Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes.
In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis. The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios.