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Resilience and sensitivity to environmental stress in the American oyster, Crassostrea virginica
Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S155–S161
dominant further north. In order to characterize the cellular response of both species to temperature and salinity stress, we are applying a proteomics approach by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Both species were acclimated to a common temperature of 13 °C for 3 weeks and subsequently exposed to 24 °C, 28 °C and 32 °C for 1 h. Mussels were either collected right after acute exposure or given a 24 h recovery period at 13 °C. We detected up to seven heat shock protein 70 isoforms that differ in isoelectric point in both congeners. The greatest differences between the congeners were found in heat induced proteins that are smaller than 30 kDa. We are currently attempting to identify these protein spots using peptide mass fingerprinting and de novo sequencing of peptides. In addition, we exposed both congeners to full-strength (100%) and diluted (85% and 70%) sea water for 4 h followed by recovery in sea water for 24 h. The goal of our ongoing analysis is to compare the acute stress proteome of gill tissue between the congeners in order to identify systems properties that determine the species' tolerance limits. doi:10.1016/j.cbpa.2008.04.405
C3.8 The metabolic consequences of immune defense coupled with the demands of exercise in marine crustaceans
S157
osmotic, or hydrostatic pressure stresses. They have been found in a variety of organisms from Archaea to metazoans, including vertebrates. Rapid accumulation of counteracting osmolytes during the onset of stress may provide a graded and reversible source of protection to cytosolic proteins, increasing the likelihood of survival in variable environments. In metazoans, however, the extent and rate of accumulation of such solutes during acclimation to environmental stress remains to be explored. We are using HPLCMS/MS to determine solute composition in tissues of mussels (genus/Mytilus/) before and after acute temperature stress. We have found that, compared to 13 °Cacclimated control individuals, gill solute composition changes dramatically in /M. galloprovincialis/ exposed to 32 °C for 1 h, with 33 of the ~120 most prevalent solutes (as measured by peak intensity in the mass spectrum) increasing or decreasing by at least two-fold. Interestingly, all of the solutes showing significant changes in peak intensity return to control levels in individuals who are allowed to recover for 24 h after the 32 °C incubation. These marked changes in gill solute composition after heat stress likely include shifts in both metabolite and counteracting osmolyte composition. The MS technique we have developed provides a rapid description of changes in tissue solute composition, but has limited ability to identify individual solutes. Thus current work focuses on developing techniques to identify those ions that show the greatest change in intensity between control and heat-exposed individuals. doi:10.1016/j.cbpa.2008.04.407
L. Burnett, K. Burnett (College of Charleston) Crustaceans have strong and rapid responses to the injection into their tissues of the bacterial pathogen Vibrio campbellii. Vibrio are removed from tissues within hours. One of the consequences of the injection of a sublethal dose of bacteria is a large 30% reduction in oxygen uptake, which occurs within hours and which is further reduced to 40% after 24 h in the penaeid shrimp Litopenaeus vannamei and the Atlantic blue crab, Callinectes sapidus. In the crab this reduction in oxygen uptake may be caused by the obstruction of hemolymph flow through the gills by hemocyte aggregations associated with the immune response. We have investigated the immunological and physiological responses of these two crustaceans under an additional stress, that of imposed activity. We have used treadmills to induce shrimp to swim and crabs to walk to see if the immune response influences their ability to perform moderate exercise. Furthermore, in the blue crab we have assessed the energy metabolism during and after walking for 30 min. The reduction in oxygen uptake is a result of an overall depression of metabolism with only small amounts of metabolism explained by anaerobic mechanisms. Metabolic rate increases during walking more than two times in both control and Vibrio-injected crabs. Small amounts of lactate accumulate in the tissues and some is excreted in both groups. In well-oxygenated water crabs and shrimp appear to tolerate exercise well. It will be interesting to see if variables such as hypoxia influence the ability to exercise. Support: NSF IBN0212921. doi:10.1016/j.cbpa.2008.04.406
