from different levels of hypoxia to nonnoxia on the survival, feeding rate, growth and m olting of C. sapidus, (4) determ ine w hether juvenile C. sapidus and C. similis can detect and avoid specific levels of dissolved oxygen under laboratory conditions, (5) determ ine the behavioral responses associated with shortte rm hypoxic exposure, (6) com pare the responses of juveniles of the two crab species to d ium ally varying oxygen tension, and (7) determ ine the difference in response to chronic hypoxia and diel variation in oxygen tension using feeding rate , survival, growth, molting and changes in the nucleic acid concentration as indices of sub le thal stress.These objectives are addressed in 3 separate chapters. In the second chap ter of th is d isserta tion I have investigated th e physiological responses of th e two species of crabs, Callinectes sapidus and C. similis, and the gastropod, Siram onita haemastoma, following exposure to long-term (28 d) hypoxia and a b ru p t changes from hypoxia to normoxia. Therefore, the second chap ter com pared th e effects of co nstan t hypoxia on the survival and feeding ra te of all th ree species as well as the growth, molting an d oxygen consum ption ra te of C. sap idus and C. similis. This chap ter h as been published in M arine Ecology Progress Series (vol 98: 263-274, 1993).The effect of hypoxia on the m ovem ent p a tte rn s of the blue crab, Callinectes sap idus and the lesser blue crab, C. similis, to and from th e es tuarine region is unknow n. M ature female C. sapidus h a tch the ir eggs in high salinity w ater (Millilcin and Williams 1984). Egg-bearing female C. sapidus m ay occur and spaw n in the coastal Gulf and estuarine w aters year-round (G unter 1950, D augherty 1952, More 1969, A dkins 1972a, Periy 1975). Egg-bearing C. similis also spaw n in high salinity w aters (Hsueh et al. in press). S tuck and Perry (1981) reported a year-round abundance of C. similis m agalopae in the w aters of M ississippi Sound with a peak during February and March. Thus, an extensive occurrence of hypoxia in nearshore and offshore w ater could cause a potential problem for the spaw ning female population of the b lue crabs as well as the larval and juvenile stages. Low dissolved oxygen m ay also act as a barrie r to th e m igration of juvenile blue crabs from high salinity to low salinity water's (Van Engel 1982). C hapter th ree of th is d issertation probes into the capacity of the juveniles of these 2 species ofcrabs to detect and avoid hypoxic w ater under laboratory conditions. This chap ter h as been subm itted to M arine Biology and is currently undergoing revision.The fourth chap ter of th is d issertation addresses physiological and biochemical responses of juvenile Callinectes sapidus and C. similis exposed to a fluctuating oxygen regime. The objective of th is chap ter is to com pare the responses of the two crab species exposed to diel variation in oxygen tension with th a t of long-term exposure to constan t hypoxia.In order to quantify the responses of the th ree species to co n stan t and diel variation in oxygen tension, survival w as used as a m easu re of resistance adaptation. LC50 w as used as a quantifiable m easure of survival. It is a veiy sensitive indicator of hypoxia tolerance because the determ ination of a LC50 value requires d a ta abou t both survival an d m ortality from a range of trea tm en ts tested. Feeding rate , growth, molting, concentration of RNA and DNA and RNArDNA ratio were used as a m easure of capacity adaptation. Feeding rate is the prim aiy determ inan t of a n anim als energy budget. Feeding rate h as been shown to vary 5-9 tim es more th a n m etabolic rate along environm ental factor gradients (Stickle 1985). Therefore, a m easu re of the feeding rate provides a direct m easure of s tress involved with exposure to hypoxia. Growth and molting are d irect consequences of changes in feeding rate. M easurem ent of the oxygen consum ption ra te provides an estim ate of m etabolic costs of adap ta tion to the hypoxia gradient. C hanges in nucleic acid concentration and the RNA:DNA ratio have been studied in relation to various biological processes in both laboratory experim ents and field studies. Since DNA content per cell nucleus rem ains constan t in som atic cells w ithin a given species (Leslie 1955) and changes in cellular RNA content I'eflect changes in cellular activity (Brachet 1960) the ratio of RNA to DNA is used as a reliable indicator of protein synthetic activity w ithin cells. RNA:DNA ratios have been show n to be correlated with growth (see review by Bulow 1987) and growth is affected byenvironm ental s tressors. Therefore, m easurem en t of the RNA:DNA ratio shou ld an useful indicator of stress.CHAPTER 2RESPONSES OF CRABS CALLINECTES SAPIDUS AND C. SIMILIS AND THE GASTROPOD STRAMONITA HAEMASTOMA TO HYPOXIA AND ANOXIA891 3 - 1 0 - 1 9 9 3 1 2 J 0 7 UOH Q . K j n n * O l d e n d o r f Qf r m n s 3N 0 0 1 5 0 4 3 3 3 1 7 6 3 S . 0 1O C T -12-1993 1 0 :1 3 PROM LSU ZOOL PHYSIOL TO 31149410239833378353 r . 01' L o u I S I. A M A S T A X H TJ N 1 V E R S I T YAMO A M » t C V h T U * A v AMO M CD e p a r t m e n t o f Z o o l o g y a n d P h y j i o / o q yOctober 12. 1993Publisher Inter-Research P.O. Box 1120 W-2124Amcltnghausen. G erm an y Dear sir.I am writing to obtain a copyright release to Include my article entitled " Sensitivity of crabs Callinectes sapidus and C. similis and the gastropod Stramordta. haemastoma to hypoxia and anoxia which was published in vol. 98: 263-274. 1993, in the Marine Ecology Progress Series, in my Ph.D dissertation. Please send written permission so that I may present it to the Louisiana State University Graduate School. I would appreciate if you FAX the permission letter to me since I would require it by the end of this week. Thank you very much for your time and consideration.Zoology & Physiology Louisiana State University3aton Rouge. LA 70803-1725--------U .5APhone (504) 388-1739 Fax (504)388-1763P e r m i s s i o n i s g r a n t e d by I n t e r - R e s e a r c h f o r t h e m a t e r i a l s t a t e d t o be u s e d f o r t h e p u r p o s e d e s c r i b e d , p r o v i d e d t h e o r i g i n a l s o u r c e o f p u b l i c a t i o n i s c o r r e c t l y s t a t e d .O c t . 1 3 , 1 9 9 3 Inter-Research•t /..v.Jsinehcusen’5t Republic df •INTRODUCTION10Seasonal depletion of dissolved oxygen in subpycnocline w aters is often observed in estuaries an d nearshore w aters of continental shelves (Sanford e t al. 1990). O ccurrence of hypoxic w ater m asses have been reported by a num ber of researchers during th e la s t decade in the C hesapeake Bay, New York harbor, New England estuaries and on the Louisiana continental shelf (Bedinger et al. 1981, T u rner and Allen 1982, Boesch 1983, Officer e ta l. 1984, Renaud 1985, 1986a, R abalais e t a l 1986a, b, Parker and O’Reilly 1991, Portnoy 1991). M ass m ortalities or m assive shorew ard m igration of dem ersal fishes and c ru staceans to shallow w ater have been reported w hen the dissolved oxygen level in the w ater w as veiy low (Loesch 1960, May 1973, Garlo e t al. 1979, Pavela et al. 1983, Pihl et al. 1991). The occurrence of hypoxic w ater m asses off the Louisiana coast is alm ost an an nual phenom ena and m ay persist for w eeks (Bedinger et al. 1981, T u rner and Allen 1982, Boesch 1983, R enaud 1985, 1986a, and Rabalais e ta l. 1986a, b). This creates a th rea t to m arine fauna, including the b lue crab, Callinectes sapidus, and th e lesser b lue crab, Callinectes similis.The b lue crab Callinectes sapidus R athbun occurs in lower reaches of rivers, estuaries, and coastal w aters along the Atlantic seaboard an d Gulf of Mexico coast of USA (Churchill 1919, G unter 1938, Odum 1953, Cam eron 1978). Juveniles and adu lts occupy h ab ita ts ranging from freshw ater to hypersaline w aters of as high a s 117°/00 S (Williams 1984) although larvae are relatively stenohaline (Costlow and Bookhout 1959). Salinity, w ater tem perature, food availability, predation, su b stra tu m , available h ab ita ts and po llu tan ts affect growth and survival of blue crabs (Gulf Coast Research Laboratory, O cean Springs, 1986; Georgia Cooperative Fishery and Wildlife R esearch Unit, A thens, 1989). Tolerances m ay vary with life stages and hypoxia m ay also be stressfu l to these species. Blue crabs do not conform to specific trophic levels and are characterized as11opportunistic benth ic omnivores. Their high abundance, diverse feeding hab its and im portance as prey species for a variety of organism s m ake them a n integral p a r t of the coastal ecosystem.Callinectes similis Williams, is a n offshore congener of the C. sap idus and occurs in the oceanic littoral zone in salin ities above 15°/00 and a t a depth of alm ost 100 m eters (Williams 1984). The distribu tional p a tte rn s and niche characteristics of these two species overlap quite frequently and in some cases the crabs com pete for food and other resources (Engel 1977). They prim arily utilize the sam e prey groups (Hsueh e ta l. 1991). This high overlap in the diet of these two crab species suggests a com petition for com mon resources.The sou th ern oyster drill, Stramonita(=Thais) haem astom a, is an im portant p redato r on oyster reefs and o ther h ard su b stra te s in the n o rthern G ulf of Mexico. It is responsible for losses of up to ha lf the yearly oyster crop in Louisiana (Cake 1983). S. haem astom a is exposed to d iu rnal fluxes of dissolved oxygen, ranging from 25 to 120% of sa tu ra tio n over a 24-h period and is veiy to lerant of hypoxia (Kapper and Stickle 1987, Stickle e ta l. 1989).The specific objectives of th is study are to : (i) com pare the hypoxia tolerance of the juvenile Callinectes sapidus, and Callinectes similis and adu lt Slramonita haemastoma; (ii) quantify the effects of long term exposure (28 days) to hypoxia on the feeding rate, growth, molting, and respiration of the juvenile C. sap idus and C. similis and feeding ra te s of adu lt S. haemastoma;(iii) determ ine the effects of ab ru p t changes from different levels of hypoxia to norm oxia on the survival, feeding rate , growth and molting of C. sapidus.MATERIALS AND METHODS12Collection and m aintenanceCallinectes sap idus were collected nearshore O cean Springs, M ississippi (Longitude 88°49.9’W; Latitude 30°24.4’N); C. similis were collected nearshore Port Fourchon, Louisiana (Longitude 90°11.4W ; Latitude 29°6.7’N); and Stramonita haem astom a were collected from bu lkheads and je tties onshore C am inada Pass, Louisiana (Longitude 90°3.0’W; Latitude 29°12.6’N). All an im als were collected between A ugust and Septem ber 1991, w hen the w ater tem peratu res a t O cean Springs, Port Fourchon, and C am inada Pass w as 28, 30, and 30°C and the salinity w as 22, 29, and 3 0 ° /oo respectively. The anim als were b rought back to the laboratory and placed into artificial sea w ater m ade from In stan t Ocean, a t 24°C and salinities m atching field conditions. All anim als were kept u n d er constan t illum ination. After a few days all an im als were acclim ated in a step wise fashion to the experim ental salinity (30°/oo). All th ree species were held for two weeks a t the final tem perature-salin ity com bination before being used in experim ents. Blue crabs and lesser b lue crabs were individually isolated as described below and were fed frozen abdom inal portions of the g rass shrim p, Palaemoneles pugio, and the oyster drills were fed live m ussels, Ischadium recurvum.Himoxia exposure sustemThe hypoxia exposure system consisted of three se ts of 12 flow-through aquaria. Two aquaria were assigned for each experim ental oxygen tension for each species. Each experim ental aquarium (38 liter) consisted of an undergravel filter overlaid with oyster chips. W ater w as pum ped from a large filtration un it into each aquarium by a peristaltic pum p a t a rate sufficient to ensu re the w ater w as completely exchanged several times13each day. The filtration un its were large (76 liter) partitioned plywood boxes lined with non-toxic fiberglass resin. A separate filtration un it w as u sed for each species.Target oxygen tensions of 119, 73, 50, 25 and 0 Torr (77%, 47%, 32%, 16% and 0% of a ir sa tu ra tio n and 5.57, 3.39, 2.31, 1.16, and 0 ppm dissolved oxygen) were created an d m ain tained by m ixing bottled nitrogen and oxygen w ith M atheson gas mixers (model 7402T) along with 0.03% carbon dioxide to m ain ta in the pH a t approxim ately 7.8. Each gas m ixer w as connected to an outlet manifold and the air m ixture w as passed th rough the undergravel filter in each aquarium . One gas m ixer w as used for each oxygen level and for each species. Ambient a ir w as used to drive the undergravel filters of the normoxic tan k s (155 Torr). P 0 2 w as always m ain tained w ithin 10% of the target value in the two higher levels, and w ithin 5% of the target value for the 50 and 25 Torr 0 2 tanks. The zero Torr tan k s were m ain tained w ithin 5% of the target value for the first 6 days and then w ithin 1% for the rem aining 22 days. A plexiglas lid w as used to cover each aquarium and then covered with a clear plastic w rapping to minimize air exchange. The w ater level w as m ain tained in each aquarium by a constant-level siphon which drained w ater from the aquaria into one of the filter boxes a t a ra te equivalent to the inflow rate. Experim ental conditions (tem perature, salinity, P 0 2, pH, ammonia) wei'e checked daily. Ammonium concentration never exceeded 25pM.The num ber of individuals used a t each P 0 2 were 16 divided into two groups of eight in each of the two aquaria designated for th a t P 0 2 for the blue crabs and the lesser b lue crabs and 12 divided into two groups of 6 for the oyster drills. W ithin each aquarium each crab w as kept in a cham ber (cham bers were m ade from two pairs of PVC tubing of 21 cm in diam eter and were 28 cm long with nylon m eshed screens on both ends) to prevent cannibalism . Animals were chosen carefully for each P 0 2 to minimize the weight and size differences w ithin and among the treatm ents.14Huvoxia bioassauFor the 28 day constan t hypoxic exposure experim ent individuals of all three species were transferred directly from normoxic w ater (155 Torr 0 2) to different hypoxic levels (119, 73, 50, 25, and 0 Torr 0 2) and were m ain tained th roughou t the experiment. In the tran sfe r experim ent the blue crabs were exposed to th ree levels of constan t hypoxia (73, 50 and 25 Torr 0 2) for 10 days and th en transferred directly to normoxic w ater (155 Torr 0 2) and were m ain tained for ano ther ten days. This experim ent was perform ed to investigate the am oun t of physiological s tre ss involved w hen the oxygen concentration in w ater drops su ddenly and re tu rn s to norrpal after staying a t th a t level for several days.Survival a t each P 0 2 w as determ ined daily for all th ree species th roughout the experim ental period. A crab w as considered dead w hen it did no t show any sign of m ovem ent after stim ulation and by a color change to straw color once it w as dead for more th a n a few hours. A snail w as considered dead w hen both the siphon and the foot failed to respond to tactile stim ulation. LC50 values were calculated for each day using th e Spearm an-K arber technique (Hamilton e t a l 1977).Determination o f food consumption, molting, and growth rateBoth crab species were fed weighed abdom inal portions of frozen g rass shrim p [Palaemonetes pugio) ad libitum so some tissue rem ained a t the end of the day. U neaten portions of th e food were removed, weighed and replaced with a new ra tion daily. Oyster drills were fed live m ussels (Ischadium recumum) a d libitum. The am oun t of d iy tissue consum ed daily by th e drills w as calculated from a d iy tissu e weight to length regression equation of the m ussels (Least square method). C rabs and drills th a t died during the hypoxic exposure were excluded from the feeding ra te analysis the week they died.15Growth ra te w as m easured for the crabs only since growth of oyster drills include a n increase in body tissue weight and incorporation of C aC 03 in the shell m aking it im possible to m easure th e increase in soft