Source of hemolymph microbiota and their roles in the immune system of mud crab

Special innate immune mechanisms against pathogens are developed in marine invertebrates such as mud crab, which is also an economically important aquaculture species in many coastal countries. Hemolymph is a critical site in host immune response, but its source of microorganisms is less known. In this study, we provided a detailed investigation of the microorganisms inhabiting various body sites of healthy mud crabs, including hemolymph, midgut, gill, subcuticular epidermis and hepatopancreas. By using fluorescence microscopy and high-throughput sequencing of the bacterial 16S rRNA genes, various abundances and kinds of microorganisms were observed in the healthy mud crabs, of which some are potential pathogens to mud crab and human. The SourceTracker analysis and oral injection experiment confirm the hypothesis that hemolymph microorganisms are derived from the digestive systems of invertebrates with open circulatory systems, indicating that these microorganisms play vital roles in crab immune response. Moreover, physiological differences (gut length), behavioral characteristics (foraging behavior), diet preferences (herbivory), and/or sex hormones (testosterone) possibly determine the unique features of the crab-associated microbiota for both sexes. These findings also contribute to the development of appropriate microbial immunoenhancers, which has potential applications for improving quality and yield during crab aquaculture.