Altered gene expression associated with epizootic shell disease in the American lobster, Homarus americanus

Abstract

Epizootic shell disease is a poorly understood condition that has significantly affected the American lobster fishery in New England (northeastern US) since the 1990s. Here we present the results of a study to identify changes in gene expression in lobsters exhibiting symptoms of epizootic shell disease. Suppressive subtractive hybridization (SSH) was used to compare gene expression between cDNA pools from diseased (symptomatic) and apparently healthy (asymptomatic) lobsters. Subsequently, quantitative real-time polymerase chain reaction (qPCR) was used to measure expression of nine genes that were differentially-expressed in the SSH analysis, in seven tissues (muscle, gill, heart, hepatopancreas, brain, branchiostegite, gonad) dissected from individual symptomatic and asymptomatic lobsters. Expression of arginine kinase (involved in cellular energetics) was significantly decreased in muscle of symptomatic lobsters. Expression of hemocyanin (a respiratory hemolymph protein involved in oxygen transport) was highest in hepatopancreas and showed highly variable expression with a trend toward higher expression in asymptomatic individuals. 2-Macroglobulin (involved in the innate immune system) was most highly expressed in the ovary, particularly of symptomatic lobsters. The ESTs produced through this study add to the fledgling field of crustacean genomics and revealed three genes that could be further evaluated in lobsters of varying shell disease severity, molt stage, and reproductive condition, for possible implication in epizootic shell disease.

Gene Expression in American Lobster (Homarus americanus) with Epizootic Shell Disease

“…AK expression was higher during late postmolt (C3) and intermolt (C4) than during premolt. Consistent with our previous results (Tarrant et al 2010), AK expression was higher in lobsters with no signs of ESD than in those exhibiting signs of disease. EcR expression showed a significant interaction (P ¼ 0.015) between molt stage and disease condition.…”

“…Sequences for AK, a2-macroglobulin, hemocyanin (Tarrant et al 2010), ecdysteroid receptor, and retinoid X receptor (Tarrant et al 2011) were described previously. The remaining sequences were identified through searches of the NCBI database, including the EST database, and annotated provisionally based on sequence identity with known genes (Table 2).…”

“…Expression of each gene was measured in thoracic muscle, ovary, and/or hepatopancreas, with tissues selected on the basis of previous studies (e.g., Snyder 1998, Chang 2005, Tarrant et al 2010) and the known or hypothesized gene function. That is, genes were amplified 18,19,20,42,45,58,67), and moderate to severe ommatidial lesions (covering $ 66% of area in crosssection, 7, 14, 21).…”

“…from the tissues in which they were previously shown to be differentially expressed (e.g., AK in muscle (Tarrant et al 2010)) or from tissues in which they were thought to be active (e.g., xenobiotic metabolism by several cytochrome P450 (CYP) enzymes in the mammalian liver, which corresponds to the lobster hepatopancreas). Ecdysteroid receptor (EcR) and retinoid X receptor (RXR) primers were designed to amplify both known isoforms of each gene (Tarrant et al 2011).…”

“…Our working hypothesis is that the identification of such genes and quantification of their expression with disease state could lead to a mechanistic understanding of ESD. We previously conducted a suppressive subtractive hybridization as a conceptually unbiased way to identify genes differentially expressed in lobsters exhibiting signs of ESD (Tarrant et al 2010). Next, we evaluated the expression of the differentially expressed genes in a small number of lobsters and identified arginine kinase (AK), hemocyanin, and a2-macroglobulin as genes of interest for further investigation.…”