Research Area 2. Response to bacteria impairs metabolism and performance in crustaceans

All organisms must adapt to changes in their environment while maintaining the ability to grow, avoid predation and reproduce. Estuarine organisms may be at particular risk from the adverse effects of low dissolved oxygen levels (hypoxia). Penaeid shrimp and brachyuran crabs, two key groups of organisms that occupy estuarine habitats, also must live in intimate contact with extremely high densities of microorganisms and particulate matter found in the water column and sediment. In addition to its critical roles in gas exchange and osmoregulation, the crustacean gill serves as a site for eliminating bacteria and other foreign particles that penetrate the exoskeleton. But the role of the gill in eliminating pathogens may compromise its other functions. We have documented that in the penaeid shrimp Litopenaeus vannamei, injection of a sublethal dose of the bacterium Vibrio campbellii caused a large and persistent decrease in aerobic metabolism, accompanied by an increase in whole body lactate (Scholnick et al., 2006). In the blue crab Callinectes sapidus, bacterial challenge induced a decrease in postbranchial oxygen tension and an increase in vascular resistance across the gills followed by a sustained decrease in oxygen uptake (Burnett et al., 2006). Based on these and related findings, we hypothesize that, even in fully air-saturated estuarine waters, oxygen uptake may be impaired in crustaceans exposed to sublethal doses of bacteria leading to alterations in energy metabolism and aerobic performance. Currently we are testing this hypothesis using several approaches. Graduate student Nat Johnson is determining whether injection of monocultures and complex mixtures of other bacteria, purified bacterial lipopolysaccharides and inert particles can replicate the changes in aerobic metabolism in the blue crab that we observe with V. campbellii. Nat is monitoring changes in oxygen uptake, hemolymph glucose and lactate and numbers of circulating hemocytes (immune cells) in blue crabs. He expects to show that a wide range of particulate antigens within a defined size range will impair respiration and that, after molting, hemolymph flow in the gills and oxygen uptake will return to pre-test rates. In another component of this research, graduate student Lindy Thibodeaux tested whether impaired gas exchange following exposure to bacteria altered metabolism and performance in crustaceans. She quantified aerobic respiration and individual endproducts of aerobic and anaerobic metabolism in hemolymph and whole body of blue crabs challenged with a dose of bacteria and then required to exercise on a treadmill. The results of her study, Thibodeaux et al., submitted) suggested a depression of metabolism in response to bacteria that persists during moderate activity. The mechanisms underlying this metabolic depression will form the basis of future investigations in our lab. In a third component of these studies, we are working with collaborating NIST scientist Dan Bearden and postdoctoral fellow Tracey Schock to explore an NMR-metabolomic approach to quantifying changes in hemolymph metabolites of blue crabs challenged with bacteria. In Summer 2009 undergraduate researcher David Stancyk worked with Drs. Bearden and Schock to show that blue crabs injected with bacteria display significant changes in small metabolites of the hemolymph that vary over time (Schock et al., submitted). In a separate effort, we plan to test whether whether the decline in aerobic metabolism following a bacterial challenge induces transcription of genes associated with the response to hypoxia. As a first step in that effort, graduate student Kolo Rathburn has used microarrays for L. vannamei to assess changes in gene expression associated with moderate hypoxia alone or in combination with high CO₂ (hypercapnia). Among the set of genes whose expression is significantly altered under these test conditions, genes regulating transcription and translation occur in high frequency. These results indicate that shrimp exposed to sublethal hypoxia experience a decline in RNA and protein synthesis, consistent with a depression of metabolism. Postdoctoral researcher Natasha Sharp has recently begun a complementary study to examine the transcriptional response of L. vannamei to sublethal bacterial challenge. From these microarray experiments we hope to identify the regulatory pathways responsible for the concordance between observed alterations in respiration, hypoxia-induced immune suppression and the host immune response to bacteria.