C3.9 Changes in the concentrations of counteracting osmolytes in response to environmental stress P. Fields, E. John (Franklin and Marshall College) Counteracting osmolytes are small molecular weight solutes that protect globular proteins from denaturation due to high temperature,
C3.10 Resilience and sensitivity to environmental stress in the American oyster, Crassostrea virginica K. Burnett (College of Charleston); H. Williams (College of Charleston); B. Macey (College of Charleston); A. Mancia (Medical University of South Carolina); P. Gross (Medical University of South Carolina); G. Warr (Medical University of South Carolina); R. Chapman (South Carolina Department of Natural Resources); L. Burnett (College of Charleston) Filter-feeding exposes oysters to large numbers of microorganisms and contaminants when they occur in the surrounding water and sediment. Recent studies from our laboratory provide some insight to the strategy that oysters use against invading pathogens. Minutes after bacteria are injected into the adductor muscle, oysters can limit the ability of the bacteria to grow; however, bacteria remain intact in tissues for days. This same immune strategy may make oysters particularly sensitive to hypoxia. Hypoxia, accompanied by high levels of high levels of CO2 (hypercapnia) reduces bacteriostatic activity in oyster tissues, increasing the opportunity for bacteria to establish infections. In the present study we asked whether long term (28 d) exposure to low levels of a metal contaminant, cadmium (50 ppb), would alter bacteriostasis and/or degradation of bacteria in oysters. Simultaneously, we looked for impacts of Cd on cells of the immune system by assessing total hemocyte count (THC) in hemolymph, and in the gills and the digestive gland by assessing gene expression profiles using cDNA microarrays. We found that, although THC decreased with Cd exposure, neither bacteriostasis nor degradation of injected bacteria was altered. Genes associated with metabolism, detoxification, tissue damage and repair were differentially expressed in the gills of Cdexposed oysters. In the digestive gland most transcriptional changes were associated with detoxification and the immune response. Thus, in response to environmental stress, each tissue examined mounted a distinct response but the oyster maintained an effective immune defense (NOAA's Center of Excellence in Oceans and Human Health). doi:10.1016/j.cbpa.2008.04.408
dominant further north. In order to characterize the cellular response of both species to temperature and salinity stress, we are applying a proteomics approach by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Both species were acclimated to a common temperature of 13 °C for 3 weeks and subsequently exposed to 24 °C, 28 °C and 32 °C for 1 h. Mussels were either collected right after acute exposure or given a 24 h recovery period at 13 °C. We detected up to seven heat shock protein 70 isoforms that differ in isoelectric point in both congeners. The greatest differences between the congeners were found in heat induced proteins that are smaller than 30 kDa. We are currently attempting to identify these protein spots using peptide mass fingerprinting and de novo sequencing of peptides. In addition, we exposed both congeners to full-strength (100%) and diluted (85% and 70%) sea water for 4 h followed by recovery in sea water for 24 h. The goal of our ongoing analysis is to compare the acute stress proteome of gill tissue between the congeners in order to identify systems properties that determine the species' tolerance limits. doi:10.1016/j.cbpa.2008.04.405
C3.8 The metabolic consequences of immune defense coupled with the demands of exercise in marine crustaceans
S157
osmotic, or hydrostatic pressure stresses. They have been found in a variety of organisms from Archaea to metazoans, including vertebrates. Rapid accumulation of counteracting osmolytes during the onset of stress may provide a graded and reversible source of protection to cytosolic proteins, increasing the likelihood of survival in variable environments. In metazoans, however, the extent and rate of accumulation of such solutes during acclimation to environmental stress remains to be explored. We are using HPLCMS/MS to determine solute composition in tissues of mussels (genus/Mytilus/) before and after acute temperature stress. We have found that, compared to 13 °Cacclimated control individuals, gill solute composition changes dramatically in /M. galloprovincialis/ exposed to 32 °C for 1 h, with 33 of the ~120 most prevalent solutes (as measured by peak intensity in the mass spectrum) increasing or decreasing by at least two-fold. Interestingly, all of the solutes showing significant changes in peak intensity return to control levels in individuals who are allowed to recover for 24 h after the 32 °C incubation. These marked changes in gill solute composition after heat stress likely include shifts in both metabolite and counteracting osmolyte composition. The MS technique we have developed provides a rapid description of changes in tissue solute composition, but has limited ability to identify individual solutes. Thus current work focuses on developing techniques to identify those ions that show the greatest change in intensity between control and heat-exposed individuals. doi:10.1016/j.cbpa.2008.04.407