tissue weight. Each time a crab m olted it w as reweighed and the difference betw een the initial and final weight w as converted into percent increase in weight.M easurem ent o f the scope for growthScope for growth for bo th blue crabs and oyster drills w as calculated (Table 2.2). Stickle et al. (1989) provided an estim ate of h ea t d issipation in the b lue crabs and the oyster drills w hen exposed to hypoxia and anoxia. A ssum ing a linear decline in the heat d issipation ra te from norm oxia to anoxia, a m easure of respiratory energy loss can be estim ated. The caloric concentration of g rass shrim p (Palaemonetes pugio) abdom inal tissu e w as taken from G uerin and Stickle (1992). Scope for grow th w as determ ined using th e balanced energy equation of W inberg (1960). The equation is P = (C-F) - (R+U) = Ab - (R+U), where, P = scope for growth; Ab = energy absorbed from food consum ed = (C, energy absorbed from food - F, energy lost as feces); R = energy lost as respiration; U = energy lost a s excretion and w as no t estim ated b u t typically only am oun ts to 4.5 - 11.9 % of energy losses a t 25°C and 3500/ 0 S (Guerin & Stickle 1992). Scope for growth for C. similis could no t be calculated because h ea t d issipation rate under various hypoxic levels were not available.Starvation experimentSixteen blue crabs were m ain tained under norm oxia for five days and monitored for daily feeding rate, then starved for 10 days and m onitored for daily food consum ption for ano ther 10 days to determ ine if starvation for a period of tim e re su lts in an enhanced feeding ra te after food is again m ade available.16M easurem ent o f oxygen consumptionOxygen consum ption ra te s a t constan t Po2’s were m easured using the flowthrough system described by Stickle et al. (1985). In cu rren t w ater w as bubbled with a gas m ixture of appropriate Po2 from a gas m ixer identical to those used in the hypoxia exposure system . W ater w as pum ped via a subm ersib le pum p from th e appropriate dosing com partm ent th rough a manifold, w ith excess w ater re tu rn ed to a separate reservoir via a re tu rn tube. Ten side ports from the m anifold were connected to separate flow-through resp iration cham bers (250 ml.) so th a t a b lank (control) cham ber was placed a t each end. W ater of appropriate oxygen tension w as pum ped from the reservoir into th e d istribu tion manifold a t a low flow ra te (~20m l/m in). Two hou rs were allowed betw een loading crabs and the initiation of the resp iration determ inations to allow for crab adap tation to the cham bers. Oxygen consum ption in each cham ber w as calculated according to the following equation :p i 0 2. h r '1 = % oxygen used by th e crab * flow ra te (1. h r '1) * 1000 * oxygen con ten t in w ater a t th a t partial pressure.Percentage oxygen used by each crab w as calculated from the difference between th e m ean oxygen partia l p ressu re of the w ater flowing ou t of the control cham bers and th e w ater flowing out of the experim ental cham ber and dividing th a t by the experim ental oxygen tension. Oxygen conten t in the w ater a t a p articu la r experim ental oxygen tension and salin ity-tem perature com bination w as calculated using the following equation:A * (Experimental oxygen tension in T o it . 155"1) where, A is the oxygen content in the normoxic w ater a t a particu la r salin ity-tem perature com bination. W ater sam ples from the cham bers were draw n anaerobically and were injected into a StrathKelvin oxygen m eter (model 781) connected to a flow-through w ater jacketed oxygen electrode.17Statistical analysisAll ra te functions were determ ined by either one-way or two-way analysis of variance (ANOVA). Differences am ong trea tm en t m eans w ere determ ined by Tukey’s range te s t or by S tuden ts-t-test. Repeated m easure analysis w as perform ed to determ ine differences in m ean weekly food consum ption between different levels an d w ithin each level of all th ree species(SAS Inst., Inc. 1989). To remove effects of body weight on th e m easu red ra te functions, all ra te functions were standard ized to a 1 g u n it body weight. Oxygen consum ption in th e two crab species w as m easured per un it ash-free d iy weight (AFDW). Different regressions were used to convert wet weight to AFDW. Respiration ra te s were m easured by a three way factorial analysis. Since oxygen consum ption ra te of different species rarely assum es either of the two ideal shapes (oxconformity or oxyregulation) th a t describe perfect conformity or regulation (Mangum and V an Winkle, 1973), m ean oxygen consum ption a t different days and w ithin each level and species w as com pared by polynomial contrasts.RESULTSR esponse to constant hupoxic exposure R esponse of Callinectes sapidus :Callinectes sapidus w as very sensitive to hypoxia (Fig. 2.1), w ith a 28 day LC50 of 106 Torr 0 2. All C. sapidus exposed to anoxia and 25 Torr 0 2 died w ithin 6 days of exposure. 100% m ortality did no t occur in any o ther hypoxic level.Feeding ra tes in Callinectes sapidus exposed to norm oxia, 119 and 73 Torr 0 2 were not significantly different from each other (Tukey’s range test) b u t were significantly different from the ra te s of crabs exposed to 50 Torr 0 2 (Fig. 2.2a). C rabs exposed to 2518Figure 2.1 LC50 of the b lue crab, Callinectes sapidus, the lesser b lue crab, C. similis, and th e oyster drill, Stram onita haem astom a, a s a function of hypoxia and anoxia a t 3 0 ° /oo S and 24°C. The shaded portions indicate the period during which m ortality rate w as too low for LC50 calculation.Torr of oxygen (LC-50)1501209 0Blue Crabt T T T T T T T T T T T T TI ^ .• —9 —9 — e —* —• —9 —9 —9 —9T/J 1 i 1 1 1 1 1 1 i i i i 160Lesser Blue Crab'(•—•" 'S ." * ' T \ /T T -i- t v * - ♦ X—♦ _L _L _LO yste r Dri0 2 4 6 8 10 12 14 16 18 2 0 2 2 2 4 2 6 28 3 0DayCD20Figure 2 .2 Callinectes sap idus and C. similis. Average feeding ra te (±SE) of crabs exposed to various levels of hypoxia for a period of 28 d. Letter’s rep resen t Tukey’s range te s t for differences betw een trea tm en t m eans. M eans with the sam e le tter are not significantly different from each other, (a) Feeding ra te of C. sap idus expressed per un it body weight, (b) M ean feeding ra te per crab in C. sapidus, (c) Feeding ra te of C. similis expressed per u n it body weight, (d) M ean feeding ra te per crab in C. similis.mg w et food .gm w et body wt -1 day ~1 mg wet food -9°° wet body wt . day .xtoro01H >01oH >H >H >OlOlCDmg w et food .crab . day . mg w et food .crab ~.1 day !—i o 3oorooou>oo*oo01oo01oo- ^ • - ^ r o t j u j j v o i o i o iC I U O O I t o t o O l t o C D O *O O I O O I O O I O O I O O I O' I 1 1 • ' ' 1 1 ■ 1 1 1 1 1 1 1 1 1 1 1 1 1aOloro •Njs ' u0a X a X X a X a hA,B119oxygenx v c S o S S S :■( >155crIZ65022and 0 Torr 0 2 were no t included in the feeding ra te analysis since 100% m ortality occurred in those two levels w ithin the first 6 days of exposure.A significant linear decline in the feeding rate w ith increasing hypoxia w as noticed w hen the feeding ra te w as m easured on a per crab basis (Fig. 2.2b). The crabs exposed to norm oxia grew faster th a n those exposed to different hypoxic levels, and the ir overall feeding ra te w as h igher though the ra te per u n it body weight rem ained unchanged (Fig. 2.2a). R epeated m easu re analysis of the weekly feeding ra te did no t show any significant tren d in the feeding ra te (Fig. 2.3a).Growth w as m easured in the blue crabs as percent increase in wet weight with respect to the initial weight of th e crab. Significant differences in the grow th of the crabs exposed to norm oxia and those exposed to hypoxia were observed (Table 2.1). Only one crab m olted in the 25 Torr 0 2. Molting rate of the crabs exposed to norm oxia w as faster th a n in th e crabs exposed to different hypoxic levels (Fig. 2.4a). Conversely, interm olt period w as sho rter in th e crabs exposed to norm oxia th a n the crabs exposed to different hypoxic levels (Table 2.1).Response of Callinectes similis :Callinectes similis exhibited w as m ore to lerant to hypoxia th a n C. sap idus with a 28 day LC50 of 43 Torr 0 2 (Fig. 2.1). All the crabs exposed to anoxic w ater died within 3 days of exposure. 100% m ortality did not occur in any o ther hypoxic levels.Feeding ra te s of Callinectes similis exposed to norm oxia (155 Torr) and 119 Torr Oa were not significantly different from each o ther (Tukey’s range test) with a m ean feeding rate of 401 and 447 mg wet food, gm wet body w t. '1 d a y '1 respectively b u t were significantly different from crabs exposed to 50 and 25 Torr 0 2 (Fig. 2.2c) with a m ean feeding ra te of 338 and 330 mg wet food, gm wet body w t. '1 day"1 respectively. C rabs exposed to anoxia were not included in the feeding ra te analysis because of 100% m ortality in th a t level w ithin 3 days.23Figure 2 .3 M ean weekly feeding rate (+SE) of (a) Callinectes sap idus an d (b) C. similis, exposed to various levels of hypoxia for a period of 28 d a t 3 0 ° /oo S and 24°C.-1mg wst food .gm wet body wt . day.W eeksb800 -----------------700 -1 2 3 4Weeks25Figure 2 .4 Molting ra te of (a) Callinectes sap idus (b) C. similis exposed to various levels of hypoxia for 28 d.No. of molts .crab . day . tslo. of molts .crab .1 day0 . 0 0 6r-i ■0 . 0 0 5 0 . 0 0 4 0 . 0 0 3 0.002 0.001 0.0000 2 5 5 0 7 3 1 1 9 1 5 5Torr of oxygen0 .0 0 3 0 0 .0 0 2 5 0.0020 0 .0 0 1 5 0.0010 0 .0 0 0 5 0.0000Torr of oxygen27A significant linear decline in the feeding ra te w ith increasing hypoxia w as noticed w hen the feeding ra te w as m easured on a per crab basis (Fig. 2.2d). The crabs exposed to norm oxia grew faster th a n those exposed to different hypoxic levels, and the ir overall feeding rate w as h igher though the rate per un it body weight w as the sam e for 155 and 119 Torr 0 2 (Fig. 2.2c). Repeated m easure analysis of the weekly feeding ra te showed a significant linear decline over time (Fig. 2.3b).Significant differences occurred in the growth ra te of Callinectes similis exposed to norm oxia (155 Torr) and those exposed to different levels of hypoxia (Table 2.1). Molting ra te in the crabs decreased linearly with the severity of hypoxia with the only exception of 119 Torr (Fig. 2.4b). The interm olt period in the crabs exposed to norm oxia w as sho rter th a n th a t of crabs exposed to hypoxia (Table 2.1).Response of SLramonita haem astom a :Stram onita haem astom a w as very res is tan t to hypoxia w ith a 28 day LC50 of 11.5 Torr Oa (Fig. 2.1). 100% m ortality occurred only u n d er total anoxia. The first death occurred after day 11, and all the oyster drills exposed to anoxia were dead by day 22.Feeding ra te s in all oyster drills exposed to hypoxic w ater were significantly different from those exposed to normoxia. Feeding ra te of Stram onita haem astom a exposed to 119 and 73 Torr 0 2 were not significantly different from each o ther (Tukey’s range test) b u t were significantly different from the di'ills exposed to 50 and 25 Torr 0 2 (Fig. 2.5a). The lowest feeding ra te w as recorded in the oyster drills exposed to anoxia. Repeated m easure analysis of feeding rate by week showed a strong in teraction with time w ithin each level (prob. < .01; Fig. 2.5b).Scope for growth in the blue crabs exposed to the two higher levels of hypoxia w as not m uch different from the normoxic scope for growth bu t, it declined linearly in theTable 2 .1. Growth and molting phenom ena in Callinectes sapidus and C. similis exposed to 28 days of hypoxia a t 3 0 ° /^ S and 24°C. C rabs exposed to 0 Torr 0 2 died w ithin a few days and did not m olt or grow. 16 crabs were used a t each P 0 2 for each species. SAP = Callinectes sapidus, SIM = Callinectes similis.Torr of 0 2155 115 73 50 25SAP SIM SAP SIM SAP SIM SAP SIM SAP SIMAverage 686 735 713 691 651 737 855 867 780initial wet wt (mg)±43 ±71 ±58 ±68 ±36 ±47 ±90 ±39 ±69% Increase in growth501 186 305 113 246 274 124 43 137Intermolt period (days)12.4 13.6 14 7.8 14.5 15 17.4 - 18.1Crabs molted 3 times2 0 0 0 0 0 0 - 0Crabs molted twice13 2 3 0 4 2 2 0 1C rabs molted once1 14 10 4 4 5 4 1 6Total molts 33 18 16 4 12 9 8 1 8hO0029Figure 2 .5 Stram onita haem astom a. (a) Average feeding ra te (±SE) per u n it dry body w eight as a function of hypoxia. Letters rep resen t Tukey’s range te s t for differences betw een trea tm en t m eans. M eans w ith the sam e letter are not significantly different from each other, (b) M ean weekly feeding ra te (±SE) as a function of hypoxia.mg dry food .gm dry body wt . day .o o o o o o o o o o o o1o cnUlHo (01 1H S 0 COH OlO O —1 N> o cn120Torr of oxygen- 1 - 1mg dry food .gm dry body wt . day .00o31oyster drills with the severity of hypoxia from 92% in 119 Torr 0 2 to 64% in 25 Torr 0 2 (Table 2.2).R esponse to direct transfer from constant hupoxic exposure to normoxiaFeeding ra te s in the crabs exposed to various levels of hypoxia were not significantly different from those exposed to norm oxia during the p re-transfer period. Feeding ra te of blue crabs transferred directly from various hypoxic levels to normoxia increased significantly (P < .01) during the post-transfer period com pared to the ir feeding ra te w hen exposed to hypoxia (pre-transfer period, Fig. 2.6a). D uring the pre-transfer period molting rate w as faster in the crabs exposed to norm oxia com pared to those exposed to various hypoxic levels. But, during the post-transfer period m olting rate was m uch faste r in the crabs previously exposed to hypoxia th a n in those exposed to norm oxia (Fig. 2.6b).R esponse o f Callinectes sap idus to starvationAfter a period of 10 days starvation, feeding rate of the b lue crabs increased sharp ly above the pre-starvation feeding rate. The m ean increase over the pre-starvation feeding rate w as 78 + 7 mg wet food, gm wet body w t. '1 d ay .'1 which equals to 123% increase and w as found to be highly significant (studen t’s t-test, P < .01). This huge increase is a ttribu tab le to the first two days of the 10 day post-starvation period (Fig. 2.6c). However, th is elevated feeding rate of the blue crabs during the 10 days poststarvation period accounted for only 31% of the calculated deficit incurred during the starvation period.Table 2 .2 . Scope for growth of Callinectes sapidus , and Stramonita haem astom a, u n d er normoxic, different levels of hypoxic, and anoxic conditions. All energy budgets are given in Kilo Jou les, gm dry body w t.'1 day .'1Callinectes sap idusOxygen C oncentration (Torr)N EnergyConsum ed(C)% of Energy normoxic Absorbed (Ab)EnergyLost(R)% o fnormoxicScopeforgrowth (P)% o fnormoxic155 16 6.15 100 2.74 0.44 100 2.3 100119 2 6.5 106 2.89 0.35 80 2.54 11073 6 6.2 101 2.76 0.23 52 2.53 11050 3 4.53 74 2.02 0.17 39 1.9 83Stram onita haem astom a155 12 1.19 100 0.97 0.21 100 0.76 100119 12 1.07 90 0.87 0.17 81 0.7 9273 12 1.04 88 0.85 0.11 52 0.74 9750 12 0.89 75 0.72 0.08 38 0.64 8425 12 0.67 56 0.54 0.05 24 0.49 64GO33Figure 2 .6 Callinectes sapidus. (a)Increase in feeding ra te during the post-transfer period in the transfer experim ent w hen m ain tained a t 3 levels of hypoxia for 10 d and th e n transferred to norm oxia for 10 d. Increase is show n by the m ean difference (±SE) in feeding ra te before and after transfer to norm oxia and plotted on the Y-axis against various oxygen concentrations on the X-axis. Letters rep resen t Tukey’s range test. M eans w ith th e sam e le tter are no t significantly different from each other.(b) Molting ra te during the transfer experim ent. Dotted b ars rep resen t the p re-transfer period and ha tched b a rs rep resen t the post-transfer period.(c) M ean daily feeding ra te during th e starvation experim ent. A clear and sh arp increase in the feeding ra te can be noticed after day 15 during the post-starvation period.Increase (mg wet food .yn wet body wt . day200180160140BA120100BA8060402050 73 119 155>. 