Environmental and Comparative Genomics Section, Department of Environmental and Population Health, Cummings School of Veterinary Medicine at Tufts University
North Grafton, MA, USA
Cadmium (Cd) is a naturally occurring toxic heavy metal with no known biological function in humans and animals. This nonessential metal is considered a carcinogen and at chronic, sublethal levels can be detrimental to humans, targeting renal epithelial cells and gonads.1,4 Cd is a hazardous element in aquatic environments where fish, crustaceans, and mollusks are found to accumulate it in their bodies.3 The effects of heavy metals on the biology and gene expression of the commercially important marine shrimp, Penaeus vannamei, remain unclear. The natural range of P. vannamei extends from northern Mexico to northern Peru and is the principle species farmed in the western hemisphere. It is hypothesized that Cd exerts effects on shrimp at both biological and molecular levels. Specific pathogen free (SPF) P. vannamei postlarvae (PL) from the US Marine Shrimp Farming Program were exposed to a range of CdCl2 concentrations (0, 0.01, 0.1, 1.0, 10.0 ppm) for a 48-hour period. This bioassay was conducted to 1) determine histological changes due to Cd exposure, 2) measure Cd concentrations in PLs after Cd exposure. Additionally, an extensive literature search was performed to 3) determine usability of Cd-related ESTs for linkage mapping in shrimp (ShrimpMap) using primers developed from published Cd-related sequences (human, mouse, fish, nematode) and a shrimp DNA test panel including samples (n = 8) of the International Resource Mapping Family (IRMF). Results showed 1) variable histologic changes in shrimp exposed to different concentrations of Cd. Histological lesions were limited to the integument, musculature, gills, hepatopancreas and midgut/hindgut. None were associated with any evident infectious etiology (i.e., viruses, bacteria, fungi and protozoa). Gills more frequently showed degeneration and necrosis of superficial/epithelial layers. Infrequent lesions were found in the midgut/hindgut, varying from mild (necrosis and sloughing of scattered epithelial cells) to severe (segmental ulceration with expansion of the lamina propria by hemocytes and distention of gut lumen by sloughed cells).2 2) P. vannamei PLs do accumulate Cd with a statistically significant (r2 = .924, p<0.0001) correlation between Cd concentrations (ppm) found in PLs and exposure concentrations. After 48 hours of exposure to 1 ppm CdCl2, PLs contained 15.82 ppm Cd compared to 0.35 ppm Cd in the control (0 ppm) group. 3) Nineteen of the 54 primers have been optimized and genotyped with the test panel at different annealing temperatures (44°C, 48°C, 52°C). Of these, 7 were polymorphic, 5 were monomorphic, and 7 need to be further optimized before genotyping is continued. The 9 polymorphic markers correlated with human genes up-regulated by Cd exposure. Four of the 9 polymorphic markers were tested with the entire IRMF panel (n = 92), and two of these were successfully genotyped. Primers flanking single or multiple SSRs of vertebrate genomes could be an efficient approach to develop markers of expressed genes useful for linkage mapping and eventually microarray studies in shrimp. Overall, the results presented here will assist not only in our understanding of the effect of Cd on shrimp gene expression and organization of the shrimp genome but also for functional and comparative genomics purposes.
This work was partially supported by the USDA-CSREES through a grant to the United States Marine Shrimp Farming Program (USMSFP, to Dr. Acacia Alcivar-Warren, Principal Investigator), the Environmental and Comparative Genomics Section at DEPH, TCSVM, and the Pathology Section of the Clinical Sciences Department at TCSVM. Martha Delaney was supported by NIH Summer Training Grant # T35DK07635.
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