Quantification of Stress in Lobsters: Crustacean Hyperglycemic Hormone, Stress Proteins, and Gene Expression

Abstract

Various methods for the quantification of stress in crustaceans have been developed in our laboratory. An ELISA was developed for the crustacean hyperglycemic hormone (CHH) from the lobster, Homarus americanus . It is sensitive to as little as 0.2 fmol of peptide. Increases in hemolymph CHH were observed following emersion. Significant levels of hemolymph CHH were also measured in lobsters that had been eyestalk-ablated. It was observed that these animals continued to produce CHH, even though the heretofore only known source of CHH had been removed. Portions of the central nervous system, from both intact and eyestalk-ablated lobsters were observed to contain significant amounts of CHH. A cDNA library was constructed from eyestalk neural tissue of H. americanus . With the use of PCR, a 171 bp probe was isolated and purified. This probe was labeled and used to examine levels of CHH expression in the central nervous system (CNS) and in eyestalk neural tissue at different periods of the lobster molt cycle. CHH mRNA is present throughout the CNS. In the eyestalk, it is undetectable in postmolt, low in intermolt, and high in premolt. Stress proteins, also known as heat shock proteins (HSPs), are a highly conserved class of proteins which show elevated transcription during periods of stress in organisms as phylogenetically divergent as bacteria and humans. Using RT-PCR, we have partially cloned the lobster HSP90 gene. A 380 bp probe was 32P-labeled and hybridized with northern blots of midgut gland total RNA from heat-shocked lobsters. A 2 hr acute heat shock from 15%C (ambient water temperature) to 28%C resulted in a 6.0-fold induction of HSP90 after 6 hr of recovery at 15%C. A northern analysis of RNA isolated from the midgut glands of lobsters injected with 10 μg of the molting hormone 20-hydroxyecdysone displayed a 2.1-fold induction of HSP90 RNA 48 hr postinjection.