In vivo model of the response of soft -shell clam (Mya arenaria) haemocytes differentially induced by two strains of Vibrio splendidus

Constantly exposed to different microorganisms, molluscs have developed means to recognize and mount defense mechanisms against potential pathogens. Conversely, pathogenic bacteria are able to subvert the host immune system by modulating their responses. Although several studies on host responses have contributed to the understanding of these interactions in various bivalves challenged with bacteria, there is no information in this respect on soft-shell clams Mya arenaria. In this study, an in vivo model of interaction between M. arenaria haemocytes and the marine bacteria Vibrio splendidus was developed by assessing a series of host responses against two bacterial strains: LGP32-GFP, derived from a pathogenic strain (LGP32) associated with oyster mortalities in France, and 7SHRW, an environmental isolate from PEI marine sediments.

Using contrast phase microscopy and flow cytometry, phenotypic responses were measured at cellular and sub-cellular levels. In addition, the expression of genes involved in physiological and immune processes was assessed with RT real time q-PCR. Also, lysozyme activity in haemolymph was studied in clams from two different origins (i.e. Ireland and Canada) challenged with the pathogenic strain. Finally, degranulation mechanisms were briefly explored in vitro. Responses of the in vivo model were assessed 24 h after injecting 4.5 x 106 bacteria/g of clam.

Increased percentages of rounded haemocytess and total numbers were found in response to both strains. However, values were significantly higher ( p ≤ 0.01) after infection with LGP32-GFP compared to 7SHRW. Also, LGP32-GFP induced markedly diminished cell adherence (p < 0.001) while no significant changes were found after infection with 7SHRW. Flow cytometry forward and side scatter profiles showed two haemocyte subpopulations: hyalinocytes and granulocytes. Granulocytes exhibited significantly higher levels of lysosomal staining (p < 0.01), revealed by the fluorescent dye LysoTracker. Following infection with LGP32-GFP, and not with 7SHRW, both subpopulations merged into a single continuous group. Also, the lysosomal content significantly decreased in hyalinocytes (p < 0.001) and granulocytes (p = 0.001), suggesting the possible occurrence of precursor cells and degranulation, respectively. Among the housekeeping genes evaluated, using the software geNorm, elongation factor 1α (EF-1α) and 2 (EF-2) had the most stable expression under in vivo challenge with LGP32-GFP, whereas EF-1α and the ribosomal protein S-18, after challenge with 7SHRW. By using these genes as internal controls, it was detected that LGP32-GFP induced significant up-regulation of γ-actin (p < 0.001), IRAK-4 homologue ( p < 0.001) and down-regulation of TLR-2 (p < 0.001) and lysozyme-2 (p < 0.05) homologues. While actin regulation is likely associated with the cytoskeleton changes, IRAK-4 regulation would suggest activation of the NF-κB pathway. Only the down-regulation of lysozyme was significant (p < 0.05) after challenge with 7SHRW.

Lysozyme activity in haemolymph of clams from Ireland significantly increased after challenge, with a peak at 12 h (p < 0.001), while lysozyme levels did not change significantly in clams from Canada. Lysozyme levels were higher in haemocytes than in plasma. In addition, haemocytes could be induced to degranulate with the compound 48/80 suggesting a signaling pathway involving G proteins that could not be confirmed as inhibitors of such pathway did not prevent degranulation.

These results suggest specific responses at a cellular and molecular level induced by different V. splendidus strains. In contrast to 7SHRW, the strain LGP32-GFP induced marked responses at both levels that would impair the defense mechanisms of haemocytes. The differential responses assessed in this model have potential to be used as a tool to evaluate health status of clam populations.