1 indicate a higher level of oxygen consum ption and values < 1 indicate a possible oxygen debt (Fig. 2.7c). M ean oxygen consum ption in C. sapidus exposed to various hypoxic levels w as less th a n the crabs exposed to normoxic w ater. The acute response of C. similis w as sim ilar to C. sap idus on day 0 bu t, the oxygen consum ption rate in C. similis exposed to hypoxia rem ained elevated for the re s t of the 28 day period. Significant (P < .01) differences in the oxygen consum ption rate between levels as well as a significant day by level in teraction w as found in both C. sapidus and36Figure 2 .7 M ean daily resp iration (±SE) a t various levels of hypoxia of (a) Callinectes sap idus and (b) C. similis. (c) Ratio of m ean hypoxic oxygen consum ption of the 2 crab species w ith in a level over 28 d to m ean normoxic oxygen consum ption over 28 d plotted against various oxygen concentrations. Normoxic oxygen consum ption ra te w as fixed a t 1 an d is represented by the heavy line. A ratio of g reater th a n 1 indicates a h igher level of oxygen consum ption com pared to th e normoxic consum ption and a ratio of less th a n 1 indicates a possible oxygen debt.ul. oxygen . hr . gm AFDW37(a) Callinectes sapidus200015001000Callinectes similis7000600040003000200010002 5 T o r r5 0 Torr1 15 Torr11 5 5 TorroroCC2Callinectes similis1Callinectes sapidus00 23 6946 92 1 15Torr of Oxygen38C. similis (Fig.2.7b). M ean oxygen consum ption over 28 days in all hypoxic levels w as > 1 com pared to the normoxic consum ption in C. similis. Three way factorial analysis showed significant dilferences (P < .001) in the oxygen consum ption ra te of the two species of crabs. All the lesser b lue crabs exposed to hypoxia consum ed more oxygen on a n average com pared to the b lue crabs exposed to the sam e levels of hypoxia.DISCUSSIONCallinectes sapidus w as very sensitive to hypoxia with a 28 day LC50 of 106 Torr Oa, C. similis w as able to to lerate m oderate hypoxia w ith a 28 day LC50 of 43 Torr 0 2. and Stramonita haem astom a w as very res is tan t to hypoxia w ith a 28 day LC50 of 11.5 Torr 0 2. Stickle et al. (1989) found th e 28 day LC50 value for C. sap idus to be 111 Torr and for S. haem astom a to be 19 Torr 0 2. Kapper and Stickle (1987) also reported S. haem astom a to be extremely to le ran t of mild hypoxia. From the above d a ta it seem s clear th a t juvenile C. similis can tolerate hypoxia better th a n juvenile C. sapidus. These two species, especially the juvenile stages, com pete for food and o ther resources m ost often (Engel 1977). D eFur e t a l (1990) qualitatively dem onstrated only 20% death of ad u lt C. sap idus a t m oderate (50 Torr) hypoxia during 25 days exposure. Findings of th is study are different for th e juvenile blue crabs. 81% m ortality w as observed a t 50 Torr over the 28 day period. All the crabs m ain tained a t 25 and zero Torr 0 2 died w ithin a short period. This m ay be due to the higher oxygen dem and per u n it body m a ss in the juvenile b lue crabs.The feeding ra te of ne ither Callinectes sapidus no r C. sim ilis varied directly with the severity of hypoxia. Feeding ra te of C. sap idus exposed to 50 Torr 0 2 w as 74% of the norm oxic feeding rate. Feeding ra te of C. similis exposed to 50 and 25 Torr 0 2 was 84 and 82% of the normoxic feeding ra te respectively. Metabolic ra te s of both species of39crabs are no t depressed in a com parable m anner to th a t of the oyster drills and th u s the ir m etabolic dem and for Q2, w hen exposed to m oderate hypoxia, rem ains close to the norm oxic level and they die quickly w hen exposed to anoxia (Stickle e t al. 1989). Blue crabs are no t veiy good anaerobes (C arpenter and Cargo 1957) and they are more dependent on the ir 0 2 tran sp o rt system th a n m ost o ther groups of anim als. Limited anaerobic m etabolism exists in c ru stacean s involving pathw ays alternative to classical glycolysis w ith the production of L-lactate during exuviation and brief exposure to hypoxia (Fields 1985 and M angum e ta l. 1985).Weekly weight specific feeding ra te of both species of c rabs exhibited a steady decline over time w ith a steeper decline in C. similis. Since th is effect w as consisten t for hypoxic a s well a s normoxic crabs, the decline in feeding rate m ay not be due to hypoxic exposure, ra th e r it m ay be due to increased body size or prolonged captivity. B u t the feeding ra te per un it body weight w as always lower in crabs exposed to hypoxia th a n those exposed to normoxia.The im portance of glucose in m etabolic processes h a s been widely recognized. B ecause aerobic m etabolic rate decreases in hypoxia-sensitive cells u n d er oxygen limiting conditions, the dem and for glucose for anaerobic glycolysis m ay rise dram atically to com pensate for the energetic shortfall (H ochachka 1986). Glucose concentration in the blood depends on th e ra te of m etabolism . The m ain source of m etabolic energy in the cru stacean s are glucose derivatives (Poolsangunan and Uglow 1974, Ram os and Fernandez 1981). Oxygen consum ed by c ru staceans is used as th e la st acceptor of electrons in th e respiratory process which usually begins w ith th e degradation of carbohydrates (Rosas e t al. 1992). T hus the feeding ra te seem s to be intim ately related to the oxygen consum ption. I found in both species of crabs the m etabolic ra te rem ains a t a veiy high level w hen exposed to various levels of hypoxia to ca ter to the g reater need40for glucose in the hypoxia sensitive cells. This creates a h igher oxygen dem and w hen the am bient oxygen tension is low.Molting ra te in both species of crabs w as considerably h igher for crabs exposed to norm oxia (155 Torr 0 2) th a n those exposed to hypoxia though th e overall molting rate of Callinectes similis w as m uch lower th a n C. sapidus. F requent m ortality while m olting w as observed in C. sap idus exposed to hypoxia. This w as no t observed in C. similis. M angum e t a l (1985) showed th a t the hem ocyanin in the hem olym ph of a blue crab tran sp o rts m ore th a n 95% of its 0 2 requirem ents and alm ost none is carried in the free form. They also reported th a t th is system does not m eet respiratory dem and during m olting w hen the m etabolic dem and is g reater th a n usual. Due to increased up take of w ater during molting, the hem ocyanin in the blood is diluted to abou t one-fifth of the interm olt concentration which in tu rn reduces the 0 2 carrying capacity of the blood. This reduction in oxygen delivery is com pounded by hypoxia an d re su lts in increased m ortality in C. sapidus exposed to hypoxia while molting. In C. sim ilis we failed to observe death during m olting w hich probably suggests either an evolutionary adap tation to lengthen the interm olt period w hen exposed to hypoxia, possibly to avoid ex tra s tress during an already existing environm ental stressor, or a slower growth rate com pared to C. sapidus. Glucose is necessary in the chitin syn thesis upon m olting (Rosas et al. 1992). Increasing the interm olt period and th u s reducing th e m olting frequency is indeed an optim al adaptive strategy to reduce s tress and th u s the need for more glucose for chitin syn thesis in C. similis. B ecause of th is high dem and for glucose, feeding ra te did not decline directly w ith th e severity of hypoxia in C. sapidus and th e ra te of oxygen consum ption rem ained quite high.Feeding ra te in the oyster drills Stramonita haem astom a varied directly with the severity of hypoxia. S uppression of the m etabolic ra te of S. haem astom a a t low oxygen tension m ay be responsible for the linear decline in their feeding ra te with declining41oxygen tension of the w ater (Stickle et al. 1989). The heat dissipation ra te in S. haem astom a h a s been reported to be very low, only 9% of norm oxia, w hen exposed to anoxia and oyster drills exhibit an oxygen debt upon re tu rn to norm oxic w ater (Stickle e t al. 1989). Metabolic rate depression in m ollusks is possible because, instead of the classical and less efficient glycolysis system , they switch to relatively m ore efficient succina te and propionate pathw ays during exposure to hypoxia and anoxia (Gade 1983, G naiger 1983a, 1987, deZwaan and Thillart 1985).Weekly food consum ption of the oyster drills w ithin each trea tm en t level showed a strong in teraction with time. O yster drills exposed to various trea tm en t levels exhibited an increase in food consum ption during the second week of th e experim ent (oyster drills exposed to 50 Torr 0 2 also exhibited increase during the th ird week); the ra te of food consum ption th en gradually declined over time in all levels. The initial shock of transfer from norm oxia to hypoxia w as probably responsible for the very low feeding ra te during the first week after which they becam e acclim ated and consum ed m ore food. B ut with prolonged exposure the ir feeding ra te could no t be m ain tained and th e ra te of food consum ption declined.An estim ate of th e scope for growth under norm oxia and different levels of hypoxia and anoxia in Callinectes sapidus and Stramonita haem astom a show s no significant difference in the scope for growth a t the two higher levels of hypoxia in C. sapidus. But in S. haem astom a a linear decline in the scope for growth occurs with the severity of hypoxia. S. haem astom a h as a lower feeding and m etabolic ra te th a t resu lts in a lower scope for growth which appears to be an adaptive advantage. W hen oyster drills are exposed to lower levels of dissolved oxygen in the w ater they restric t the ir metabolic dem ands for m aintenance. As is true with exposure of m arine invertebrate carnivores to o ther environm ental s tresso r gradien ts (Stickle 1985), variation in energy consum ption is m uch greater th a n variation in m etabolic rate; 6; 1 in juvenile C. sapidus and 3.3:1 in42S. haem astom a. After a lower threshold limit of m etabolic ra te is reached in S. haem astom a a t interm ediate w ater tem perature (24°C), th is m etabolic ra te reduction becom es futile a s w hen oyster drills were exposed to anoxic w ater.C rustaceans com pensate for sho rt term hypoxia by increasing ventilation (B atterton and Cam eron 1978, Taylor 1982, Pease and D eFur 1987), increased extraction efficiency (H agerm an and Uglow 1985), a n d /o r oxygen affinity of the hem ocyanin (H agerm an and Uglow 1985, D eFur eta l. 1990). An increase in oxygen consum ption w as observed a t all hypoxic levels in Callinectes similis a t least by day 1 com pared to the norm oxic crabs. Oxygen consum ption rem ained a t th is elevated ra te for the res t of the 28 day hypoxic exposure, which dem onstrates a clear over com pensation in th is species. In C. sapidus, oxygen consum ption in the hypoxic crabs varied w ithin a narrow limit around the normoxic consum ption showing a partial com pensatory acclim ation over the 28 days hypoxic exposure. The increased oxygen consum ption in C. similis m ay be due to both increased ventilation and an increased extraction efficiency. Sanchez eta l. (1991) dem onstrated higher extraction efficiency in C. similis per un it of metabolically active biom ass am ong six different c ru stacean species studied.In decapod crustaceans, hem ocyanin in the blood is responsible for a veiy large fraction of th e oxygen supplied to metabolizing tissue. The tran sp o rt m echanism is pertu rbed during and im m ediately following a molt (Mangum et al. 1985). During the p rem olt period a decrease in blood calcium , which acts as an allosteric effector m ay increase oxygen affinity of hem ocyanin, th a t accom panies hardening of the new shell lowers the hem ocyanin oxygen affinity (Mangum et al. 1985). T hus an increased supply of oxygen is necessary a t th is stage of the m olt cycle. In our experim ent u n d er hypoxic exposure, oxygen consum ption in Callinectes sapidus showed no increase w ith extreme of hypoxia. Average oxygen consum ption w as lower th a n normoxic consum ption, b u t the feeding rate rem ained unchanged. Thus, the metabolic dem and during m olting w as higher and the43supply of oxygen w as low. This probably w as the m ain cause of death during molting in C. sapidus. In C. similis the opposite is true. They m ain tained a higher ra te of oxygen consum ption th roughou t the hypoxic exposure and the frequency of molting w as less com pared to C. sapidus. The ex tra am oun t of oxygen w as probably used for m etabolic m ain tenance, increasing the likelihood of survival.In the direct transfer experim ent no significant difference in the ra te of food consum ption existed w ith hypoxia over 10 days though the hypoxic crabs always exhibited lower food consum ption ra te s com pared with the crabs exposed to normoxia. B ut w hen the crabs were transferred back to norm oxia (post-transfer period), the food consum ption ra te of the crabs previously exposed to hypoxia increased sharply. It is probable th a t the ra te of adap ta tion from hypoxia to norm oxia is m uch faster th a n from norm oxia to hypoxia since th e feeding adap tations in the b lue crabs associated with hypoxic exposure seem to be very minor. The starvation experim ent created a sim ilar type of response as w as noticed after the transfer of the crabs from hypoxic to normoxic w ater in th e tran sfe r experim ent. The food consum ption ra te increased very sharp ly and stayed a t th a t elevated level.Tolerance to hypoxia is possibly also related to the developm ental history of the b lue crab. Complete acclim ation to hypoxia h as not been reported for either of the two species in the laboratory. B ut partia l acclim ation to frequently occurring patchy hypoxia in th e Gulf of Mexico m ay be possible. O ccurrence of w idespread hypoxia is a regular phenom enon in the no rthern G ulf of Mexico during the sum m er m onths. C rustaceans can avoid hypoxia by swimming away from deeper to shallow er w ater (Loesch 1960, May 1973, Pavela et al. 1983, R enaud 1986b and Pihl et al. 1991), b u t some exposure to hypoxia m ay be unavoidable w hen the hypoxic w ater m ass is veiy wide spread. It is reasonable th a t some acclim ation occurs in blue crabs and the lesser b lue crabs to these fi'equently occurring events which probably helps them to res is t sh o rt term hypoxia.44D epending on th e acclim ation h istory of the crab population, hypoxia to lerance will vary an d so will o ther responses related to it.CHAPTER 3DETECTION AND AVOIDANCE OF HYPOXIC WATER BY JUVENILE CALLINECTES SAPIDUS AND C. SIMILIS45INTRODUCTION46M ass m ortality of m arine and estuarine benthic organism s due to hypoxia has been widely reported (Santos and Sim on 1980a, b, H arper e t al. 1981, R abalais et al. 1985, R abalais and H arper 1991b, 1992a). Commercial crab-pot fisherm en have frequently reported dead blue crabs in pots (Carpenter an d Cargo 1957, T atum 1979, V an Engel 1982) which were probably subjected to low dissolved oxygen in the w ater. The occurrence of hypoxic bottom w ater off the Louisiana coast w est of the M ississippi Delta is a com m on and recu rren t (Bedinger et a l 1981, S tun tz et a l 1982, Boesch 1983, Leming and S tun tz 1984, R enaud 1985, 1986a, Rabalais et al. 1986a, b) phenom enon locally know n as th e occurrence of dead w ater (Turner and Allen 1982, Boesch 1983, R enaud 1985, 1986a). Hypoxic w ater m asses m ay persist for w eeks and are usually associated with stratification of the w ater column, phytoplankton blooms, and stable high p ressu re w eather system s (Ragan et al. 1978, Bedinger e t a l 1981, H arper et a l 1981, Boesch 1983, R enaud 1985). Reports suggest th a t fish and cru staceans can avoid hypoxic w aters (Loesch 1960, May 1973, Garlo el a l 1979, Pavela et a l 1983, Officer et a l 1984, Pihl et a l 1991). Juven iles of two species of shrim p, Penaeus aztecus and P. setiferus are capable of detecting and avoiding hypoxic w ater (Renaud 1986a) as can the san d goby, Pom atoschistus m inutus (Petersen and Petersen 1990). Pihl et a l (1991) reported a general m igration of cru staceans from deeper to shallow er w ater (including the ad u lt b lue crab, Callinectes sapidus) in association with hypoxia .The effect of hypoxia on the m ovem ent p a tte rn s of the blue crab, Callinectes sap idus R ath b u n and the lesser b lue crab, C. similis Williams, to and from the estuarine region is unknow n. M ature female C. sapidus hatch the ir eggs in high salinity w ater (Millikin and Williams 1984). Egg-bearing female C. sap idus m ay occur and spaw n in the coastal Gulf and estuarine w aters year-round (G unter 1950, D