Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(−) Vibrio anguillarum and V. splendidus, Gram(+) Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) andhemolymphsampled from3to48hpost-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus N V. anguillarum N M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decreasewas observed for V. splendidus. Bacterial challenge also induced a rapid (from3 h p.i.) and significant increase in serumlysozyme activity; the effectwas persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluatewhether in vivo challengemay affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples fromV. anguillarum-injectedmussels showed a significant increase in E. coli killing (+35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populationswere evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease inTHC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocyteswas observed (V. splendidus N V. anguillarum N M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria.