A novel LPS binding /bactericidal permeability-increasing protein (LBP/BPI) from the scallop Argopecten purpuratus plays an essential role in host resistance to Vibrio infection.

Lipopolysaccharide binding proteins (LBPs) and bactericidal permeability increasing proteins (BPIs) play significant roles in the immune response of vertebrates against bacterial pathogens. These soluble proteins produced by immune cells, specifically interact with and bind to bacterial lipopolysaccharides (LPS), with BPIs also displaying antibacterial activity. In Argopecten purpuratus scallop larvae resistant to Vibrio bivalvicida VPAP30, we identified a significant overexpression of a transcript displaying molecular features of an LBP/BPI protein, both before and after infection. Therefore, in the present work we aimed to understand the role of this novel LBP/BPI, named ApLBP/BPI3, in the scallop resistance to this Vibrio. The ApLBP/BPI3 open reading frame encodes a putative protein of 506 amino acids, with a molecular weight 56.78 kDa. The protein contains a C-terminal domain of 403-amino acid that, after theorical cleavage, displays a soluble form of 44.99 kDa, featuring two BPI/LBP/CETP domains, an apolar binding pocket, a single disulfide bond and a BPI dimerization interface. Phylogenetic analysis reveals high similarity between ApLBP/BPI3 and other mollusk LBP/BPI proteins. Aplbp/bpi3 transcripts were constitutively and highly expressed in hemocytes, gills, mantle, and digestive gland tissues, and were induced following VPAP30 infection in scallop larvae and adult hemocytes. We characterized ApLBP/BPI3 protein using a polyclonal antibody against a synthetic peptide. ApLBP/BPI3 was secreted to the media by infected cultured hemocytes, detected by ELISA. ApLBP/BPI3 was spotted inside non-infected hemocytes, bound to the cell wall of V. bivalvicida after in vitro hemocyte infection, and coating the gills and mantle epithelial barriers before and after an in vivo immune challenge, with stronger detection after VPAP30 injection, detected by immunofluorescence. Infected scallop larvae showed increased ApLBP/BPI3 levels, with slightly higher production in resistant larvae, determined by Western blot. Finally, silencing the Aplbp/bpi3 transcript through RNA interference and and subsequently infecting scallop juveniles with an LD50 of V. bivalvicida resulted in 100 % mortality. Altogether, results demonstrate the essential role of this immune effector in the resistance of A. purpuratus.

Crassostrea gigas oysters from a non-intensive farming area naturally harbor potentially pathogenic vibrio strains.

Farming intensification and climate change are inevitably linked to pathogen emergence in aquaculture. In this context, infectious diseases associated with vibrios span all developmental stages of the Pacific Oyster Crassostrea gigas. Moreover, virulence factors associated with pathogenicity spread among the vibrio community through horizontal gene transfer as part of the natural eco-evolutive dynamic of this group. Therefore, risk factors associated with the emergence of pathogens should be assessed before the appearance of mass mortalities in developing rearing areas. In this context, we characterized the vibrios community associated with oysters cultured in a non-intensive area free of massive mortalities located at Tongoy bay, Chile, through a culture-dependent approach. We taxonomically affiliated our isolates at the species level through the partial sequencing of the heat shock protein 60 gene and estimated their virulence potential through experimental infection of juvenile C. gigas. The vibrio community belonged almost entirely to the Splendidus clade, with Vibrio lentus being the most abundant species. The virulence potential of selected isolates was highly contrasted with oyster survival ranging between 100 and 30 %. Moreover, different vibrio species affected oyster survival at different rates, for instance V. splendidus TO2_12 produced most mortalities just 24 h after injection, while the V. lentus the most virulent strain TO6_11 produced sustained mortalities reaching 30 % of survival at day 4 after injection. Production of enzymes associated with pathogenicity was detected and hemolytic activity was positive for 50 % of the virulent strains and negative for 90 % of non-virulent strains, representing the phenotype that better relates to the virulence status of strains. Overall, results highlight that virulence is a trait present in the absence of disease expression, and therefore the monitoring of potentially pathogenic groups such as vibrios is essential to anticipate and manage oyster disease emergence in both established and under-development rearing areas.

A g-type lysozyme from the scallop Argopecten purpuratus participates in the immune response and in the stability of the hemolymph microbiota.