augherty 1952, More 1969,47A dkins 1972a, Periy 1975). Egg-bearing C. similis also spaw n in high salinity w aters (Hsueh et a l in press). S tuck and Perry (1981) reported a year-round abundance of C. similis m agalopae in the w aters of M ississippi Sound w ith a peak during February and M arch. T hus, an extensive occurrence of hypoxia in nearshore and offshore w ater could cause a potential problem for the spaw ning female population of b lue crabs as well as th e larval and juvenile stages. Low dissolved oxygen m ay also ac t as a barrie r to the m igration of juvenile b lue crabs from high salinity to low salinity w aters (Van Engel 1982). Juvenile blue crabs are estuarine-dependent and regularly utilize the m arsh creek hab ita t (Orth and van M ontirans 1987, Williams e t al. 1990, van M ontfrans e l al. 1991, McClintock et al. 1993). D iurnal variation in the dissolved oxygen tension of w ater in the m arsh es of the Louisiana Gulf coast is frequent, w ith w ater becom ing alm ost anoxic during the early m orning period in sum m er (Das and Stickle, unpublished observation). We th in k sho rt duration of hypoxic exposure m ay also s tress the blue crabs and the lesser blue crabs and m ay be equally im portan t to the ir d istribution and survival.The objectives of th is study are (i) to determ ine w hether juvenile C. sap idus and C. similis can detect and avoid specific levels of dissolved oxygen u n d er laboratory conditions, (ii) to observe the behavioral responses associated w ith sho rt term hypoxic exposure and (iii) to consider how th e behavioral responses m ay affect field populations of the two species of blue crabs.MATERIALS AND METHODSCollection arid m aintenanceCallinectes sapidus and C. similis were collected nearshore a t Port Fourchon, Louisiana (90°11.4W , 29°6.7’N) between Ju n e and Septem ber, 1992, w hen the w ater tem peratu re w as 28 - 30°C and the salinity w as 28 ° /00. The anim als were brought back48to the laboratory and placed Into artificial sea w ater m ade from In stan t Ocean® a t room tem peratu re (23°C) and salinity m atching field conditions. All the crabs were kept under co n stan t illum ination and acclim ated to the laboratory conditions for two weeks before being used in the experim ents. C rabs were individually isolated in cham bers m ade from a pair of PVC pipe sections th a t fit together (one piece with 21 cm and the o ther piece w ith 23 cm inside diam eter and both 28 cm in length) w ith nylon m eshed screens on the free end of each piece to prevent cannibalism . Anim als were chosen to minimize weight an d size differences w ithin an d am ong the trea tm en ts (average weight 750 - 850 mg; average carapace w idth 20 - 25 mm). C rabs were fed frozen abdom inal portions of the g rass shrim p Palaemonetes pugio. Avoidance w as no t analyzed with respect to sex because it is difficult to sex th e juvenile blue crabs unequivocally.Experimental design and procedureThe experim ent w as conducted in an avoidance ap p ara tu s modified from Renaud (1986b; Fig. 3.1), consisting of a glass cham ber 100 cm long and 10 cm in diam eter m arked a t 1 cm intervals. A raised floor of tran sp a ren t white plastic m esh w as placed across the cen ter of the cham ber to facilitate m ovem ent of the crabs w ithin the tube. There were two w ater inlets, one a t each end of the tube, and a centrally located outlet a t th e top of the cham ber. Two anim al entry po rts a t each end of the cham ber n ear the inlet tubes, allowed in troduction of the crabs into the cham ber. The inlets were connected to both the control and experim ental w ater ta n k s and a gravity flow system controlled w ater flow into the cham ber. W ater flow from either w ater source could be directed to either end of the cham ber w ith in-line valves. Two flow m eters, one on each side, were installed before the inlets were connected to the cham ber to m ain tain a co n stan t flow rate. W ater from th e outlet w as controlled by a valve and th u s required flow ra te s could be achieved by altering the outflow. The outlet opened into a bio-filter from49Figure 3 .1 Schem atic d iagram of the avoidance behavior ap p ara tu s and the experim ental setup . Figures are no t draw n according to scale.To Gas MixerIntermediateSaturationTank (Hypoxic Water)ControlWaterTankHypoxic Water TankInlet Valves | g j ^ Inlet ValvesFlow MeterAnimal Entry PortTest Chamber Outlet100 cmNylon Mesh PlatformSampling Ports h§l*» — Outlet ValveTo Gas MixerAirControlWaterTankHypoxicWaterTankBiofilter51which w ater w as pum ped back to the control and experim ental tanks. There were nine sam pling ports located along the length of the ap p a ra tu s for m onitoring dissolved oxygen tension. W ater sam ples from th e sam pling ports were draw n anaerobically and injected into a S trathkelvin oxygen m eter (model 781) connected to a flow -through w ater jacketed oxygen electrode.Target oxygen tensions of 90, 75, 50, 25, and 0 Torr (60%, 50%, 35%, 16%, and 0% of a ir sa tu ra tio n or 4.2, 3.5, 2.3, 1.2, and 0 ppm of dissolved oxygen) were created and m ain tained by mixing bottled nitrogen and oxygen with a M atheson gas m ixer (model 7402T) along w ith 0.03% carbon dioxide to m ain ta in the pH a t approxim ately 7.8. A total of 76 crabs (41 Callinectes sap idus and 35 C. similis) of both species were tested a t various POas. B ecause of technical difficulties, only C. sap idus w as exposed to the 75 Torr oxygen tension. Each crab w as used only once in a n experim ent.C rabs were initially in troduced from one end of the avoidance cham ber and allowed to acclim ate inside the cham ber for one hou r while normoxic w ater (155 Torr at 23°C and 30 ° / GO S) flowed th rough the cham ber from both ends. E n tiy of the crabs into th e cham ber, either through th e right or left anim al en tiy port, w as sw itched for each experim ent to avoid any possibility of starting preference. Following one h o u r of acclim ation, crabs were allowed to stay inside the cham ber for ano ther 30 min, which served a s a control period. The position of the crabs w ith in the cham ber during the control period w as noted every 3 min. Upon com pletion of the control period, trea tm ent w ater of desired dissolved oxygen tension w as in troduced into th e end of the cham ber n eare st to the crab while normoxic w ater flow w as m ain tained from the o ther end of the cham ber. This created an oxygen gradient inside the cham ber so th a t the side of hypoxic w ater entry w as m ost hypoxic and oxygen tension gradually increased tow ards the m iddle of the cham ber, while the o ther side w as completely normoxic. The middle region of the cham ber (50 cm mark) served as a hypothetical boundary line betw een the hypoxic52and norm oxic (control) zone. This boundary line w as no t very sharp ly dem arcated and there w as always some mixing of hypoxic and norm oxic w ater in th is region. The oxygen grad ien t w as m easured for each experim ent. The te s t period continued for 60 m in and th e position of the crab w ithin the cham ber w as noted every m inu te along w ith their behavioral responses. Behavior w as observed th rough slits in cu rta in to prevent outside m ovem ents and shadow s from influencing crab behavior. Lam inar w ater flow, as determ ined with dye tests , of 450 ± 50 m l/m in w as m ain tained by the inline valves. Alter every experim ent the cham ber w as flushed with normoxic water.The following response variables were m easured for each crab:1) Total Tim e - The am oun t of time spen t by each crab a t the hypoxic end of the cham ber during the final 50 m in of each 60 m in treatm ent. The first 10 m in of each experim ent were allowed to estab lish a stable oxygen gradien t inside the cham ber. M ovement of crabs inside the cham ber w as m onitored constantly to m easure the am ount of tim e spen t by each crab a t the hypoxic end of the cham ber.2) R esponse Tim e - The time in m inu tes before a c rab ’s initial entry into the control (normoxic) side of the cham ber after the introduction of hypoxic water.3) L ocation - Location w as scaled from 0 to 100, based on the side of hypoxic w ater en try being 0, for the position of each crab w ithin the cham ber.4) A ctiv ity Index - Activity index w as calculated based on the total d istance (cm) travelled by a crab during the final 50 m in of the 60 m in experim ental period. This index w as calculated for each crab w ithin each trea tm en t and the m ean activities a t various oxygen tensions were com pared.T ests for significant differences between trea tm en ts for the above four response variables were determ ined by either one-way or two-way analysis of variance (ANOVA; SAS Inst., Inc. 1989) and split plot design. Levene’s te s t was used to determ ine the hom ogeneity of variances w ithin trea tm ents. All analysis were performed on both53untransform ed and log transform ed data. Differences am ong trea tm en t m eans were determ ined by W aller-D uncan m ultiple range test.5) P re fe rred O xygen T en sio n - Oxygen tension m ost preferred by a crab or the oxygen tension a t which a crab sp en t m ost of its time w as m easured. Movement of crabs w as m onitored constan tly to m easure the am oun t of tim e sp en t by each crab a t the hypoxic or norm oxic (control) side of the cham ber. A ctual oxygen tensions were m easured from th e 9 sam pling ports located along the length of the avoidance cham ber for each experim ent and a predicted oxygen value w as generated from the actual gradient for each location of a crab used in a particu la r experim ent. The ac tu a l oxygen gradient in the cham ber followed a sigmoidal p a tte rn (See results). The sigmoidal function w as m ade linear using a n a tu ra l log transform ation:Y, = In [oxyj + 1 / 155 - (oxy4 + 1)] w here, oxy = ac tua l oxygen tension m easurem en t from a particu la r sam pling port, and 155 is the asym ptotic normoxic oxygen tension a t 23°C tem peratu re and 30 ° / HO S. A value of 1 w as added to each oxygen value because the n a tu ra l log of "0" does not exist. A simple linear regression w as th en ru n between these transform ed oxygen values (Yj) and the actual locations obtained for each crab during the final 50 m in of a 60 m in experim ental period using PROC REG (SAS Inst., Inc., 1989) to obtain predicted oxygen values for each location of a crab. The linear regression m odel used is as follows:Yj = a + (3 * location j + Ej w here, Ys = transform ed oxygen tension, a = estim ated intercept, (3 = estim ated slope, Locatiorij = observed location in cm for each crab, and e, = random error term for each crab. The average R2 value, which indicates the proportion of variability observed in Ys th a t is explained by the simple linear regression model, w as 0 .8847 (n = 76) indicating a good fit of the d a ta to the linear model. The n a tu ra l log of these transform ed values w ere th e n back transform ed into actual oxygen values. A m ean preferred oxygen value.54the oxygen tension a t which a crab spen t m ost of its time, w as calculated for each crab from these transform ed oxygen values.Percent avoidance of th e hypoxic side w as also used to calculate avoidance for each crab w ithin each trea tm ent. Percent avoidance w as calculated based on a crab’s position w ithin th e avoidance cham ber with respect to the 50 cm m ark , ie . hypoxic side or norm oxic (control) side. Significant percent avoidance w as determ ined with analysis of variance.O ur null hypothesis w as th a t blue crabs cannot detect hypoxia, and will spend abou t th e sam e am oun t of time a t the control and trea tm en t side of the cham ber based on the assum ption th a t there is no side preference. The alternative hypothesis w as th a t b lue crabs can detect and avoid hypoxia and will spend m ore tim e a t th e control side of the cham ber through avoidance of hypoxic water.RESULTSThe average positions of Callinectes sapidus and C. similis were equally d istribu ted between the left and right side of the cham ber during the control periods, indicating th a t there w as no preference for either side (P < 0.7). Since a crab w as capable of travelling the entire d istance w ithin the cham ber several tim es in one m inute, the position of a crab w ithin the cham ber a t a particu la r in s tan t w as no t dependent on its previous position. Therefore, crab behavior during the trea tm en ts w as only affected by changes in dissolved oxygen concentration. Fig. 3.2 show s the typical avoidance and n on avoidance responses of two crabs w ithin the cham ber during two different experim ents, one a t a low P 0 2 (0 Torr, Fig. 3.2a) and ano ther a t a higher P 0 2 (90 Torr, Fig. 3.2b).W hen average crab position, trea tm en t versus control side of th e cham ber, was used to deteim ine avoidance, Callinectes similis w as found to detect and avoid hypoxia55Figure 3 .2 (a) A typical avoidance response of Callinectes similis w hen the dissolvedoxygen level w as very low. The crab spen t m ost of its time a t the normoxic end of the cham ber, (b) A typical non-avoidance response of C. sap idus w hen the dissolved oxygen level w as higher. The crab m ovem ent h a s no detectable p a tte rn an d is fairly random .Location (cm) Location (cm)56Control T reatment1009 0807060504 030201001 16 31 36 41 46 51 56 61 66 71T im e (m in u te )C ontro l T rea tm ent1009 080706050403020101 16 31 36 41 46 51 56 61 6 6 71 76 86T im e (m in u te )57a t zero (72 ± 2.2 percent avoidance) and 25 Torr oxygen (70 ± 4.4 percen t avoidance) tension (P < 0.004). No significant avoidance w as detected a t 50 Torr (57 ± 5 . 1 percent avoidance) and 90 Torr (52 ± 3 . 9 percent avoidance) oxygen tension in C. similis. Avoidance w as no t detected in any trea tm en t (62 ± 2.3 percen t avoidance a t 0 Torr ; 49 ± 7.3 percen t avoidance a t 25 Torr ; 60 ± 6.3 percent avoidance a t 50 Torr ; 52 ± 6.4 percen t avoidance a t 75 Torr ; an d 46 ± 7.5 percent avoidance a t 90 Torr) in C. sapidus.A linear decline in response time with increasing oxygen tension w as observed in C. similis (Fig. 3.3). Response tim e for C. similis exposed to 0 and 25 Torr oxygen w as significantly (P < 0.05) longer th a n the response tim e of crabs exposed to 50 and 90 Torr oxygen (W aller-Duncan m ultiple range test). No definite trend w as noticed in C. sapidus (Fig. 3.3). Response time of C. sap idus exposed to 75 Torr oxygen tension w as shorter them all o ther trea tm ents. This sudden drop can not be explained.A significant (P < 0.001) linear increase in total tim e, the am oun t of time spent a t the hypoxic end, with increasing oxygen tension w as observed in C. similis (Fig. 3.4). The am oun t of time spen t a t th e hypoxic end by C. similis exposed to 0, 25, and 50 Torr oxygen tension w as significantly less th a n the ones exposed to 90 Torr oxygen tension (W aller-Duncan m ultiple range test). No significant trend w as noticed in the am ount of tim e spen t a t the hypoxic end by C. sapidus (Fig. 3.4).Both species of crabs were found to be m ore active a t the higher oxygen tensions (75 and 90 Torr) and were significantly different (P < 0.005) from the m ore hypoxic levels (0, 25, and 50 Torr) where the crabs were found to be m uch less active (W aller-Duncan m ultiple range test; Fig. 3.5).The preferred oxygen tension for both species increased w ith increasing oxygen tension (Table 3.6). The m ean oxygen gradient a t each trea tm en t and the preferred oxygen level of the two species of crabs a t those trea tm en ts are plotted in Fig. 3.2. Mean preferred oxygen tension for C. similis ranged from 82 - 121 Torr with a m ean of 108 To it58Figure 3 .3 Callinectes sapidus and C. similis. Response tim e (±SE), the tim e in m inu tes before a crab ’s initial entry into th e control side of the cham ber after the in troduction of hypoxic w ater plotted against various levels of oxygen tension. Letter’s rep resen t Waller- D uncan m ultiple range te s t for differences betw een trea tm en t m eans.Response Time (minutes)2 0181614121086420AC. sapidusAA/4AC. sim ilisAAB0 25 50 75 90Oxygen Tension (torr)0 25 50 90CJlCO60Figure 3 .4 Callinectes sap idus and C. similis. Total time (±SE), the am oun t of time sp en t by each crab a t the trea tm en t side of the cham ber plotted against various levels of oxygen tension. Letter’s rep resen t W aller-duncan m ultiple range te s t for differences betw een trea tm en t m eans.