Mussels (Mytilus galloprovincialis) are filter feeder bivalves that are constantly in contact with a wide range of microorganisms, some of which are potentially pathogenic. How mussels recognize and respond to pathogens has not been fully elucidated to date; therefore, we investigated the immune mechanisms that these animals employ in response to a bacterial bath infection from the surrounding water, mimicking the response that mussels mount under natural conditions. After the bath infection, mussels were able to remove the bacteria from their bodies and from the water tank. Accordingly, antibacterial activity was detected in gill extracts, demonstrating that this tissue plays a central role in removing and clearing potential pathogens. A transcriptomic study performed after a bath infection with Vibrio splendidus identified a total of 1,156 differentially expressed genes. The expression levels of genes contributing to a number of biological processes, such as immune response activation pathways and their regulation with cytokines, cell recognition, adhesion and apoptosis, were significantly modulated after infection, suggesting that the gills play important roles in pathogen recognition, as well as being activators and regulators of the mussel innate immune response. In addition to RNA-seq analysis, long non-coding RNAs and their neighboring genes were also analyzed and exhibited modulation after the bacterial challenge. The response of gills against bath infection was compared with the findings of a previous transcriptomic study on hemocytes responding to systemic infection, demonstrating the different and specific functions of gills. The results of this study indicate that recognition processes occur in the gill, thereby activating the effector agents of the immune response to overcome bacterial infection.

Mediterranean mussels ( Mytilus galloprovincialis ) are sessile filter feeders that live in close contact with numerous marine microorganisms. As all invertebrates, they lack an adaptive immune response and how these animals are able to respond to a bacterial infection and discriminate it from their normal microbiome is difficult to understand. In this work, we conducted Illumina sequencing of the transcriptome of individual mussels before and after being infected with Vibrio splendidus . The control mussels were injected with filtered seawater. We demonstrate that a great variability exists among individual transcriptomes and that each animal showed an exclusive repertoire of genes not shared with other individuals. The regulated genes in both the control and infected mussels were also analyzed and, unexpectedly, the sampling before the injection was considered a stress stimulus strong enough to trigger and modulate the response in hemocytes, promoting cell migration and proliferation. We found a clear response against the injection of filtered seawater, suggesting a reaction against a tissue injury in which the myticins, the most expressed antimicrobial peptides in mussel, appeared significantly up regulated. Functional experiments with flow cytometry confirmed the transcriptomic results since a significant alteration of hemocyte structures and a decrease in the number of hemocytes positive for myticin C were found only after a Vibrio infection and not observed when mussels were bled before, generating a tissue injury. Therefore, we report the involvement of myticins in the response to a danger signal such as a simple injection in the adductor muscle.

Mediterranean mussels ( Mytilus galloprovincialis ) are sessile filter feeders that live in close contact with numerous marine microorganisms. As is the case in all invertebrates, mussels lack an adaptive immune system, but they respond to pathogens, injuries or environmental stress in a very efficient manner. However, it is not known if they are able to modify their immune response when they reencounter the same pathogen. In this work, we studied the transcriptomic response of mussel hemocytes before and after two consecutive sublethal challenges with Vibrio splendidus . The first exposure significantly regulated genes related to inflammation, migration and response to bacteria. However, after the second exposure, the differentially expressed genes were related to the control and inhibition of ROS production and the resolution of the inflammatory response. Our results also show that the second injection with V. splendidus led to changes at the transcriptional (control of the expression of pro-inflammatory transcripts), cellular (shift in the hemocyte population distribution), and functional levels (inhibition of ROS production). These results suggest that a modified immune response after the second challenge allowed the mussels to tolerate rather than fight the infection, which minimized tissue damage.

Mediterranean mussels (Mytilus galloprovincialis) are marine bivalve molluscs with high resilience to biotic and abiotic stress. This resilience is one of the reasons why this species is such an interesting model for studying processes such as the immune response. In this work, we stimulated mussel hemocytes with poly I:C, β-glucans, and LPS and then sequenced hemocyte mRNAs (transcriptome) and microRNAs (miRNome) to investigate the molecular basis of the innate immune responses against these pathogen-associated molecular patterns (PAMPs). An immune transcriptome comprising 219,765 transcripts and an overview of the mussel miRNome based on 5,175,567 non-redundant miRNA reads were obtained. The expression analyses showed opposite results in the transcriptome and miRNome; LPS was the stimulus that triggered the highest transcriptomic response, with 648 differentially expressed genes (DEGs), while poly I:C was the stimulus that triggered the highest miRNA response, with 240 DE miRNAs. Our results reveal a powerful immune response to LPS as well as activation of certain immunometabolism- and ageing/senescence-related processes in response to all the immune challenges. Poly I:C exhibited powerful stimulating properties in mussels, since it triggered the highest miRNomic response and modulated important genes related to energy demand; these effects could be related to the stronger activation of these hemocytes (increased phagocytosis, increased NO synthesis, and increased velocity and accumulated distance). The transcriptome results suggest that after LPS stimulation, pathogen recognition, homeostasis and cell survival processes were activated, and phagocytosis was induced by LPS. β-glucans elicited a response related to cholesterol metabolism, which is important during the immune response, and it was the only stimulus that induced the synthesis of ROS. These results suggest a specific and distinct response of hemocytes to each stimulus from a transcriptomic, miRNomic, and functional point of view.