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Mass mortality events caused by vibriosis have emerged in hatchery-reared scallop larvae from Chile, threatening scallop aquaculture. In an attempt to mitigate this emerging infectious disease and provide candidates for marker-assisted selective breeding, we tested here the existence of a genetic component of Argopecten purpuratus scallop resistance to the pathogen Vibrio bivalvicida. Through a dual RNA-seq approach we analyzed the basal transcriptome and the transcriptional response to infection in two resistant and two susceptible families as well as the pathogen transcriptomic response to host colonization. The results highlighted a genetic basis in the resistance of scallop larvae to the pathogen. The Vibrio response was characterized by a general metabolic adaptation to the host environment, along with several predicted virulence factors overexpressed in infected scallop larvae with no difference between resistant and susceptible host phenotypes. On the host side, several biological processes were enriched in uninfected resistant larvae. Within these enriched categories, immune-related processes were overexpressed, while morphogenesis, biomineral tissue development, and angiogenesis were under expressed. Particularly, genes involved in immune recognition and antimicrobial response, such as lipopolysaccharide-binding proteins (LBPs), lysozyme, and bactericidal permeability-increasing protein (BPI) were overexpressed in uninfected resistant larvae. As expected, immune-related biological processes were enriched in Vibrio-infected larvae, but they were more numerous in resistant larvae. Overexpressed immune genes in response to infection included several Toll-like receptors, TNF and NF-κB immune signaling genes, and the antimicrobial peptide Big defensin ApBD1. Results strongly suggest that both a front-loading of immune genes and an enhanced antimicrobial response to infection contribute to the resistance, while pathogen infective strategy does not discriminate between host phenotypes. Overall, early expression of host immune genes appears as a strong determinant of the disease outcome that could be used in marker-assisted selective breeding.

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Crustins represent the largest and most diverse family of antimicrobial peptides (AMPs) found in crustaceans. They are classically defined as disulfide-rich peptides/polypeptides holding a typical Whey Acidic Protein (WAP) domain at the C-terminal end. This WAP domain has eight cysteine residues forming a tightly packed structure, the four-disulfide core (4DSC) motif, that is also found in other proteins displaying protease inhibitory properties. Crustins are highly diverse in terms of primary structure, size and biochemical features, thus exhibiting a series of biological functions beyond their antimicrobial properties. In order to better categorize the distinct crustin members, different classification systems have been proposed. In this review, we discuss the current classification systems and explore the biological implication of the impressive molecular diversity of this unique AMP family. We also summarize the recent findings on the role of these effectors in crustacean immunity and homeostasis as well as in host-microbe interactions.

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The interaction between host immune response and the associated microbiota has recently become a fundamental aspect of vertebrate and invertebrate animal health. This interaction allows the specific association of microbial communities, which participate in a variety of processes in the host including protection against pathogens. Marine aquatic invertebrates such as scallops are also colonized by diverse microbial communities. Scallops remain healthy most of the time, and in general, only a few species are fatally affected on adult stage by viral and bacterial pathogens. Still, high mortalities at larval stages are widely reported and they are associated with pathogenic Vibrio. Thus, to give new insights into the interaction between scallop immune response and its associated microbiota, we assessed the involvement of two host antimicrobial effectors in shaping the abundances of bacterial communities present in the scallop Argopecten purpuratus hemolymph. To do this, we first characterized the microbiota composition in the hemolymph from non-stimulated scallops, finding both common and distinct bacterial communities dominated by the Proteobacteria, Spirochaetes and Bacteroidetes phyla. Next, we identified dynamic shifts of certain bacterial communities in the scallop hemolymph along immune response progression, where host antimicrobial effectors were expressed at basal level and early induced after a bacterial challenge. Finally, the transcript silencing of the antimicrobial peptide big defensin ApBD1 and the bactericidal/permeability-increasing protein ApLBP/BPI1 by RNA interference led to an imbalance of target bacterial groups from scallop hemolymph. Specifically, a significant increase in the class Gammaproteobacteria and the proliferation of Vibrio spp. was observed in scallops silenced for each antimicrobial. Overall, our results strongly suggest that scallop antimicrobial peptides and proteins are implicated in the maintenance of microbial homeostasis and are key molecules in orchestrating host-microbiota interactions. This new evidence depicts the delicate balance that exists between the immune response of A. purpuratus and the hemolymph microbiota.

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Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.