(f)CD4-115C0I -C0oI—403530252015100C. sapidusAA/I0 25 50 75C. sim ilisA90Oxygen TensionA0 25 50 9062Figure 3 .5 Callinectes sap idus and C. similis. Activity (±SE), a n index of the d istance (cm) travelled by a crab during th e final 50 m in u tes of a 60 m inu te experimented period, plotted against various levels of oxygen tension. Letter’s rep resen t W aller-D uncan m ultiple range te s t for differences betw een trea tm en t m eans.cE"D00>cou>-M’>+->o<35302520151050C. sapidusBBX0 25 50 75C. sim ilis90 0 25 50 90Oxygen Tension (torr)64oxygen. M ean preferred oxygen tension for C. sapidus ranged from 98 - 125 Torr with a m ean of 112 Torr oxygen.Noted behavioral responses of both crab species during the trea tm en ts were: (1) an initial increase in the ir activity with the in troduction of hypoxic w ater into the cham ber. This initial b u rs t in activity w as m ore prom inent in C. sap idus com pared to C. sim ilis, (2) frequent m ovem ent of the eye-stalk, (3) avoidance of hypoxic zone by slowly crawling out of the area (when avoidance w as present), (4) restless and erratic m ovem ents showing clear signs of escape behavior, (5) rapid m ovem ent of the ir antennae. These behaviors were a clear sign of s tress due to low oxygen tension in the w ater. These behaviors were no t p resen t a t th e 90 Torr oxygen tension in either species.DISCUSSIONWe have used different response variables in th is s tudy to analyze avoidance. Justifica tion for using these variables is to avoid the lim itation im posed on the experim ent by a con tinuous oxygen gradient. We found th a t none of the three variables, location, response time, and to tal time can be used as a robust m easure of avoidance because of the n a tu re of the oxygen gradient w ithin the cham ber as d iscussed below.Calculation of the average crab position (percent avoidance), trea tm en t versus control side of the cham ber showed clear avoidance of hypoxic w ater by CaU.inecl.es similis a t 0 and 25 Torr oxygen tension. Significant avoidance w as not detected a t any o ther levels by either species. R enaud (1986b) reported significant avoidance of hypoxic w ater by Penaeus aztecus and P. seliferus w hen the dissolved oxygen concentration was <1.5 ppm (~ 32 Torr a t 23°C and 30 ° / 00 S). Petersen and Petersen (1990) reported a linear relationship between avoidance and oxygen concentration in the sand goby, Pomatoschisius m inutus with significant avoidance a t 30% oxygen sa tu ra tio n (~ 46 Torr65Figure 3 .6 The m ean oxygen tension recorded from th e nine sam pling ports for each trea tm en t are plotted against location of those sam pling ports. M ean preferred oxygen tensions, oxygen tension m ost preferred by a crab or th e oxygen tension a t which a crab sp en t m ost of its time, are superim posed on the oxygen gradient to show the oxygen preference of crabs a t various trea tm ents. M ean preferred oxygen tension for C. similis is 108 Torr and for C. sapidus 112 Torr oxygen.OXYGEN TENSION (Torr)66A = C. sapidiis 0 = 0 . similisTREATMENT = 0 TORRTREATMENT = 25 TORR125 -1 0 0 - 7 5 - 50 = 2 5 - 01 5 0 - 125 - 1 0 0 - 7 5 - 5 0 - 2 5 -01 5 0 r 125 - 1 0 0 - 7 5 : 5 0 - 25 - 010 20 30TREATMENT = 50 TORR10 20 30TREATMENT = 75 TORR50 100J_10 20 30TREATMENT = 90 TORR10020_ l I l !50-j L100LOCATION (cm)67Table 3 .1 . M ean preferred oxygen tension of Calltnectes sap idus and C. similis a t various trea tm en t levels. S.E. = S tandard error.Oxygen Tension Preferred oxygen (Torr)(Torr)C. sap idus S.E. C. similis S.E.0 98.16 2.41 82.32 2.8725 100.40 2.46 116.30 1.9250 121.95 1.76 111.48 2.2075 116.21 1.2490 125.58 1.27 120.82 1.05a t 23°C and 30 ° / 00 S). A nalysis of total time, am ount of time sp en t by each crab a t the hypoxic end, however revealed significant avoidance by C. similis a t 0, 25, and 50 Torr oxygen tension. W hen th e to tal time w as calculated for C. sap idus no significant avoidance w as detected. We conclude th a t to tal time is a m ore sensitive m easurem ent of hypoxia avoidance since it gives an exact estim ation of tim e spen t by each crab a t the hypoxic end (time spen t by each crab a t the hypoxic and normoxic end for each experim ent w as m onitored constantly) in com parison w ith location. Location of each crab w ith in the cham ber w as noted every m inute during each experim ent b u t a crab w as capable of travelling the entire length of the cham ber several tim es w ithin one m inute. T hus th e position of a crab between two successive m easurem ent rem ained undefined w hereas the to tal time gave a complete m easurem ent of the am oun t of time spen t a t the hypoxic end of the cham ber during an experiment.M easurem ent of response time, the time in m inu tes before a crab ’s initial entry into the control side of the cham ber alter the in troduction of hypoxic w ater, showed a significant linear decline w ith increasing oxygen tension in Callinectes similis b u t no such tren d w as obvious in C. sapidus. Contrary to our expectation, the response tim e of C. similis exposed to 0 and 25 Torr oxygen w as significantly longer th a n in c rabs exposed to 50 and 90 Torr oxygen. After th e initial introduction of hypoxic w ater into the cham ber to the achievem ent of a final stable gradient there w as always a time lag of abou t 10 min. This w as the time required for the mixing of hypoxic and normoxic w ater to occur. Therefore w hen anoxic or acutely hypoxic w ater w as introduced from one end of the cham ber, w hich w as initially filled up with normoxic w ater, the dissolved oxygen level did not drop im mediately a t th a t end. I th ink the response tim e is a reflection of the time required to achieve a stab le gradient and for the crab to detect and then avoid the hypoxic w ater zone. However, a t less stressfu l higher oxygen tension (50 and 90 T o it oxygen), which does not induce im m ediate avoidance, response time m ay not be a69reflection of avoidance b u t ra th e r the random m ovem ent p a tte rn of the crabs. Response tim e can be a good indicator of avoidance w hen hypoxia is very acute b u t, a t m oderate hypoxia it is no t a veiy dependable m easure of avoidance because of variability in individual crab to crab behavior.Use of average location, to tal time, and response tim e have dem onstrated clearly th a t C. sim ilis is able to detect and avoid hypoxia a t zero and 25 Torr oxygen tension and C. sap idus is no t as sensitive in initiating an avoidance behavior even a t the acute hypoxic condition. In using the above three variables as a m easure of avoidance we assum ed the following: (i) the avoidance cham ber w as hypothetically divided into two sides - trea tm en t side and control side, (ii) the trea tm en t side w as completely hypoxic and the control side w as completely normoxic and there w as a sharp boundary between the hypoxic and normoxic zone which w as the m id point of the cham ber where the outlet w as located. B ased on these assum ptions we inferred th a t if a crab stayed a t the trea tm ent side it showed non-avoidance and if it stayed a t the control side it showed avoidance. B ut, th e p ic tu re w as no t so sim ple w hen we m easured oxygen gradients from the nine sam pling ports located along the length of the cham ber. Oxygen gradient in the cham ber followed a sigmoidal pa tte rn gradually increasing from the hypoxic side to the normoxic side (See results). Therefore, the assum ption of a hypothetical sh arp boundary between the hypoxic an d normoxic zone w as invalid. As a resu lt the crab location and time spent a t the trea tm en t side (total time) could not be used as a completely reliable m easure of avoidance because in both cases th is distinction would have been biased. Instead, I used the crab locations for each experim ent to predict an oxygen value for each location and from those oxygen values calculated the m ost preferred oxygen value for each crab or the oxygen tension a t which a crab sp en t m ost of its time. The preferred m ean oxygen value for C. similis w as 108 Torr and for C. sapidus w as 112 Torr. T hus use of m ean preferred oxygen tension, which is an unbiased m easure of avoidance, proved th a t both species of70crabs were able to detect hypoxic w ater and choose an optim um oxygen tension which helped them to conserve the ex tra energy required for hyperventilation.Activity, a s m easured by the d istance travelled by a crab during the final 50 m in of a 60 m in experim ent, of the crabs w as used as a m easure of s tress during the experim ent. Both species of crabs were found to be significantly less active a t the severe levels of hypoxia (0, 25, and 50 Torr oxygen). C rustaceans com pensate for short term hypoxia by increasing ventilation (Batterton and C am eron 1978, Taylor 1982, Pease and D eFur 1987). This requires an ex tra supply of energy which is obtained from the energy available for locomotion, food gathering etc. Therefore a t severe hypoxic levels (<25 Torr) th e crabs become less active because they are energy limited com pared to the crabs exposed to the higher levels of oxygen where the activities are higher too.M inim um dissolved oxygen requirem ent for various aquatic species varies widely and th e effect of hypoxia depends on their tolerance limit and duration of exposure. Survival of an organism exposed to stressfully low dissolved oxygen depends on the an im al’s capability of detecting the event, directly or indirectly, and physiologically acclim ating or altering their behavioral pattern . W hen the degree of hypoxia is stressfully low, behavioral a lterations, su ch as avoiding th e hypoxic w ater m a ss or moving around th e spatially and tem porally isolated hypoxic w ater m ass, are of m ore im portance th an physiological tolerance. This is especially true for Cattinectes sap idus because of their inefficient anaerobic capacity (Carpenter and Cargo 1957). The inability to detect severe hypoxia or anoxia m ay limit the d istribu tion of the species by reducing the energy available for locomotion, growth, and reproduction (Brett and Groves 1979). The ability to detect and avoid hypoxia in the field h as been reported in m any species including the K um ura praw n, Penaeus japonicus (Egusa and Yamamoto 1961), European brown shrim p, Crangon crangon (Hagerman and Uglow 1982), adu lt blue crabs, Callinecies sap idus and several unidentified species of c ru staceans and fish (Loesch 1960, May1973, and Pihl et al. 1991). D as and Stickle (1993) reported th a t C. sap idus is more sensitive to hypoxia, w ith a 28 day LC50 of 106 Torr oxygen, th a n C. similis w ith a 28 day LC50 of 43 Torr oxygen. They also reported th a t severe hypoxia re ta rd s th e growth rate of both species of juvenile b lue crabs. T h u s th e ability to detect and avoid hypoxia in th ese two species of blue c rabs is im portant for the ir survival, su sta ined growth and distribution.CHAPTER 4PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF JUVENILE BLUE CRABS, CALLINECTES SAPIDUS AND C. SIMILIS, TO DIURNALLY VARYING DISSOLVED OXYGEN TENSION72INTRODUCTION73Hypoxia is one of the m ajor environm ental variables affecting the perform ance and ultim ately survival of es tuarine dependent b lue crabs (Das an d Stickle 1993). Previous investigations were m ainly concerned with the physiological and biochemical responses following exposure to long term hypoxia (Pease and D eFur 1987, DeFur et al. 1990, Phil e t al. 1991, D as and Stickle 1993) of the b lue crabs Callinectes sap idus and C. similis. In th e p resen t study, however, we m easured several physiological and biochem ical responses of these two species exposed to a fluctuating oxygen regime. Published observations docum enting diel variation in dissolved oxygen tension are lacking. Diel variation in the dissolved oxygen tension in es tuarine w ater along the Louisiana G ulf coast occurs frequently, especially during the sum m er m onths, with w ater becom ing extremely hypoxic during the early m orning period (see results). Both C. sap idus and C. similis spend a portion of their life cycle in the vegetated h ab ita t of the coastal G ulf and estuarine w aters (G unter 1950, D augherty 1952, More 1969, Adkins 1972a, Perry 1975, O rth and van M ontfrans 1987, Williams e t al. 1990, H sueh et al. in press) and are th u s exposed to d ium ally varying levels of dissolved oxygen.D as and Stickle (1993) u sed survival, feeding rate , growth and molting to m easure responses of Callinectes sapidus and C. similis w hen exposed to chronic hypoxia. Survival is a very sensitive indicator of hypoxia tolerance as is feeding rate , which varies 5-9 tim es m ore th a n m etabolic rate along environm ental factor grad ien ts (Stickle 1985). Deoxyribonucleic acid (DNA) is m ainly found in the cell nucleus associated with chrom osom al m aterial and the total quantity of DNA rem ains constan t in the som atic cells w ithin a given species (Leslie 1955). On the o ther hand, ribonucleic acid (RNA) is produced during gene ti'anscription and correlates positively with the rate of protein syn thesis and th u s inflects changes in the cellular activity (Brachet 1960). Since the74am oun t of DNA rem ains constan t for a given num ber of cells, the ratio of RNAto DNA is u sed a s a reliable indicator of th e protein synthetic activity w ithin cells (see review by Bulow 1987). Nucleic acid concentration and th e ratio of RNA to DNA have been used to s tu d y cell growth and developm ent in c ru staceans (Sulkin et al. 1975), m olluscs (e.g. Collier 1976) and fishes (see review by Bulow 1987). It h as also been used to m easure th e effects of various stresso rs and toxicant on fishes (e.g. B arron and A delm an 1984, Loweiy and Somero 1990, W ang et al. 1993) and the blue crabs (Wang and Stickle 1986, W ang and Stickle 1988). In th is study we have used the change in concentrations of nucleic acids an d RNA:DNA ratio to m easure the am oun t of s tre ss resulting from exposure to constan t and d ium ally fluctuating dissolved oxygen.The objectives of th is s tudy are to (i) com pare th e responses of juvenile Callinectes sap idus and C. similis to d ium ally varying oxygen tension, and (ii) determ ine the difference in response to chronic hypoxia and diel variation in oxygen tension using the following indices: survival, feeding rate, growth, molting and changes in the RNA and DNA concentration as well as in the RNA:DNA ratio.MATERIALS AND METHODSCollection and maintenanceCallinectes sapidus and C. similis were collected nearshore a t Port Fourchon, L ouisiana (90°11.4’ W, 29°6.7’ N) or n ear C am inada Pass, Louisiana, USA (90°3.0’ W, 29°12.6’ N) during Ju n e , 1992, when the w ater tem peratu re w as 28 - 30°C and the salinity w as 28 ° / 00. The crabs were b rought back to the laboratory and placed into artificial sea w ater m ade from Instan t Ocean® a t room tem peratu re (23 - 25°C) and salinity m atching field condition. All the crabs were kept u n d er constan t illum ination and were acclim ated in a step wise fashion to the experim ental salinity (30°/oo). C rabs were75individually isolated in cham bers m ade from a p a ir of PVC pipe sections (one with 21 cm inside d iam eter and the o ther piece with 23 cm inside d iam eter and both were 28 cm in length) th a t fit together w ith nylon m eshed screens on the free ends of each piece to res tra in the crabs. C rabs were chosen carefully to minimize weight and size difference. All the crabs were held for 2 w k a t the final salinity before being used in the experiment. C rabs were fed frozen abdom inal portions of the g rass shrim p Palaemonetes pugio.Field ObservationDiel variation in oxygen tension w as m onitored using a m ultiparam eter w ater quality m onitoring instrum en t (DataSonde 3, Hydrolab Corp., A ustin, Texas) a t Port Fourchon and C am inada Pass, Louisiana, a t the beginning and end of Ju n e , 1992, for a period of 7 d each w hen th e anim als were collected. D ata were recorded every 15 m inu tes during each collection period. The oxygen probes were installed a t a depth of abou t 6 ft and 1.5 ft a t C am inada Pass and Port Fourchon respectively.Experimental designC onstan t Hypoxia Exposure System :C rabs of both species were m ain tained a t 155 (normoxic water) and 50 Torr oxygen tension th roughout the 28 d experim ental period as control trea tm ents. In an earlier study (Das and Stickle 1993) we found th a t 100% m ortality occurred in Callinectes sap idus exposed to 25 Torr oxygen tension w ithin 6 days of exposure. 