Lysozymes are antimicrobial acid hydrolases widely distributed in nature. They are located inside the cells in lysosomes, or they are secreted to the extracellular space, where they can lyse the cell wall of certain species of bacteria via hydrolysis of the peptidoglycan. Thus, lysozymes are bacteriolytic enzymes and play a major biological role in biodefense, as these enzymes can act as antibacterial and immune-modulating agents. In this study, we characterized a g-type lysozyme from the scallop Argopecten purpuratus named ApGlys. The cDNA sequence comprises an open reading frame (ORF) of 600 nucleotides, codifying for a putative protein of 200 amino acids with a signal peptide of 18 amino acids. The deduced mature protein sequence displays a molecular weight of 20.07 kDa and an isoelectric point (pI) of 6.49. ApGlys deduced protein sequence exhibits conserved residues associated with catalytic activity and substrate fixation in other g-type lysozymes. The phylogenetic analysis revealed a high degree of identity of ApGlys with other mollusk g-type lysozymes, which form a restricted and separated clade from the vertebrate lysozymes. ApGlys transcripts were constitutively and highly expressed in the digestive gland, and it was induced in hemocytes and gills of scallops after an immune challenge. Furthermore, the ApGlys protein was located inside hemocytes of immunostimulated scallops, determined by immunofluorescence analysis. Finally, the transcript silencing of ApGlys by RNA interference led to an increase of total culturable bacteria from the scallop hemolymph. Furthermore, we detected a higher diversity of the bacterial community in ApGlys-silenced scallops and an imbalance of certain bacterial groups present in the hemolymph by 16S rDNA deep amplicon sequencing. Overall, our results showed that ApGlys is a new member of scallop lysozymes that is implicated in the immune response and in the microbial homeostasis of A. purpuratus hemolymph.

Big defensin from the scallop Argopecten purpuratus ApBD1 is an antimicrobial peptide which entraps bacteria through nanonets formation.

Big defensins is a large family of antimicrobial peptides found in restricted groups of invertebrates, in particular mollusks where they have highly diversified. Big defensins are composed of a highly hydrophobic N-terminal region and a C-terminal region containing six cysteine residues whose arrangement is identical to that of vertebrate ß-defensins. They have been shown to be active against both Gram-positive and Gram-negative bacteria and fungi. Antimicrobial aggregates called nanonets entrapping and killing bacteria have been recently described for the hydrophobic N-terminal region of the Cg-BigDef1 from the oyster Crassostrea gigas. To determine whether nanonets formation is a conserved trait of mollusk big defensins, we assessed the potential entrapping of bacteria through nanonets of the big defensin from the scallop Argopecten purpuratus, ApBD1. Recombinant ApBD1 was produced with a thrombin-cleavable N-terminal His6 tag, followed by the mature peptide carrying a mutation of the last cysteine residue of the C-terminal region by and arginine, named rApBD1(C87R). This mutation did not apparently affect the three-dimensional structure and the biological properties of rApBD1(C87R), as evidenced by in silico modeling and in vitro antimicrobial assays. Strong immune staining of rApBD1(C87R) in numerous areas surrounding bacteria was observed by confocal microscopy, suggesting that rApBD1(C87R) entraps bacteria in peptide aggregates similar to those reported to the oyster big defensin. This study suggests the conservation of bactericidal activity and nanonet formation across big defensins from bivalve mollusks.

Molecular characterization and expression patterns of two LPS binding /bactericidal permeability-increasing proteins (LBP/BPIs) from the scallop Argopecten purpuratus.

Lipopolysaccharide-binding proteins (LBPs) and bactericidal permeability-increasing proteins (BPIs) are effectors of the innate immune response which act in a coordinated manner to bind and neutralize the LPS present in Gram negative bacteria. The structural organization that confers the function of LBPs and BPIs is very similar, however, they are antagonistic to each other. In this work, we characterized two LBP/BPIs from the scallop Argopecten purpuratus, namely ApLBP/BPI1 and ApLBP/BPI2. The molecular and phylogenetic analyses of ApLBP/BPIs indicated that both isoforms display classic characteristics of LBP/BPIs from other invertebrates. Additionally, ApLBP/BPIs are constitutively expressed in scallop tissues and their transcript expression is upregulated in hemocytes and gills in response to an immune challenge. However, some structural characteristics of functional importance for the biological activity of these molecules, such as the net charge differ substantially between ApLBP/BPI1 and ApLBP/BPI2. Furthermore, each isoform displays a specific profile of basal expression among different tissues, as well as specific patterns of expression during the activation of the immune response. Results suggest that functional specialization of ApLBP/BPIs might happen, with potential role as LBP or BPI in this species of scallop. Further research on the biological activities of ApLBP/BPIs are necessary to elucidate their participation in the scallop immune response.

