Gene Expression Profile of Grass Shrimp Palaemonetes pugio Exposed to Chronic Hypoxia

DNA microarrays have become an important tool to measure global gene expression changes and genetic pathways involved in response to environmental stressors and toxicants. In this study a cDNA microarray was designed and constructed from six libraries of expressed sequence tags generated in a previous study (Li, T., Brouwer, M., 2009. Bioinformatic analysis of expressed sequence tags from grass shrimp Palaemonetes pugio exposed to environmental stressors. Comp. Biochem. Physiol. Part D Genomics Proteomics. doi:10.1016/j.cbd.2009.03.001). The microarrays were used to examine differentially expressed genes in hypoxic vs. normoxic groups at 6 (H6), 12 (H12), 24 (H24), 48 (H48), 120 (H120), and 240 (H240) h exposure to chronic hypoxia (dissolved oxygen (DO) 1.5 mg/L). The initial response to hypoxia was an up-regulation of 29 genes. Only 6 h later, a dramatic down-regulation of 47 genes was observed. There was another reversal with 19 genes being up-regulated and none down-regulated at 24 h. After 2 and 5 days 34 and 22 genes were up-regulated, respectively, and 24 genes were down-regulated and 6 up-regulated by day 10. Cluster analysis confirmed two response patterns, one composed of an up-regulated dominated cluster, including H6, H24, and H120, the other composed of a down-regulated dominated cluster, including H12, H48, and H240. Venn diagrams of differentially expressed genes showed there was no gene up- or down-regulated common to all six groups. Hemocyanin transcription was up-regulated after 24, 48, and 120 h, but down-regulated after 12 h. Some genes appeared unique for specific time points. Phosphoenolpyruvate carboxykinase was up-regulated in the H120 and H240 groups. Cytochrome c oxidase subunit I and C-type lectin were uniquely up-regulated in H12, whereas vitellogenin and trachealess were uniquely down-regulated in H48. GOstats and org.Dm.eg.db packages from R were used to assign GO terms to significantly expressed genes. A total of 291, 129, and 219 genes were assigned to biological process, cellular components, and molecular function, respectively. The most abundant groups of genes were associated with transport, metabolic process, defense response, and proteolysis. Pathways were analyzed using Drosophila metabolic pathways in the KEGG database. Oxidative phosphorylation/Citrate cycle and Ribosome were the most abundant categories for chronic hypoxic exposure. Of 19 selected genes that showed differential expression on the microarrays, 17 showed similar up- or down-regulated patterns in both microarray and qPCR. In conclusion, the custom cDNA microarray is a valid and useful tool to investigate the changes in gene expression of grass shrimp during chronic hypoxia exposure. Some genes, such as those coding for hemocyanin, ATP synthase, phosphoenolpyruvate carboxykinase, vitellogenin, trachealess, cytochrome c oxidase subunit I, lysosomal thiol reductase, and C-type lectin, could be used as molecular indicators of chronic hypoxia at specific time points. However, changes of these significant genes were too dynamic to serve as generic biomarkers of hypoxia stress in grass shrimp for the whole duration of the chronic hypoxia exposure.