In-vitro model of interaction between Vibrio splendidus and hemocytes of soft-shell clams, Mya arenaria.

摘要

The main goal of this thesis is to develop an in-vitro model of interaction between hemocytes of soft-shell clams and Vibrio splendidus LGP32, and investigate the early immune response of soft-shell clams both at cellular and molecular levels.;In hemocytes challenged for two hours, phagocytosis and respiratory burst activities were investigated to understand the early response of hemocytes against V. splendidus. Hemocytes of soft-shell clams showed the ability to phagocytose and produce reactive oxygen/nitrogen species. This phagocytosis and respiratory burst activity of hemocytes was significantly reduced (p0.001) post-Vibrio exposure. The reduction in phagocytosis with an increasing percentage of rounded cells suggests that hemocytes pseudopodia (cytoplasmic extensions) could play a key role in internalizing microbes. Consequently, V. splendidus seems to affect the function of hemocytes by modifying their morphology.;Besides the morphological modifications, this research also focused on the expression of genes in hemocytes post Vibrio challenge. The selected candidate genes included ribosomal proteins (rpS-15, rpS-18 and L-37), ubiquitin, receptor for activated C kinase (RACK), elongation factor 1 (EF-1) and elongation factor 2 (EF-2) as well as 18S-rRNA and actin. Our data analysis showed that actin and 18S-rRNA, usually used as housekeeping genes, were among the least stable transcripts, whereas EF-1, rpS-18 and ubiquitin were the most stable transcripts and were used as housekeeping genes in this in-vitro interaction model. Genes associated with cytoskeleton structural stability (actin and EF-2) were among least stable genes. Consequently, the expression of both genes was quantified and showed significant up-regulation (actin p0.001; EF-2 p0.05) in hemocytes challenged for two hours at a ratio 1:1.;To explore the effect of V. splendidus at the molecular level, suppression subtractive hybridization (SSH) technique was used to identify the up- and down-regulated genes in hemocytes of soft-shell clams. Both forward and reverse subtracted cDNA were constructed and a total of 16,000 reads were obtained and analyzed. Identity searches in protein databases were performed using BlastX program and the transcripts were clustered to different physiological processes including structural proteins, immunity, stress proteins, apoptosis, cell process, general metabolism and signal transduction. The expression level of transcripts associated with immunity such as ficolin, killer cell lectin-like receptor, natural resistance-associated microfiche protein 1, MAPK, ferritin, HSP90 and cathepsin were quantified using RT-qPCR. Some of these genes play a key role in pathogen recognition, phagocytosis, respiratory burst activity and activating innate immune response in general. The expression pattern in most transcripts showed an up-regulation at one hour followed by a down-regulation at two and three hours.;Hemocytes of soft-shell clams are actively involved in immune defence. However, they lost their pseudopodia and became rounded after V. splendidus challenge. This phenotypic response of hemocytes (measured in percentage of rounded cells) shows a positive correlation between the ratio of hemocytes:bacteria (1:20, 1:10, 1:5 and 1:1) and the time of exposure. At a high number of bacteria per hemocyte (1:20, 1:10 and 1:5), the percentage of rounded (without pseudopodia) hemocytes increased dramatically from less than 20% in one hour to 80-90% in two. At 1:1 hemocyte to Vibrio ratio however phenotypic response was gradual (4.3% at 1 hour, 44.2% at 2 hours and 92% at 3 hours) and consequently, the ratio 1:1 was used in all the following experiments. Hemocyte viability in challenged (1:1 hemocyte to Vibrio) and control groups was very high (>91%) suggesting morphologically modified cells were alive.;This study showed that hemocytes of soft-shell clams are actively involved in immune defence. However, exposure to V. splendidus impaired their cellular functions suggesting the virulent nature of the bacterium. Moreover, several novel immune associated genes such as Nramp 1, ficolin, T cell receptor and Killer cell lectin-like receptor were identified in this study. Further investigations are needed to characterize and understand the role of those transcripts in the immune defence of soft-shell clams. (Abstract shortened by UMI.)