Molecular indicators of hypoxia in the blue crab Callinectes sapidus

Abstract

Occurrence of hypoxia in estuarine waters is increasing, and recovery of impacted estuaries is slow. Detection of early effects of hypoxia is needed for timely remedial action to be taken. Here we examine the use of hypoxia-responsive gene and protein expression profiles in the blue crab Callinectes sapidus as early indicators of impacts of hypoxia. We cloned 23 potential hypoxiaresponsive genes, which were used to construct gene macroarrays. Crabs exposed to chronic hypoxia (2.5 ppm dissolved oxygen [DO]) for 15 d showed significant (p < 0.05) changes in gene expression of heat shock protein 70 (Hsp70), copper metallothionein (CuMt3), cytosolic MnSOD (cyt-MnSOD) and ribosomal proteins S15 and L23. In all cases except for CuMt3, gene expression decreased after 5 d exposure to hypoxia. Expression of Hsp70, CuMt3 and cyt-MnSOD also increased (p < 0.05) in normoxic crabs held for 15 d, suggesting confounding effects from confinement stress. Hemocyanin protein concentrations changed significantly (p = 0.005) across the 15 d chronic hypoxia exposure. Hemocyanin in crabs exposed to 10 d intermittent hypoxia (2.5 to 8 ppm DO over a 24 h cycle) did not change, but cyt-MnSOD gene expression increased significantly (p = 0.037), whereas cytochrome c oxidase subunit 1 (ccox1) showed a 2.2-fold downregulation. Blue crabs collected from Pensacola Bay, Florida, showed significant (p < 0.006) downregulation of ccox1 and cyt-MnSOD gene expression as well as hemocyanin protein levels at a diurnally hypoxic marsh site. Several hypoxiaresponsive genes (elongation factor 2, cryptocyanin, hemocyanin) were also significantly elevated (p < 0.006) in intermolt versus premolt normoxic crabs. The identification of hypoxia-responsive genes and proteins in the blue crab is a promising first step towards the development of sensitive molecular tools for the detection of sublethal effects of hypoxia in estuarine-resident species.