100% m ortality did not occur a t any higher levels of dissolved oxygen tension. I have used 50 Torr as a control exposure so I can have representative sam ples from th is level th roughou t the experim ental period. Two aquaria were assigned for each control trea tm ent. Each aquarium (76 1) consisted of an undergravel filter overlaid with crushed oyster shells. W ater from a large filtration u n it w as pum ped constantly into each76aquarium w ith peristaltic pum ps a t a ra te sufficient to recycle th e w ater several tim es a day.An oxygen tension of 50 Torr w as created and m ain tained by mixing bottled nitrogen and oxygen with a M atheson gas m ixer (model 7402T) along w ith 0.03% carbon dioxide to m ain ta in th e pH a t approxim ately 7.8. The gas m ixer w as connected to an ou tlet m anifold an d the air m ixture w as passed th rough the undergravel filter in each of th e two aquaria assigned for the 50 Torr control treatm ent. Am bient a ir w as used to drive th e undergravel filters of the normoxic tan k s (155 Torr). Each aquarium w as covered with a plexiglas lid and th en w ith a clear plastic w rapping to minimize a ir exchange. The w ater level in each ta n k w as m ain tained with a co nstan t level siphon which drained w ater from the aquarium into the filtration unit. Experim ental conditions (tem perature, salinity, P 02, am monia) were checked daily. The am m onium concentration in the ta n k s never exceeded 25 pM. A group of 15 norm oxia acclim ated crabs of each species were transferred to each of th e replicate aquaria for each oxygen tension (a total of 30 crabs of each species) and were m onitored for survival, feeding rate, growth and molting.D ium ally F luctuating Oxygen System :Diel fluctuation of dissolved oxygen sim ilar to the fluctuations noticed in the field w as sim ulated in the laboratory with a com puter program th a t controlled a m ass flow gas m ixer (Model GF-3, Cam eron Instrum en t Company, Port A ransas, Texas) and a m ultiparam eter w ater quality m onitoring device (Scout 3, Hydrolab Corp., A ustin, Texas). This fully au tom ated system used a simple feedback control algorithm to sinusoidally vary the oxygen tension in the water. Oxygen tension varied from 25 Torr (16% air saturation) to 155 Torr (100% a ir saturation) a t 23 - 25°C and 3 0 ° /oo S (Fig. 4.1). All o ther experim ental conditions were m aintained as in the co nstan t hypoxia exposure system . A group of 30 norm oxia acclim ated crabs of each species were transferred separately.77Figure 4 .1 Plot of th e d iu rna l variation in oxygen tension in th e laboratory for a period of 28 d.TORR OXYGEN2 5 0200150100500242016 28121 8DAYS79replicates of 15 each, to th e fluctuating oxygen system and m onitored for 28 d for survival, feeding rate , growth and molting.Determination o f food consumption, molting and growth rateBoth crab species were fed weighed abdom inal portions of frozen g rass shrim p, Palaemonetes pugio, ad libitum so some tissue rem ained a t the end of th e day. U neaten food w as removed, weighed and replaced w ith a new ration daily. C rabs th a t died during th e experim ent were removed from the feeding ra te analysis from the week they died. E ach tim e a crab m olted it w as reweighed and the difference betw een the initial and final w eight w as converted into percen t increase in weight.M easurem ent o f DNA and RNADNA and RNA analyses were perform ed according to the procedure developed by M unro and Fleck (1966). All analyses were performed on whole crabs ra th e r th a n on specific tissues because of the sm all size of the crabs. Six crabs of each species from each of all 3 trea tm en ts (155, 50 Torr and F luctuating 0 2) were sacrificed after 0, 7, 14, 21 and 28 d exposure. However, som e sam ples were lost due to crab deaths in the hypoxic trea tm ent, in the fluctuating 0 2 trea tm en t or during sam ple preparation. The m inim um sam ple size w as four. C rabs wei'e sacrificed and frozen in liquid nitrogen on each sam pling date and th en stored a t -80°C. Individual frozen crabs were pulverized in a m ortar and pestle and homogenized in 10 volum es of ice-cold distilled w ater using a Tekm ar homogenizer. 200 pi of hom ogenate w as dried a t 80°C in pre-weighed alum inum p an s for 24 h rs to determ ine the d iy weight of each sam ple. 200 pi of hom ogenate w as used separately for DNA and RNA analysis. M acromolecules including nucleic acids were precipitated from the hom ogenate by adding 100 pi of 0 .6 N and 500 pi of 0.2 N perchloric acid and incubating the m ixture on ice for a t least 10 m inu tes. The sam ples80were th en centrifuged a t 5600 X g a t 4°C for 15 min. The su p e rn a ta n t w as discarded and th e pellet w as w ashed twice in 750 pi of ice-cold 0.2 N perchloric acid. The pellet w as saved for DNA and RNA analysis. DNA content of the pellet w as determ ined using the diphenylam ine procedure (Burton 1956) according to S hatk in (1969). Two vol of diphenylam ine reagent and 1 vol of 0.5 N perchloric acid were added to the pellet, mixed an d incubated a t 25°C for 20 h rs . DNA content of each sam ple w as m easured a t an absorbance of 600 n m and com pared w ith the s tan d ard prepared from calf thym us DNA (Sigma Chem ical Co.). Analyses were perform ed in duplicate.RNA content of the pellet w as determ ined using th e modified Schm idt- T h an n h au se r procedure recom m ended by M unro and Fleck (1966). Homogenates were hydrolyzed by adding 500 pi of 0.3 N po tassium hydroxide and the m ixture w as incubated a t 37°C for 30 min. After cooling the digest on ice m acrom olecules were precipitated by adding 250 pi of 1.5 N perchloric acid and the m ixture w as incubated on ice for a t least 10 min. The sam ples were th en centrifuged a t 5600 X g a t 4°C for 15 min. The su p ern a ta n t w as saved and the pellet w as w ashed twice w ith 150 pi 0.2 N perchloric acid. RNA content of the pooled su p ern a tan t w as m easured a t 260 nm and com pared with the s tan d ard prepared from yeast RNA (Sigma Chem ical Co.). Analyses were perform ed in duplicates.Statistical analysisTo remove the effects of body weight, the feeding ra te s were standardized to a 1 g u n it wet body weight. Feeding rate w as analyzed with a repeated m easure analysis. Differences in feeding ra te w ithin and am ong trea tm en ts were determ ined with polynomial contrasts. Nucleic acid concentration was standard ized for each crab within each trea tm en t for 1 g un it dry body weight. Total nucleic acid in each crab w as also m easured . Nucleic acid concentration and RNA:DNA ratio were analyzed as factorial81design. Differences am ong trea tm en t m eans were determ ined by using least square m eans. Regression analysis w as used to determ ine the effect of crab body size on the nucleic acid con ten t and RNA: DNA ratio for the crabs exposed to normoxia.RESULTSThe d iu rnal trend in dissolved oxygen tension along the collection sites of coastal Louisiana is plotted in Fig. 4.2. D ata presented in th e figure are the traces over each 7 days a t each location (Cam inada Pass and Port Fourchon) and the average of the two locations over the 14 d period.The overall feeding rate (feeding ra te per day over the 28 days of exposure) in the two species of crabs followed distinct trends (Table 4.1). The overall feeding rate of Callinectes sap idus exposed to diurnally varying oxygen tension w as higher (p < 0.001) th a n the trea tm en t groups m ain tained a t 155 and 50 Torr oxygen. Overall feeding rate of C. sapidus exposed to 50 Torr oxygen tension w as lower (p < 0.001) th a n the crabs exposed to normoxic water. In con trast, the overall feeding ra te of C. similis exposed to d ium ally varying oxygen tension w as lower (p < 0.001) th a n in the crabs exposed to 155 and 50 Torr oxygen. Overall feeding rate of C. similis exposed to normoxic w ater w as h igher (p < 0.001) th a n the crabs exposed to 50 Torr oxygen tension.Feeding ra te of both species of crabs exposed to the d ium ally varying oxygen tension dropped (p < 0.001) from the first week to the second week and then stayed at th e sam e level for the res t of the 28 d experim ental period w hen it w as analyzed on weekly b asis (Fig. 4.3).Growth w as m easured in both species of crabs as percent increase in wet weight w ith respect to th e initial weight of the crab. Growth ra te in the crabs exposed to the d ium ally varying oxygen tension w as significantly higher th a n the crabs exposed to 5082Figure 4 .2 D iurnal trend in oxygen tension along the collection sites of coastal Louisiana. D ata presented in th e figure are the recordings a t the two collection s ites (Port Fourchon and Cam inada Pass) and the average of the two collection s ites over the 14 days of recording period.1801601401201008060402020015010050 ■0 •20015010050 ■0 -CAMINADA PASSPORT FOURCHON7 11DaysAVERAGE . .tT m r tm r i iT i i im in im ir m m n i iT T m iim m T T n ir i i i i i i i iM i i i i i i . i i i i r i i i i iK i i - r i r i i in i i i i i i in l 'm i i i i i i i i im T n ir . in i i im n n n T im r r T m n n T n m r r : : "1 0 0 0 1 6 0 0 Time (hrs)22:00nrimiiiiiiiniin rmm4 0 084Table 4 .1 . Callinectes sa p id u s and C. similis. Overall feeding ra te (expressed a s food consum ed / day over the 28 d exposure) of the two species of crabs. 30 crabs were used a t each P 0 2 for each species.Species T reatm ents Overall food consum ption (mg)Std. error p valuesCallinectes Fluctuating 492.5 30.1 0.001sa p id u s 155 Ton- 388.4 16.9 0.00150 Ton- 286.2 16.2 0.001Callinectessim ilisFluctuating 275.4 17.2 0.001155 Ton- 400.5 23.8 0.00150 Ton- 337.5 29.1 0.001Table 4 .2 . Callinectes sa p id u s and C. similis. Growth and m olting phenom ena the two species of crabs exposed to various hypoxia regime for 28 days. 30 crabs were used a t each P 0 2 for each species. Sap = C. sa p id u s ; Sim = C. similis.Oxygen (Torr) TensionSap155Sim Sap50SimFluctuating Sap SimAverage 655 574 635 632 564 659initial w et wt (mg)i+ 00 o ± 52 ± 52 ± 5000+i ± 73Increase in wet weight (%)98 76 67 60 95 73No. of crabs m olted once10 15 5 3 14 1485Figure 4 .3 Callinectes sap idus and C. similis. M ean daily feeding ra te (± SE) of the two species of c rab s exposed to th e fluctuating oxygen regim e an d 155 and 50 Torr oxygen tension du ring each week.mg wet food gm wet body wt dayC all inec tes sapidus>(0T>$0D0-oooEWeeksCall inec tes similisWeeks87Torr oxygen tension (Table 4.2). Growth ra te of C. sapidus exposed to d ium ally varying oxygen tension w as higher th a n the growth rate of C. similis. No significant differences were noticed in growth rate of crabs exposed to norm oxia and d ium ally varying oxygen tension.Only 3% m ortality in Callinectes sapidus and 11% m ortality in C. similis occurred in th e crabs exposed to the fluctuating oxygen tension. M ortality in the crabs exposed to 50 Torr oxygen tension w as 82% and 50% for C. sap idus and C. similis respectively. There w as no m ortality in the crabs exposed to 155 Torr oxygen tension.The ratio of RNA to DNA of both species of crabs exposed to 155 Torr oxygen tension increased with increasing body size over the 28 days (p < 0.01, Fig. 4.4). Slope of th e regression for either species did not differ significantly from each o ther (p = 0.43).D ium ally varying oxygen tension strongly affected the nucleic acid concentration of bo th species of crabs. The RNA concentration of Callinectes sapidus and C. similis decreased by 36% and 35% respectively on day 28 in the crabs exposed to d iurnal oxygen variation com pared to the control crabs of each species from day 0 (Table 4.3). The DNA concentration of C. sapidus and C. similis decreased by 40% and 21% respectively on day 28 in the crabs exposed to d iurnal oxygen variation com pared to the control crabs of each species from day 0 (Table 4.3). In com parison, the RNA concentration of C. sapidus and C. similis decreased by 27% in both species on day 28 in the crabs exposed to the 50 Torr oxygen tension com pared to the control crabs of each species from day 0 (Table 4.3). The DNA concentration of C. sapidus and C. similis decreased by 31% an d 23% respectively on day 28 in the crabs exposed to the 50 Torr oxygen tension com pared to th e control crabs of each species from day 0 (Table 4.3). There w as a significant effect of tim e (p < 0.01) on the RNA and DNA concentration of bo th species of crabs.The RNA:DNA ratio of C. sapidus increased by 9% while th a t of C. similis decreased by 18% on day 28 in the crabs exposed to d iu rnal oxygen variation com pared88Figure 4 .4 Relationship betw een th e RNA:DNA ratio and body weight in juvenile Callinectes sapidus and C. similis. N = 22 in C. sap idus (filled circles) an d N = 19 in C. sim ilis (empty triangles).RNA:DNA Ratio4.00 Callinectes similis1.7386903 + 0 .0081678 X3.202.401.600.80 Callinectes sapidus Y = 1 .1 2 1 2 4 4 8 0 .0 0 8 1 6 7 8 X0.000 34 47 51 68 107 141 176 237 323Dry w t (mg)00CD90Table 4 .3 . Callinectes sap idus and C. similis. C hanges in the RNA and DNA concentration and RNA:DNA ratio in the two species of crabs a t th e end of the experim ent (day 28) a s com pared to the control c rabs from day 0. Nucleic acid concentration is given in p g /m g dry body wt.T reatm ent Day RNA % of day 0DNA % of day 0RNA: DNA Ratio% of day 0Calltnecles sapidus155 Torr 0 1296 ± 71100 696 ± 30100 1.93 ± 0.12810028 1091 ± 7084 697 ± 67100 1.64 ± 0.10585F luctu ating28 825 ± 11264 419 ± 7460 2.11 ± 0.13010950 Torr 28 941 ± 11073 481 ± 5869 1.97 ± 0.057102Callinectes similis155 Torr 0 1111 ± 109100 456 ± 56100 2.56 ± 0.19210028 1142 ± 86103 785 ± 58172 1.49 ± 0.14558F luctu ating28 719± 2365 361 ± 2579 2.09 ± 0.1458250 Torr 28 714± 4873 353 ± 3477 2.02 ± 0.1007991to the control c rab s of each species from day 0 (Table 4.3). The RNA:DNA ratio of C. sapidus increased by 2% and of C. similis decreased by 21% on day 28 in the crabs exposed to 50 T orr oxygen tension com pared to the control crabs of each species from day 0 (Table 4.3). The RNA:DNA ratio of both species changed significantly (p < 0.001) over tim e an d w as also significant (p < 0.001) a t the species level (Fig. 4.5, 4.6).Both species of c rab s m ain tained a t 155 Torr oxygen tension showed an overall increase in DNA an d RNA synthesis over time. There w as a 72% increase in DNA concentration in Callinectes similis w hen it w as com pared to th e control crabs from day 0. Increase in th e DNA concentration in C. sap idus w as negligible.DISCUSSIONFood consum ption of CaUinectes sapidus over th e 28 d period of exposure w as significantly h igher in th e d ium ally varying oxygen tension th a n the two control (155 and 50 Torr oxygen tension) trea tm ents. On the o ther h an d , C. similis exposed to the d ium ally varying oxygen tension consum ed significantly less food as com pared to the two control trea tm en ts . D as and Stickle (1993) reported a 123% increase in feeding rate in C. sap idus after 10 days of starvation. In the p resen t s tu d y C. sap idus was exposed to oxygen tensions high enough to allow elficient feeding for a sufficient period of time during the d iu m a l varia tion in dissolved oxygen. However, a lower feeding rate of C. similis m ay be due to a variable m etabolic ra te over the d iu m al cycle due to hypoxic stress. The m etabolic ra te of C. similis exposed to chronic hypoxia w as elevated over the rate of crabs exposed to norm oxia indicating overcom pensation (Das and Stickle 1993). The growth ra te , m easu red a s percent increase in wet weight, of both species of crabs exposed to th e fluctuating oxygen regime w as com parable to th e crabs of both species exposed to 155 Torr. W ang (1986) reported a lower tissu e content with growth in92Figure 4 .5 Callinectes sapidus. C oncen tration of RNA, DNA an d RNA:DNA ratio (± SE) of th e blue crabs exposed to fluctuating oxygen regime and 155 and 50 T orr oxygen during the four sam pling dates. The m ean concentration of the nucleic acids and the RNA: DNA ratio on day 0 w as u sed a s a s ta n d a rd starting point.93Call inec tes sapidusa 1 5 0 0? 