L. Burnett, K. Burnett (College of Charleston) Crustaceans have strong and rapid responses to the injection into their tissues of the bacterial pathogen Vibrio campbellii. Vibrio are removed from tissues within hours. One of the consequences of the injection of a sublethal dose of bacteria is a large 30% reduction in oxygen uptake, which occurs within hours and which is further reduced to 40% after 24 h in the penaeid shrimp Litopenaeus vannamei and the Atlantic blue crab, Callinectes sapidus. In the crab this reduction in oxygen uptake may be caused by the obstruction of hemolymph flow through the gills by hemocyte aggregations associated with the immune response. We have investigated the immunological and physiological responses of these two crustaceans under an additional stress, that of imposed activity. We have used treadmills to induce shrimp to swim and crabs to walk to see if the immune response influences their ability to perform moderate exercise. Furthermore, in the blue crab we have assessed the energy metabolism during and after walking for 30 min. The reduction in oxygen uptake is a result of an overall depression of metabolism with only small amounts of metabolism explained by anaerobic mechanisms. Metabolic rate increases during walking more than two times in both control and Vibrio-injected crabs. Small amounts of lactate accumulate in the tissues and some is excreted in both groups. In well-oxygenated water crabs and shrimp appear to tolerate exercise well. It will be interesting to see if variables such as hypoxia influence the ability to exercise. Support: NSF IBN0212921. doi:10.1016/j.cbpa.2008.04.406
C3.9 Changes in the concentrations of counteracting osmolytes in response to environmental stress P. Fields, E. John (Franklin and Marshall College) Counteracting osmolytes are small molecular weight solutes that protect globular proteins from denaturation due to high temperature,
C3.10 Resilience and sensitivity to environmental stress in the American oyster, Crassostrea virginica K. Burnett (College of Charleston); H. Williams (College of Charleston); B. Macey (College of Charleston); A. Mancia (Medical University of South Carolina); P. Gross (Medical University of South Carolina); G. Warr (Medical University of South Carolina); R. Chapman (South Carolina Department of Natural Resources); L. Burnett (College of Charleston) Filter-feeding exposes oysters to large numbers of microorganisms and contaminants when they occur in the surrounding water and sediment. Recent studies from our laboratory provide some insight to the strategy that oysters use against invading pathogens. Minutes after bacteria are injected into the adductor muscle, oysters can limit the ability of the bacteria to grow; however, bacteria remain intact in tissues for days. This same immune strategy may make oysters particularly sensitive to hypoxia. Hypoxia, accompanied by high levels of high levels of CO2 (hypercapnia) reduces bacteriostatic activity in oyster tissues, increasing the opportunity for bacteria to establish infections. In the present study we asked whether long term (28 d) exposure to low levels of a metal contaminant, cadmium (50 ppb), would alter bacteriostasis and/or degradation of bacteria in oysters. Simultaneously, we looked for impacts of Cd on cells of the immune system by assessing total hemocyte count (THC) in hemolymph, and in the gills and the digestive gland by assessing gene expression profiles using cDNA microarrays. We found that, although THC decreased with Cd exposure, neither bacteriostasis nor degradation of injected bacteria was altered. Genes associated with metabolism, detoxification, tissue damage and repair were differentially expressed in the gills of Cdexposed oysters. In the digestive gland most transcriptional changes were associated with detoxification and the immune response. Thus, in response to environmental stress, each tissue examined mounted a distinct response but the oyster maintained an effective immune defense (NOAA's Center of Excellence in Oceans and Human Health). doi:10.1016/j.cbpa.2008.04.408