Assessment of morphological and cellular responses after infection with living bacteria in the marine mussel Mytilus galloprovincialis

Abstract

Bacterial strains of Vibrio genus associated with temperate regions are linked to mussels-borne infections. The sedentary nature of marine mussels, Mytilus galloprovincialis, together with their filter feeding combine to ensure that they have the potential for considerable exposure to infective agents. Bacterial strains of Vibrio genus associated with temperate regions are linked to mussels-borne infections. The sedentary nature of marine mussels, Mytilus galloprovincialis, together with their filter feeding combine to ensure that they have the potential for considerable exposure to infective agents. The primary mechanism of bivalve internal defense involves hemocytes responsible for cell-mediated immunity through a panel of activities such as phagocytosis, the release of cytotoxic molecules, reactive oxygen intermediates, lysosomal enzymes, PO enzyme and lysozyme. In this work in vivo infection of M. galloprovincialis with living V. splendidus to collect hemolymph from the posterior adductor muscle 1, 3, 6, 9, 12, 24 and 48 h post-injection was carried out. Previously we have found that when bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased already after thirty minutes, suggesting intense phagocytosis, then decreasing until 24 h. The quantification by flow cytometry indicated a variation of proportions of the three cell categories. In our study, injection of living bacteria resulted in total hemocyte count (THC) higher than normal 24 - 48 h post-injection, suggesting proliferation and/or recruitment of hemocytes which are mainly concentrated in the site of infection. To compliment this, here histological and immunohistochemical assessment was performed using adductor musclein order to evaluate the morphological features and cellular response spost bacterial infection. The morphological analysis showed changes in tissue organization, with an altered cell volume and recruitment of hemocytes among fibers. The change of osmotic equilibrium across muscle cell membranes was observed by increased the staining of Na-K ATPase during the entire period of stimulation, and reduced immunopositivity of aquaporin (AQP) 1 h post infection but with an increasing trend in the all experimental steps. The investigation on cellular turnover showed a tendency to recover a regular tissue structure, as highlighted by an intense immunopositivity of proliferating cell nuclear antigen (PCNA) from 24 h to 48 h post injection, as well as cell surface death receptor (FAS) and the cysteine-aspartic acid protease (CASP3) untilto 72 h post bacterial injection.