Identification and expression of immune-related genes in hemocytes of soft-shell clams, Mya arenaria, challenged with Vibrio splendidus.

Although the mollusc immune system has been studied at the cellular level, the response to pathogens at gene expression level has not been thoroughly investigated. This study aimed to investigate the early molecular response of hemocytes of soft-shell clams, Mya arenaria, to Vibrio splendidus strain LGP32 by identification of transcripts involved in immune defense. The Suppression Subtractive Hybridization (SSH) was used to selectively identify differentially expressed genes in hemocytes exposed to V. splendidus at a ratio 1:1 for 2 h. Both forward and reverse subtracted cDNA were constructed and a total of 16,000 reads were obtained and analyzed. Identity searches in genome databases were performed using BlastX program and transcripts were clustered to cellular functions including structural proteins, immunity, stress proteins, apoptosis, cell process, metabolism and signal transduction. Among the differentially expressed immune associated genes were ficolin, killer cell lectin-like receptor, natural resistance-associated macrophage protein 1 (Nramp-1), mitogen-activated protein kinases (MAPK), ferritin, heat shock proteins 90 (HSP90) and cathepsin and their expressions were quantified using Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) at 1, 2 and 3 h post-Vibrio challenge. These genes showed similar expression patterns, up-regulation at 1 h, followed by a down-regulation at 2 and 3 h. These data corroborates our previous observations of cell rounding, reduced phagocytosis and respiratory burst activity. To our knowledge, this is the first study to demonstrate an effect of V. splendidus on expression of genes related to immune system in soft-shell clams M. arenaria. However, further investigations are needed to unravel the molecular mechanisms of hemocytes subjected to V. splendidus.

Selection and evaluation of housekeeping genes for haemocytes of soft-shell clams (Mya arenaria) challenged with Vibrio splendidus.

Gene expression studies have opened a tremendous field of investigation in biological research over the last decades. Expression of genes is most frequently quantified by real time PCR (RT-qPCR), as this method has proven to be highly sensitive. One of the critical steps, however, in comparing transcription profiles is the availability of selected housekeeping genes. Expression of these genes should be steadily stable across the conditions under study so that they provide a baseline for gene expression comparison. Such a baseline is best established using a set of few housekeeping genes. Usually, those genes are involved in maintaining homeostasis and cell viability. In our study, nine candidate genes were used, including some commonly used housekeeping genes, such as ribosomal RNA (18S, S-15, S-18 and L-37), beta actin, ubiquitin, receptor activated C kinase (RACK) and elongation factor 1 and 2, in order to determine the most stable housekeeping genes, after haemocytes of Mya arenaria were exposed to Vibrio splendidus for 2 h. Our results showed that EF-1, S-18 and ubiquitin appear to be the most stable genes for this experimental condition. On the other hand, both 18S and beta actin, the most widely used housekeeping genes, turned out to be the least stable. This demonstrates the absolute need for preliminary assessment of housekeeping genes in gene expression studies.