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Stylicins are anionic antimicrobial host defense peptides (AAMPs) composed of a proline-rich N-terminal region and a C-terminal portion containing 13 conserved cysteine residues. Here, we have increased our knowledge about these unexplored crustacean AAMPs by the characterization of novel stylicin members in the most cultivated penaeid shrimp, Litopenaeus vannamei. We showed that the L. vannamei stylicin family is composed of two members (Lvan-Stylicin1 and Lvan-Stylicin2) encoded by different loci which vary in gene copy number. Unlike the other three gene-encoded antimicrobial peptide families from penaeid shrimp, the expression of Lvan-Stylicins is not restricted to hemocytes. Indeed, they are also produced by the columnar epithelial cells lining the midgut and its anterior caecum. Interestingly, Lvan-Stylicins are simultaneously transcribed at different transcriptional levels in a single shrimp and are differentially modulated in hemocytes after infections. While the expression of both genes showed to be responsive to damage-associated molecular patterns, only Lvan-Stylicin2 was induced after a Vibrio infection. Besides, Lvan-Stylicins also showed a distinct pattern of gene expression in the three portions of the midgut (anterior, middle and posterior) and during shrimp development. We provide here the first evidence of the diversity of the stylicin antimicrobial peptide family in terms of sequence and gene expression distribution and regulation.

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Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with a central ß-hairpin structure able to bind to microbial components. Mining sequence databases for ALFs allowed us to show the remarkable diversity of ALF sequences in shrimp. We found at least seven members of the ALF family (Groups A to G), including two novel Groups (F and G), all of which are encoded by different loci with conserved gene organization. Phylogenetic analyses revealed that gene expansion and subsequent diversification of the ALF family occurred in crustaceans before shrimp speciation occurred. The transcriptional profile of ALFs was compared in terms of tissue distribution, response to two pathogens and during shrimp development in Litopenaeus vannamei, the most cultivated species. ALFs were found to be constitutively expressed in hemocytes and to respond differently to tissue damage. While synthetic ß-hairpins of Groups E and G displayed both antibacterial and antifungal activities, no activity was recorded for Group F ß-hairpins. Altogether, our results showed that ALFs form a family of shrimp AMPs that has been the subject of intense diversification. The different genes differ in terms of tissue expression, regulation and function. These data strongly suggest that multiple selection pressures have led to functional diversification of ALFs in shrimp.

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Crustins form a large family of antimicrobial peptides (AMPs) in crustaceans composed of four sub-groups (Types I-IV). Type II crustins (Type IIa or "Crustins" and Type IIb or "Crustin-like") possess a typical hydrophobic N-terminal region and are by far the most representative sub-group found in penaeid shrimp. To gain insight into the molecular diversity of Type II crustins in penaeids, we identified and characterized a Type IIb crustin in Litopenaeus vannamei (Crustin-like Lv) and compared Type II crustins at both molecular and transcriptional levels. Although L. vannamei Type II crustins (Crustin Lv and Crustin-like Lv) are encoded by separate genes, they showed a similar tissue distribution (hemocytes and gills) and transcriptional response to the shrimp pathogens Vibrio harveyi and White spot syndrome virus (WSSV). As Crustin Lv, Crustin-like Lv transcripts were found to be present early in development, suggesting a maternal contribution to shrimp progeny. Altogether, our in silico and transcriptional data allowed to conclude that (1) each sub-type displays a specific amino acid signature at the C-terminal end holding both the cysteine-rich region and the whey acidic protein (WAP) domain, and that (2) shrimp Type II crustins evolved from a common ancestral gene that conserved a similar pattern of transcriptional regulation.

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Reactive oxygen intermediates (ROI) are metabolites produced by aerobic cells which have been linked to oxidative stress. Evidence reported in vertebrates indicates that ROI can also act as messengers in a variety of cellular signaling pathways, including those involved in innate immunity. In a recent study, an inhibitor of NF-kB transcription factors was identified in the scallop Argopecten purpuratus, and its functional characterization suggested that it may regulate the expression of the big defensin antimicrobial peptide ApBD1. In order to give new insights into the messenger role of ROI in the immune response of bivalve mollusks, the effect of ROI production on gene transcription of ApBD1 was assessed in A. purpuratus. The results showed that 48 h-cultured hemocytes were able to display phagocytic activity and ROI production in response to the ß-glucan zymosan. The immune stimulation also induced the transcription of ApBD1, which was upregulated in cultured hemocytes. After neutralizing the ROI produced by the stimulated hemocytes with the antioxidant trolox, the transcription of ApBD1 was reduced near to base levels. The results suggest a potential messenger role of intracellular ROI on the regulation of ApBD1 transcription during the immune response of scallops.

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Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In previous studies, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide in vitro e in vivo. Hyperimmune serum was obtained from rabbits immunized with a P. argus -NOS fragment of 31 kDa produced in Escherichia coli, which specifically detected the recombinant polypeptide and the endogenous NOS from lobster hemocytes by western blotting and immunofluorescence. In the present work, we demonstrate that the hyperimmune serum obtained against P. argus NOS also recognizes Litopenaeus vannamei NOS in hemocytes by western blotting and immunofluorescence. Our data also show that while the hemolymph of L. vannamei has a strong antibacterial activity against the Gram negative bacteria Aeromonas hydrophila, the administration of the anti NOS serum reduce the natural bacterial clearance. These results strongly suggest that NOS is required for the shrimp immune defense toward Gram negative bacteria. Therefore, the monitoring of induction of NOS could be an important tool for testing immunity in shrimp farming.

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