PROTEIN-LEVEL CHARACTERIZATION OF HEMOCYANIN ISOFORMS IN THE PACIFIC WHITELEG SHRIMP, Litopenaeus vannamei AND ISOFORM EXPRESSION FOLLOWING HYPOXIA
Hypoxia is common in estuaries and aquaculture systems, and recently, hypoxic zones in coastal waters have been growing in severity. Crustaceans in these environments may adapt to hypoxia by changing their respiratory pigment hemocyanin. Recent transcriptomic work has found several hemocyanin transcripts not previously seen in penaeid shrimp, but their protein-level expression in hypoxia is not well understood. In the present study, classical and proteomic approaches were used to characterize hemocyanin oligomers and monomers in the Pacific whiteleg shrimp, <i>Litopenaeus vannamei</i>. Shrimp were exposed to hypoxia (4.0 kPa Po<sub>2</sub>) for 1 or 14 days with time-matched normoxic controls. Hemocyanin forms and O<sub>2</sub> binding properties were compared between treatments. <i>L. vannamei</i> hemocyanin is dominantly hexameric (98%) as opposed to dodecameric, and hexamers utilize a significantly higher ratio (2.05:1) of small subunit (HcS) to large subunit (HcL) compared to dodecamers (1.04:1). Five full length hemocyanins predicted by RNA sequencing were identified by LC-MS/MS in both hemocyanin oligomers including three isoforms of HcL and the first protein-level evidence of a β-type subunit in penaeid shrimp. Several immune-related proteins were also identified, mostly related to the phenoloxidase cascasde. Exposure to hypoxia for 1 day resulted in a significantly higher HcS:HcL ratio compared to normoxia, but this ratio returned to normoxic levels after 14 days. <i>L. vannamei</i> did not change its O<sub>2</sub> binding properties over the exposure periods, suggesting that this species is well adapted to hypoxia. The present study provides protein-level confirmation of newly discovered hemocyanin isoforms and shows that their use is different after 1 day exposure to hypoxia compared to normoxia; however future quantitation of isoforms using proteomic techniques such as iTRAQ can further elucidate their roles in hypoxia.