1200 >.■fe& 9 0 06 0 03 0 07 14 21Sampling Period (days)2 85 0Torrb5 0 07 14 21Sampling Period (days)155TorrFluctu a tin gc3 ->•b& 900s5^ 6003007 14 21Sampling Period (days)2850TorrFluctu a tin g14 2Sampling Period (days)7 14 21Sampling Period (days)2896juvenile b lue crabs exposed to w ater soluble fraction of crude oil com pared to the control crabs and h a s suggested size increase un d er stressfu l environm ental condition is a possible an ti-predation adaptation. In the p resen t experim ent, growth w as m easured as increase in wet weight. Therefore, a sim ilar growth ra te in the two species of crabs exposed to fluctuating and 155 Torr oxygen m ay be due to th e ex tra am oun t of w ater p resen t in th e crabs exposed to diel oxygen variation and the size increase may essentially an an ti-predation strategy. The increase in size w as not as great for either species of crabs exposed to 50 Torr oxygen tension because of th e s tre ss involved with co n stan t hypoxic exposure.W ang and Stickle (1986, 1988) reported the ratio of RNA to DNA a s a reliable and sensitive index of s tress in juvenile Callinectes sapidus. W ang et al. (1993) used both RNA:DNA ratio and RNA content as a reliable m easure of fish growth fed crude oil contam inated food. They have also indicated th a t RNA content m ay be a m ore useful ind icator of fish growth th a n the ratio of RNA to DNA. B arron and Adelm an (1984) used RNA, DNA and RNA:DNA ratio to m easure the growth oflarval fish exposed to sublethal concentrations of toxicant. RNA, DNA concentration and the RNA:DNA ratio have been show n to be a m easure of grow th ra te in fishes (Buckley 1984, Bulow 1987).Growth ra te in Callinectes siniilis exposed to d ium al oxygen variation was com parable to th e crabs exposed to 155 Torr oxygen tension bu t, the feeding rate was lower in the crabs exposed to diel oxygen variation. A high growth rate and low intake of food m ight cause a situa tion sim ilar to starvation which would resu lt in decreased RNA syn thesis leading to a selective cell catabolism as suggested by W ang and Stickle 1986. As a direct resu lt of a decrease in cell num ber the DNA concentration per un it m ass of crab also decreased. On the o ther hand , a poor nutritional s ta tu s can not be inferred from the app aren t decrease in (he RNA and DNA concentration in C. sapidus. Since nucleic acid concentrations were m easured as pg nucleic acid per un it body weight, a97decrease in the nucleic acid concentration per u n it body weight with increasing body size w as possible especially if the crabs were growing by increasing the cell size or relative carapace size and no t cell num ber. M ustafa (1978) proposed growth by increase in size of cells ra th e r th a n cell nu m b er in the catfish, H eteropneustesfossilis. A response sim ilar to C. similis exposed to the d ium ally varying oxygen tension h a s been observed in both species exposed to the 50 Torr oxygen tension. C rabs exposed to 50 Torr oxygen consum ed significantly less food com pared to the crabs exposed to normoxic w ater and there w as a significant decrease in both RNA and DNA concentration by 28 d. In addition the grow th ra te of the crabs of both species exposed to 50 Torr oxygen tension w as significantly lower th a n the crabs exposed to normoxia. Therefore, the explanation for the app aren t decrease in RNA and DNA concentration in C. similis exposed to d iurnal oxygen fluctuations m ay no t hold for the crabs exposed to 50 Torr oxygen tension. The fluctuating oxygen regime had a sim ilar elfect on the nucleic acid concentration of both species of crabs bu t, in C. similis the elfect w as probably com pounded by a low consum ption of food which w as a direct elfect of hypoxia. The tren d of decreasing nucleic acid concentration in crabs exposed to the 50 Torr oxygen tension w as sim ilar to the response of the crabs exposed to the fluctuating oxygen regime. W ang and Stickle (1986) found a 73% decrease in RNA concentration and 31% decrease in DNA concentration in the b lue crabs starved for 30 days. In the p resen t study RNA concentration in neither species decreased m ore th an 35% of the concentration in crabs a t 155 Torr on day 0.Sulkin eta l. (1975) report ed a cyclic activity p a tte rn in the RNA:DNA ratio during larval development of the X anthid crab Riihropanopeus hairisii. They have reported a peak of protein synthesis during the interm olt period followed by a drop before molt. In the p resen t study the RNA:DNA ratio for both species of crabs w as variable. The fluctuating n a tu re of the RNA:DNA ratio w as m ainly due to the fluctuation in the RNA and DNA concentration in all the trea tm en ts over time. This ap paren t fluctuation in thenucleic acid concentration m ay be due to the molting activity in the crabs. However, the changes in th e nucleic acid concentration in relation to the m olt cycle w as no t m easured in th is study. W hen the effects of environm ental and biological factors on th e nucleic acid syn thesis are unknow n for a n a tu ra l population the utility of RNA:DNA ratio becom es limited (Dagg an d Littlepage 1972, O ta and Landry 1984). C hanges in the nucleic acid concentration in both species of crabs due to hypoxic s tre ss were no t of the sam e m agnitude as reported by W ang and Stickle (1986) for juvenile b lue crabs starved for 30 d. The RNA:DNA ratio in the ir s tudy decreased from 2.92 for the control crabs to 1.15 in th e experim ental c rabs after 30 days of starvation, a 61% decline from the control level. The ratio increased by 51% after 5 days of feeding which dem onstrates the sensitivity of th e RNA:DNA ratio to changes in food availability. In th is s tudy th e m agnitude of decrease in the concentration of either RNA or DNA and the RNA:DNA ratio is m uch less com pared to those m entioned by W ang and Stickle (1986). This probably indicates th a t th e d ium al variation in oxygen tension did not s tress the crabs enough to cause an equivalent am oun t of change in the concentration of RNA and DNA or the RNA:DNA ratio. M ortality in the blue crabs due to hypoxic s tress is no t as m uch due to bio-energetic constra in ts a s it is because of the s tress during molting w hen dem and for oxygen goes up by several folds (Perry e t a t 1979).CHAPTER 5SUMMARY AND CONCLUSIONS99The Louisiana con tinen ta l shelf is the largest, m ost severe, and p ersis ten t zone of hypoxia in th e U nited S ta tes coastal w aters (Rabalais and H arper 1992a). Hypoxia is a m ajor environm ental factor which can cause trem endous s tre ss to the ab u n d an t dem ersal fau n a (fish, penaeid shrim p and swimming crabs) which characterize the Gulf of Mexico she lf environm ent (Boesch and Rabalais 1991). Reports suggest th a t low levels of dissolved oxygen cause m ortality of Callinectes sap idus and can im pede the ir m igration (Tatum 1982, V an Engel 1982). The distributional p a tte rn of C. similis, a n offshore congener of C. sapidus in the G ulf of Mexico region overlaps frequently. Spaw ning hab its of C. sap idus and C. similis overlap tem porally and spatially (Perry 1975). T hus some p a rts of th e life cycle of these two species are exposed to th e sam e set of environm ental variables. O ccurrence of diel variation in dissolved oxygen h a s no t been reported before in the coastal G ulf of Mexico. In the present study diel variation in dissolved oxygen has been show n to occur a t the two collection sites w here the juveniles of these two species co-occur. M igration to higher salinity w aters th a t are favorable for larval developm ent has been reported for both C. sap idus and C. similis (Millikin and Williams 1984, Williams 1984, H ines et al. 1987). Therefore, both of these species are exposed to low dissolved oxygen during different p arts of their life cycle.Tolerance to hypoxia is probably related to the developm ental history of the organism . C hanges in the resistance and capacity adap tation of an organism over it’s lifetime is related to the degree of exposure to the s tresso r gradient a s well as to its evolutionary history. C. similis is exposed to constan t hypoxia in the offshore w ater for a m ajor p a rt of its life cycle w hereas, C. sapidus is m ore often exposed to the diel variation in dissolved oxygen in the coastal vegetated habitat. Only the ovigerous female C. sapidus m igrate to the h igher salinity w ater w hen they are exposed to the constan t hypoxia for only a short period. A direct consequence of th is m ay be a g reater tolerance to constan t hypoxia in C. similis com pared to C. sapidus.101Since hypoxia is a com mon and recu rren t phenom enon along th e Louisiana Gulf coast a n ability to properly detect and avoid hypoxia is of im portance in the survival to these estuarine-dependent species. Diel variation in the dissolved oxygen tension in e s tu a iin e w aters along th e Louisiana Gulf coast h a s also been show n to be of frequent occurrence, especially during th e sum m er m onths w ith w ater becom ing alm ost anoxic during th e early m orning period of th e day. Therefore, ability to avoid hypoxia becom es even m ore im portan t in daily activities like foraging, diel m igration etc. The inefficient anaerobic capacity of the blue crabs m ake them vulnerable to hypoxic exposure and timely detection becom es necessary. This s tudy dem onstrated th a t although both species of c rabs can detect and avoid hypoxic w ater, Callinectes similis is m ore efficient in detecting an d avoiding hypoxia than C. sapidus. Avoidance of hypoxic w ater by larger motile invertebrates were photodocum ented by Stachow itsch (1984). R abalais el al.(199 lb) docum ented sim ilar behavior am ong motile m arine invertebrates and vertebrates in the G ulf of Mexico con tinen ta l shelf off Louisiana. Tolerance to hypoxia and ability to detect and avoid th e phenom enon in a timely m anner m ay also be related to resource partitioning w ithin th e environm ent shared by these two species. Such differences in to lerance and avoidance ensure the ir survival and coexistence in a com m on hab itat, at least for a portion of the ir life cycle, w ithout excluding each o ther by competitive interaction.LITERATURE CITEDAdkins, G. (1972a). A study of the blue crab fishery in Louisiana. La. Wildl. Fish. Comm. Tech. Bull. 3: 1-57.Barron, M. G. and Adelman, I. R. (1984). Nucleic acid, pro tein content, and growth of larval fish sublethally exposed to various toxicant. Can. J . Fish. Aquat. Sci. 41: 141-150.B atterton , C. V., and Cam eron, J . N. (1978). C haracteristics of resting ventilation and response to hypoxia, hypercapnia, and em ersion in the b lue crab Callinectes sap idus (Rathban). J . Exp. Zool. 203: 403-418.Bedinger, C. A., Childers, R. E., Cooper, J . W., Kimball, K. T. and Kwok, A. (1981). Pollution fate an d elfect studies. In: Bedinger, C. A. (ed.) Ecological Investigations of Petroleum Production Platform s in the C entral Gulf of Mexico, Vol. 1, Pt. 1,Final Report to the B ureau of Land M anagem ent, New O rleans, LA, Contract No. A5551 - CT9 - 17, pp 1-53.Boesch, D. F. (1983). Implication of oxygen depletion on the continental shelf of the N orthern Gulf of Mexico. Coastal O cean Pollut. A ssess. News. 2: 25-28.Boesch, D. F. and Rabalais, N, N. (1991). Effects of hypoxia on continental shelf benthos: com parisons betw een the New York Bight and the no rth ern Gulf of Mexico. In Tyson, R. V. and Pearson, T. H. (eds). M odem and ancien t continental shelf anoxia. Geological Society Spec. Publ. 58: 27-34Brachet, J . (1960). The biological role of ribonucleic acids, Elsevier, New York, pp. 1-144.Brett, J . R. and Groves, T. D. D. (1979). Physiological energetics. In: W. S. Hoar, D. J . Randall, and J . B rett (eds.). Fish Physiology, Vol. VIII., Academic Press, New York, pp 279-352.Buckley, L. J . (1984). RNA-DNA ratio: an index oflarval fish growth a t the sea. Mar. Biol. 80: 291-298.Bulow, F. J . (1987). RNA-DNA ratios a s indicators of growth in fish: a review. In: R. C. Sum m erfelf and G. E. Hall, eds., The age and growth of fish, Iowa University Press, Ames, Iowa, pp. 45-64.B urton, K. (1956). A study of the conditions and m echanism of th e diphenylam ine reaction for the colorimetric estim ation of deoxyribose nucleic acid. Biochem. J . 62: 315-323.Cake, E. W. (1983). H abitat suitability index m odels : Gulf of Mexico Am erican Oyster. FW S/OBS -82 /10 .57 . U. S. Dept, of Interim', Fish and Wildlife Seivice. W ashington, DC. 37 pp.Cam eron, J . N. (1978). NaCl balance in blue crabs, Callinectes sapidus, in fresh water. J . Comp. Physiol. 123: 127-135.102103C arpenter, J . H., and Cargo, D. G. (1957). Oxygen requirem ent and m ortality of the blue crab in the C hesapeake Bay. C hesapeake Bay Inst. Tech. Rep. 13: 22.Churchill, E. P., J r . (1919). Life history of the blue crab, Fish and Wildlife Service, U. S. Dept, of Commerce, Fish. Bull. 36: 95-128.Collier, J . R. (1976). Nucleic acid chem istry of the Ilyanassa embryo. Am. Zool. 16: 483- 500.Costlow, J . D., J r . and Bookhout, C. G. (1959). The larval developm ent of Callinectes sa p id u sR athbun reared in the laboratory. Biol. Bull. m ar. biol. Lab., Woods Hole. 116: 373-396.Das, T and Stickle, W. B. (1993). Sensitivity of crabs Callinectes sap idus and C. similis and the gastropod Stramonila haem aslom a to hypoxia and anoxia. Mar. Ecol. Prog. Ser. 98: 263-274.D augherty, F. M., J r . (1952). The blue crab investigation, 1949-50. Tex. J . Sci. 4(1): 77- 84.DeFur, P. L., M angum, C. P. and Reese, J . E. (1990). Respiratory responses of the blue crab Callinectes sapidus to long term hypoxia. Biol. Bull. 178: 46-54.deZwann, A., and Thillart, G. V. D. (1985). Low and high power o u tp u t m odes of anaerobic m etabolism : invertebrate and vertebrate strategies. In: Gilles, R. (ed.) C irculation, Respiration and M etabolism : Current Comparative Approaches, Springer-Verlag, Berlin, Heidelberg, pp 166-192.Egusa, S. and Yamamoto, T. (1961). S tudies on the resp iration of the "Kumura" praw n Penaeus japonicus Bate. I. Burrowing behavior with special reference to its relation to environm ental oxygen concentration. Bull. Jap n . Soc. Sci. Fish. 27:22-27.Engel, D. W. (1977). Com parison of the osm oregulatory capabilities of two Portunid C rabs, Callinectes sapidus and C. similis. Mar. Biol. 41: 275-279.Fields, J . H. A. (1985). A note on anaerobic m etabolism in Callinectes sap idus during the interm olt cycle. J . C rust. Biol. 5(2): 242-248.Gade, G. (1983). Energy m etabolism of arthropods and m ollusks during environm ental and functional anaerobiosis. J . Exp. Zool. 228: 415-429.Garlo, E. V., Milstein, C. B. and Ja h n , A. E. (1979). Im pact of hypoxic conditions in the vicinity of Little Egg Inlet, New Jersey in Sum m er 1976. E stuarine & Coastal Mar.Sc. 8: 421-432.Georgia Cooperative Fishery and Wildlife Research Unit, A thens. (1989). Species profiles : life histories and environm ent al requirem ents of coastal fishes and invertebrates (Mid Atlantic). Blue Crab Biological Report. 27 pp.104Gnaiger, E. (1983a). H eat dissipation and energetic efficiency in anim al anoxibiosis : economy con tra power. J . Exp. Zool. 228: 471-490.Gnaiger, E. (1987). O ptim um efficiencies of energy transform ations in anoxic metabolism . The strategies of power and economy. In: Calow, P. (ed.) Evolutionary Physiological Ecology, Cambridge University Press, Cambridge, pp 7-36.G uerin, J . L., and Stickle, W. B. (1992). The effects of salinity on the tolerance and bioenergetics of juvenile blue crabs (Callinectes sapidus R athbun) from different salinities. Mar. Biol. 114: 391-396.Gulf C oast Research Lab., O cean Springs, M ississippi. (1986). Species profiles : life histories and environm ental requirem ents of coastal fishes and invertebrates. Gulf of Mexico. Blue Crab Biol. Report. 29 pp.G unter, G. (1938). The common blue crab in fresh w ater (Callinectes sapidus). Science. 