Identification of the molecules involved in zebra mussel (Dreissena polymorpha) hemocytes host defense

Abstract

The invasion of North American waterbodies by the zebra mussel (Dreissena polymorpha) has caused ecological catastrophies in North America. Unfortunately, little is known about this nuisance mollusk and its host defense mechanisms. In this study, 32 expressed sequence tags (ESTs) associated with hemocyte stimulation were obtained from a suppression subtractive hybridization (SSH) cDNA library. This SSH-cDNA library was produced by using a cDNA library of naïve hemocytes as the driver and a cDNA library of hemocytes stimulated with a mixture of microbial antigens namely, lipopolysacchride (LPS), peptidoglycan (PGN), and zymosan (ZYM), as the tester. The driver cDNA was subtracted from the tester to increase the relative abundance of the cDNAs that were induced by stimulations. The putative function of 27 ESTs were obtained by using the homologue searching program BLASTx and BLASTn. Four ESTs encoding the protein product homologous to matrilin (Matrn, AM503947), heat shock protein 70 (HSP70, EU835391), seryl-tRNA synthetase (STS, AM503950), and glycine-rich protein (GRP, AM502279) were selected for a subsequent study using quantitative PCR (qPCR) assays with the RNA extracted from hemocytes stimulated with LPS alone. The results of quantitative PCR with stimulated hemocyte RNA demonstrated that the four candidate genes were upregulated by LPS stimulation. The expression levels of both HSP70 and Matrn genes between naïve and 1-h stimulated hemocyte samples are the most significant with 2.78 and 2.20 fold increases, respectively. The significant changes of GRP and STS genes were observed after 2-h stimulation. The phylogenetic analysis of HSP70 molecule indicated that this protein is phylogenetically close to the HSP70 identified from other mollusks. This study shed light on hemocyte-mediated host defense mechanisms of D. polymorpha.