TA2 Molecular Indicators for Ecological Exposure
255 Portland Ballroom
8:00 AM - 12:00 PM, Tuesday
() Molecular and organismal indicators of effects of chronic and intermittent hypoxia in estuarine crustacea.
Brouwer, M1, Larkin, P3, Brown-Peterson, N1, King, C1, Manning, S1, Denslow, N2, 1 University of Southern Mississippi, Ocean Springs, MS, USA3 EcoArray LLC, Alachua, FL, USA2 University of Florida, Gainesville, FL, USA
ABSTRACT- To test the hypothesis that hypoxia-induced changes in gene expression may serve as molecular indicators of hypoxic stress we cloned gene transcripts from blue crab and grass shrimp proteins, predicted to be responsive to the intracellular redox state. Additional hypoxia-responsive genes were identified using PCR-select subtractive hybridization. The cDNAs were used to construct macroarrays. Next we exposed blue crabs and grass shrimp to chronic and intermittent hypoxia in a flow-through system and examined effects on gene expression in both species, while grass shrimp were also used to examine reproductive effects. The macroarrays were hybridized with 33P- labeled cDNA prepared from RNA from control and hypoxia-exposed crabs and shrimp and from crabs and shrimp collected at hypoxic and normoxic field sites. Analysis of crab tissues has been completed. Transcript levels of many genes, including those for ribosomal proteins, hemocyanin, MnSOD, CuMT, and Hsp 70 change significantly in response to chronic hypoxia, but not in response to diurnal hypoxia. Interpretation of results of field studies was complicated by changes in gene expression associated with molt cycle changes. At the protein level, Western Blots showed significantly increased hemocyanin concentrations in crabs exposed to hypoxic conditions for 10 days, whereas hemocyanin in crabs from field sites with diurnal hypoxia showed reduced hemocyanin concentrations. An unexpected, and as yet unexplained, crosslinking of MnSOD protein was observed upon hypoxia treatment of both crabs and shrimp. Reproductive studies showed grass shrimp fecundity was significantly elevated in animals exposed to moderate and severe hypoxia. In addition, eggs produced by hypoxic animals had higher triglycerides content than eggs from normoxic animals. These results suggest that hypoxia-responsive macroarrays and Westerns might be useful for monitoring effects of hypoxia in estuarine crustacea and that it might be possible to link these molecular signals to reproductive fitness. Supported by EPA-STAR Grant R 82945801.
Key words: gene arrays, hypoxia, crabs