87: 87-88.G unter, G. (1950). Seasonal population changes and d istribu tions as related to salinity, of certain invertebrates of the Texas coast, including the commercial shrim p. Publ. Inst. Mar. Sci. Univ. Tex. 1(2): 7-51.H agerm an, L. and Uglow, R. F. (1982). Effects of hypoxia on osmotic and ionic regulation in the brow n shrim p Crangon crangon (L.) from brackish water. J . Exp. Mar. Biol. Ecol. 63: 93-104.Hagerm an, L. and Uglow, R. F. (1985). Effects of hypoxia on the respirato iy and circulatoiy regulation of Nephrops noruegicus. Mar. Biol. 87: 273-278.Ham ilton, M. A., Russo, R. C. and T hurston, R. V. (1977). Trim m ed Spearm an-K arber m ethod for estim ating m edian lethal concentrations in toxicity bioassay. Environ. Sci. Technol. 11: 714-719.H arper, D. E., McKinney, L. D. Salzer, R. R. and Case, R. J . (1981). The occurrence of hypoxic bottom w ater off the upper texas coast and its effect on the benthic biota. Contrib. Mar. Sci. 24: 53-79.Hines, A. H., Lipicus, R. N. and I laddon, A. M. (1987). Population dynam ics and hab ita t partitioning by size, sex, and moll stage of blue crabs, Callinectes sapidus, in a su b estu ary of Central C hesapeake Bay. Mar. Ecol. Prog. Ser. 36: 55-64Hochachlta, P. W. (1986). Defense strategies against hypoxia and hypotherm ia. Science. 231: 234-241.H sueh, P., McClintock, J . B. and Hopkins, T. S. (1993). Population dynam ics and life history characteristics of the blue crabs Callinectes similis and C. sapidus in bay environm ents of the northern Gulf of Mexico. P.Z.S.N.I. Marine Ecology. 3: in press.105H sueh, P., McClintock, J . B. and Hopkins, T. S. (1992). Com parative study of the diets of the blue crabs Callinectes similis and C. sapidus from a m ud-bottom hab ita t in Mobile Bay, A labam a. J . C rust. Biol. 12(4): 615-619.H sueh, P., McClintock, J . B. and Hopkins, T. S. (1991). N atural d iets of the b lue crabs Callinectes similis and C. sapidus in Mobile Bay, A labam a. Am. Zool. 31(5): 27A.Kapper, M. A., and Stickle, W. B. (1987). Metabolic responses of th e estuarine gastropod Thais haem astom a to hypoxia. Physiol. Zool. 60(1): 159-173.Leming, T. D. and S tuntz, W. E. (1984). Zones of coastal hypoxia revealed by satellite scann ing have im plications for strategic fishing. N ature (London). 310: 136-138.Leslie. I. (1955). The nucleic acid content of tissues and cells. In: E. Chargaff and I. N. Davidson, eds. The nucleic acids, chem istry and biology, Academic Press, New York. vol. 2, pp. 1-50.Lowery, M. S. and Somero, G. N. (1990). S tarvation effects on protein synthesis of red and white m uscle of the barred sand bass. Paralabrax nebulifer. Physiol. Zool. 63: 630-648.Loesch, H. (1960). Sporadic m ass shorew ard m igrations of dem ei'sal fish and cru staceans in Mobile Bay, Alabama. Ecology. 41(2): 292-298.M angum , C. P. and Van Winkle, W. (1973). Responses of Aquatic invertebrates to declining oxygen conditions. Am. Zool. 13: 529-541.M angum, C. P., McMahon, B. R .. deFur, P. L. and Wheatly, M. G. (1985). G as exchange, acid-base balance, and the oxygen supply to the tissues during a m olt of the blue crab Callinectes sapidus. J . Crus. Biol. 5(2): 188-206.M angum, C. P., deFur, P. L., Fields, J. H. A., Henry, R. P., Kkormanik, G. A., McMahon, B. R., Ricci, J . , Towle, D. W. and Wheatly, M. G. (1985). Physiology of the blue crab Callinectes sapidus R alhban during a molt. In: Perry, H. M. and R. F. Malone (ed.) Proc. Nat. Syinp. on the Soft-shelled Blue Crab Fishery, February 12- 13, pp 1-12.May, E. B. (1973). Extensive oxygen depletion in Mobile Bay, A labam a. Limnol. Oceanogr. 18(3): 353-366.McClintock, J . B., Marion, K. R., Dindo, J ., H sueh, P. W. and A ngus, R. (1993). Population stud ies of blue crabs in soft-bottom , unvegitated h ab ita ts of a su b estu ary in the no rthern Gulf of Mexico. J . C rust. Biol. 13(3): 000-000.Millikin, M. R. and Williams, A. B. (1984). Synopsis of biological d a ta on the blue crab, Callinectes sapidus R athbun. FAO Fisheries Synopsis No. 138. NOAATech. Rep. NMF: 1-38.More, W. R. (1969). A contribution to (he biology of the blue crab (Callinectes sapidus R athbun) in Texas, wit h a description of the fishery. Tex. Parks Wildl. Dep. Tech. Ser. 1: 1-31.106M unro, H. N. and Fleck, A. (1966). Recent developm ents in the m easu rem en t of nucleic acids in biological m aterials. Analyst. 91: 78-88.M ustafa, S. (1978). Deoxyribose nucleic acid in the m u scu la tu re of freshw ater cat-fish H eteropneustes fo ssilis (Bloch). Broteria C iencias N aturais. 48: 83-92Norse, E. A. and Fox-Norse, V. (1979). Geographical ecology and evolutionary rela tionsh ips in Callinecies spp. (Brachyura: Portunidae). In: Perry, H. M. and V an Engel, W. A. (eds.). Proc. Blue Crab Colloq, O ctober 18-19, 1979. pp. 1-9Norse, E. A. (1977). A spects of tire zoogcographic d istribu tion of Callinectes (Brachyura: Portunidae). Bull. Mar. Sci. 27(3): 440-447O dum , H. T. (1953). Factors controlling m arine invasion into florida freshw ater. Bull. Mar. Sci. G ulfC arib . 3: 134-156.Officer, C. B., Biggs, R. B., Taft, J . L., Cronin, L. E., Tyler, M. A. an d Boynton, W. R. (1984). C hesapeake Bay anoxia : origin, developm ent and significance. Science. 223: 22-27.O rth, R. J . and van M ontfrans, J . (1987). Utilization of seagrass m eadow and tidal m arsh creek by blue crabs Callinecies sapidus. I. Seasonal and an n u a l variations in abundance with em phasis on post-setl lenient juveniles. Mar. Ecol. Prog. Ser. 41: 283-294.Parker, C. A., and O’Reilly, J . E. (1991). Oxygen depletion in Long Island Sound : a historical perspective. E stuaries. 14(3): 248-264.Pavela, J . S., Ross, J . L. and C hillenden J r ., M. E. (1983). Sharp reductions in abundance of fishes and bent hie m acro-invertebrates in the Gulf of Mexico off Texas associated with hypoxia. Northwest Gulf Sci. 6(2): 167-173.Pease, A. L. and DeFur, P. L. (1987). Effect of long term hypoxia on resp irato iy and circulato iy function of blue crabs, Callinecies sapidus. Am Zool. 7(4): 109A.Perry, H. M. (1975). The blue crab fishery in M ississippi. G ull'Res. Rep. 5(1): 39-57.Perry, H. M., Ogle, J . T. and Nicholson, L. C. (1979). The fishery for soft shelled crabs w ith em phasis on the developm ent of a closed recirculating sea w ater system for shedding crabs. In: Perry, H. M. and Van Engel, W. A. (eds.). Proc. Blue Crab Colloq., O ctober 18-19, pp 137-152Petersen, J . K .and Petersen, G. I. (1990). Tolerance, behavior and oxygen consum ption in the san d goby, Pomaloschislus m inuius (Pallas), exposed to hypoxia. J . Fish Biol. 37: 921-933.Pihl, L., Baden, S. P. and Diaz, R. J . (1991). Effects of periodic hypoxia on distribu tion of dem ersal fish and crustaceans. Mar. Biol. 108: 349-360.107Poolsanguan B. and Uglow, R. F. (1974). Q uantitative changes in blood su g ar levels of Crangon vulgaris. J . Comp. Physiol. 93: 1-6.Portnoy, J . W. (1991). Sum m er oxygen depletion in a Diked New England estuaiy . E stuaries. 14(2): 122-129.R abalais, N. N. and H arper, D. E. (1992b). D istribution and characteristics of hypoxia on th e Louisiana Shelf in 1990 and 1991. Proceedings of W orkshop, Louisiana U niversities M arine Consortium , O ctober 1991, pp. 15-20.Rabalais, N. N. and H arper, D. E. (1992a). S tudies of benth ic biota in areas affected by m oderate and severe hypoxia. Proceedings of W orkshop, Louisiana U niversities M arine Consortium , O ctober 1991, pp. 150-153.R abalais, N. N. and Harper, D. E. (1991b). S tudies on the benth ic oxygen depletion phenom enon off Louisiana. In: II. J . Krock and D. E. H arper, J r ., eds.. Proceedings of the American Academy of U nderwater Sciences Eleventh A nnual Scientific Diving Sym posium , 25-30 Septem ber, 1991, Honolulu, Hawaii, pp. 57- 63.R abalais, N. N., Turner, R. E., W isem an, J r . W. J . and Boesch, D. F. (1991a). A brief sum m ary of hypoxia on the northern Gulf of Mexico continental shelf: 1985-1988. In: M odem and Ancient Continental Shelf Anoxia, R. Tyson and T. H. Pearson, eds.. Geological Society of London Special Publication No. 58. pp 38-48.R abalais, N. N., Turner, Rt E., W iseman, J r . W. J . and Boesch, D. F. (1986b).Hydrographic, biological, and nutrien t characteristics of the w ater colum n on the Louisiana Shelf, Ju ly and Septem ber, 1985. D ata Report No. 3, Louisiana Universities M arine Consortium , Chauvin, LA 70344. 150 pp.R abalais, N. N., Turner, R. E., W iseman, J r . W. J . and Boesch, D. F. (1986a). H ydrographic, biological, and nutrien t characteristics of the w ater colum n in the M ississippi River Delta Bight, Ju n e , 1985 to December, 1985. D ata Report No. 2, Louisiana Universities Marine Consortium , Chauvin, LA 70344. 167 pp.Rabalais, N. N., Dagg, M. J . and Boesch, D. F. (1985). Nationwide review of oxygen depletion and eutrophication in estuarine and coastal w aters: Gulf of Mexico (Alabama, M ississippi, Louisiana, and Texas). Final Rep. NOAA, National O cean Service, O cean A ssessm ent Division. Rockville, M aryland. 60 pp.Ragan, J . G., H arris, A. H. and Green, J . H. (1978). Tem perature, salinity and oxygen m easurem en ts of surface and bottom w aters on the continenta l shelf off Louisiana during portions of 1975 and 1976. Nicholls S tate Univ. Prof. Pap. Ser. (Biol.). 3: 1-29.Ram os, L. and Fernandez, I. (1981). V ariaciones del m etabolism o glucidico duran te el ciclo reproductor en la espccie Penacus notialis Perez Farfante, 1967. Revta Investnes M arinas. 5: 35-53.108R enaud, M. L. (1985). Annotated bibliography on hypoxia and its effects on m arine life, w ith em phasis on the Gulf of Mexico. NOAA Technical Report NMFS 21, U. S. Dept, of Commerce, W ashington, DC. 9 pp.R enaud, M. L. (1986a). Hypoxia in Louisiana coastal w aters during 1983 : im plications for fisheries. Fish Bull. 84: 19-26.R enaud, M. L. (1986b). Detecting and avoiding oxygen deficient sea w ater by brown shrim p, Penaeus aztecus (Ives), and w hite shrim p, Penaeus setiferus (Linnaeus). J . Exp. Mar. Biol. Ecol. 98: 283-292.Rosas, C., A. Sanchez, E. Escobar, L. Soto, and A. Bolongaro-Crevenna. (1992). Daily variations of oxygen consum ption and glucose hem olym ph level related to m orphophysiological and ecological adap ta tions of Crustacea. Comp. Biochem. Physiol. 101A (2): 323-328.Rosenberg, R. (1986). A review. Eutrophicalion in m arine w aters su rround ing Sweden. National Swedish Environm ental Protection Board Report. 3054. [translation of SNVPM 1808 (1984)). Solna. SwedenSanford, P. L., Sellner, K. G. and Breitburg, D. L. (1990). Co-variability of dissolved oxygen w ith physical processes in the sum m ertim e C hesapeake Bay. J . Mar. Res. 48: 567-590.Santos, S. L. and Simon, J . L. (1980a). Marine soft-bottom com m unity establishm ent following annual delaunation: larval or adu lt recru itm ent? Mar. Ecol. Prog. Ser. 2: 233-241.Santos, S. L. and Simon, J . L. (1980b). Response of soft-bottom ben thos to annual catastrophic d istu rbance in a south Florida estuaiy . Mar. Ecol. Prog. Ser. 3: 347- 355.SAS Institu te Inc. (1989), SAS/STAT Usei'’s Guide, Version 6, Fourth Edition, Cary, NC.Shatk in , A. J . (1969). Colorimetric reactions for DNA.RNA, and protein determ inations. In: K. Habel and N. P. Salzman, eds.. Fundam ental techniques in virology. Academic Press, New York, N.Y., pp. 231-237.Sanchez, A., Rosas, C., Escobar, E. and Solo, L. (1991). Skeleton weight-free oxygen consum ption related to adap tations to environm ent and hab its of six cru stacean species. Comp. Biochem. Physiol. 100A (1): 69-73.Stachow itsch, M. (1984). M ass m ortality in the Gulf of Trieste: The course of com m unity destruction. Mar. Ecol. 5(3): 243-264Stickle, W. B., Kapper, M. A., Liu, L. L., Gnaiger, E. and Wang, S. (1989). Metabolic adap ta tions of several species of cru staceans and m ollusks to h ypox ia : tolerance and m icrocalorimetric studies. Biol. Bull. 177: 303-312.Stickle, W. B. (1985). Effects of environm ental factor gradien ts on scope for growth in several species of carnivorous m arine invertebrates. In: J . S. Gray and M. E.109C hristiansen (ed.) M arine Biology of Polar Regions and Effects of S tress on Marine O rganism s. J o h n Wiley & Sons Ltd., pp 601-616.S tuck, K. C. and Perry, H. M. (1981). O bservations on the d istribu tion and seasonality of portun id m egalopae in M ississippi coastal w aters. G ulf Res. Rep. 7(1): 93-95.S tun tz , W. E., Sanders, N., Leming, T. D., Baxter, K. N. and Barazotto, R. N. (1982). Area of hypoxic bottom w ater found in the northern G ulf of Mexico. Coastal Oceanogr. Climatol. News. 4: 1-2.Sulkin, S. D., M organ II, R. P. and M inasian jr ., L. L. (1975). Biochemical changes during larval developm ent of t he xant hid crab RhiUvopanopeus harrisii II. Nucleic acids. Mar. Biol. 32: 113-117.T atum , U. M. (1979). The blue crab fishery of Alabama. In: H. A. Loyacano and J . P. Sm ith (eds.). Sym posium on I he n a tu ra l resources of the Mobile Bay estuary , A labam a, pp 211-220.T atum , U. M. (1982). The blue crab fishery of Alabama. Proc. Blue Crab Colloq. Oct. 18- 19, 1979, Biloxi, M ississippi. Gulf S ta tes Marine F isheries Commission. 7: 23-28Taylor, E. W. (1982). Control and co-ordination of ventilation and circulation in crustaceans: response to hypoxia and exercise. J . Exp. Biol. 100: 289-319.T urner, R. E., and Allen, R. L. (1982). Bottom w ater oxygen concentration in the M ississippi River Delta Bight. Contr. Mar. Sci. 25: 161-172.van M ontfrans, J . , Ryer, C. H. and Orth, R. J . (1991). Population dynam ics of blue crabs Callinectes sapidus R athbun in a lower C hesapeake Bay tidal m arsh creek. J . Exp. Mar. Biol. Ecol. 153: 1-14.V an Engel, W. A. (1982). Blue crab m ortalities associated w ith pesticides, herbicides, tem perature, salinity, and dissolved oxygen. In: H. M. P eny an d W. A. Van Engel (eds.). Proc. Blue Crab Colloq., O ctober 18-19, 1979. pp 89-92.Wang, S. Y. and Stickle, W. B. (1988). Biochemical com position of the blue crab Callinectes sapidus exposed to w ater-soluble fraction of crude oil. Mar. Biol. 98:23-30.Wang, S. Y. and Stickle, W. B. (1986). Changes in nucleic acid concentration with starvation in th e blue crab Callinectes sapidus R athbun. J . C rust. Biol. (Lawrence, Kansas). 6: 49-56.Wang, S. Y. (1986). Tolerance, bioenergetics and biochem ical com position of the blue crab, Callinectes sapidus R athbun, exposed to the w ater soluble fraction of south Louisiana crude oil. Ph.D. diss. 1.011181000 S tate Univ. pp. 103Wang, S. Y., Lum, J . L., Carls, M. G. and Rice, S. D. (1993). Relationship between growth and to tal nucleic acids in juvenile pink salm on, Oncorhynchus gorbuscha, fed crude oil contam inated food. Can. J . Fish. Aquat. Sci. 50(5): 996-1001.110Williams, A. H., Coen, L. D. and Stoelting, M. S. (1990). Seasonal abundance, d istribution, and h ab ita t select ion ofj uvenile Callinectes sap idus (Rathbun) in the n o rth ern G ulf of Mexico.J. Exp. Mar. Biol. Ecol. 137: 164-183.W illiams, A. B. (1984). Shrim ps, lobsters and crabs of the Atlantic coast of th e E astern US M aine to Florida. Sm ithsonian Institu tion Press, USA.W inberg, C. G. (1960). Rate of m etabolism and food requirem ents of fishes. Fish. Res. Bd. C an., Transl. Ser. 194: 202.VITAT apash Das w as b o m in B ihar, India, on Ju ly 3rd, 1961. He g rad u a ted from B urdw an University, India, in 1981 with a B.S. degree in Zoology. He also received his M.S. in Zoology from Kalyani University, India, in 1983. He en tered th e Ph.D. program in th e D epartm ent of Zoology a n d Physiology a t the Louisiana S ta te U niversity in 1989. H is research publications include a s tu d y on the responses of b lue c rab s and sou th ern oyster drill to hypoxia and anoxia (Marine Ecology Progress Series 98: 263-274, 1993).I l lDOCTORAL EXAMINATION AND DISSERTATION REPORTCandidate:Major Field:Title of Dissertation:Date of Examination:Tapash Das ZoologyPhys io logical and Biochemical R e s p o n s e s to Hypoxiain the Blue Crab, Call inectes s ap id us Rathbun , theL esser Blue Crab, (T. similis Williams, and the SouthernO y s te r Dril l, Stramonita haemastoma Linnaeus Approved:Major Professor and ChairgftSnEXAMINING COMMITTEE:/ ' / / ' A' / // — v ;------------ r _ ^ -----------------------------ur~r-' ' ■' yj /TT —/Jfr'SXfU- W; C'V^//\ i S M /! s i s ' / s i ,October 29, 1993

Gene

Abstract

Extracted data

Gene

Abstract

Extracted data

Related items

Related items