De novo assembly, characterization of tissue-specific transcriptomes and identification of immune related genes from the scallop Argopecten purpuratus.

The scallop Argopecten purpuratus is one of the most economically important cultured mollusks on the coasts from Chile and Peru but its production has declined, in part, due to the emergence of mass mortality events of unknown origin. Driven by this scenario, increasing progress has been made in recent years in the comprehension of immune response mechanisms in this species. However, it is still not entirely understood how different mucosal interfaces participate and cooperate with the immune competent cells, the hemocytes, in the immune defense. Thus, in this work we aimed to characterize the transcriptome of three tissues with immune relevance from A. purpuratus by next-generation sequencing and de novo transcriptome assembly. For this, 18 cDNA libraries were constructed from digestive gland, gills and hemocytes tissues of scallops from different immune conditions and sequenced by the Illumina HiSeq4000 platform. A total of 967.964.884 raw reads were obtained and 967.432.652 clean reads were generated. The clean reads were de novo assembled into 46.601 high quality contigs and 32.299 (69.31%) contigs were subsequently annotated. In addition, three de novo specific assemblies were performed from clean reads obtained from each tissue cDNA libraries for their comparison. Gene ontology (GO) and KEGG analyses revealed that annotated sequences from digestive gland, gills and hemocytes could be classified into both general and specific subcategory terms and known biological pathways, respectively, according to the tissue nature. Finally, several immune related candidate genes were identified, and the differential expression of tissue-specific genes was established, suggesting they could display specific roles in the host defense. The data presented in this study provide the first insight into the tissue specific transcriptome profiles of A. purpuratus, which should be considered for further research on the interplay between the hemocytes and mucosal immune responses.

Litopenaeus vannamei stylicins are constitutively produced by hemocytes and intestinal cells and are differentially modulated upon infections.

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.

Identification of Antimicrobial Peptides from the Microalgae Tetraselmis suecica (Kylin) Butcher and Bactericidal Activity Improvement.

The outburst of microbial resistance to antibiotics creates the need for new sources of active compounds for the treatment of pathogenic microorganisms. Marine microalgae are of particular interest in this context because they have developed tolerance and defense strategies to resist the exposure to pathogenic bacteria, viruses, and fungi in the aquatic environment. Although antimicrobial activities have been reported for some microalgae, natural algal bioactive peptides have not been described yet. In this work, acid extracts from the microalga Tetraselmis suecica with antibacterial activity were analyzed, and de novo sequences of peptides were determined. Synthetic peptides and their alanine and lysine analogs allowed identifying key residues and increasing their antibacterial activity. Additionally, it was determined that the localization of positive charges within the peptide sequence influences the secondary structure with tendency to form an alpha helical structure.

Massive Gene Expansion and Sequence Diversification Is Associated with Diverse Tissue Distribution, Regulation and Antimicrobial Properties of Anti-Lipopolysaccharide Factors in Shrimp.

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.

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes.

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due to their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Molecular characterization and protein localization of the antimicrobial peptide big defensin from the scallop Argopecten purpuratus after Vibrio splendidus challenge.

Big defensins are antimicrobial peptides (AMPs) that are proposed as important effectors of the immune response in mollusks, chelicerates and chordates. At present, only two members of the big defensin family have been identified in scallop. In the present work, a cDNA sequence encoding a new big defensin homologue was characterized from the scallop Argopecten purpuratus, namely ApBD1. ApBD1 cDNA sequence comprised 585 nucleotides, with an open reading frame of 375 bp and 5'- and 3'-UTRs of 41 and 167 bp, respectively. The deduced protein sequence contains 124 amino acids with a molecular weight of 13.5 kDa, showing characteristic motifs of the big defensin family and presenting 76% identity with the big defensin from the scallop A. irradians. Phylogenetic analysis revealed that ApBD1 is included into the cluster of big defensins from mollusks. Tissue-specific transcript expression analysis by RT-qPCR showed that ApBD1 was present in all tissues tested from non-immune challenged scallops but it was most strongly expressed in the mantle. The transcript levels of ApBD1 were significantly up-regulated in gills at 24 and 48 h post-injection with the heat-attenuated bacteria Vibrio splendidus. Additionally, immunofluorescence analysis using a polyclonal anti-ApBD1 antibody showed that this protein was abundantly located in epithelial linings of gills and mantle; and also in digestive gland showing ApBD1-infiltrating hemocytes from immune challenged scallops. This is the first time that a big defensin is detected and located at the protein level in a mollusk. These results suggest an important role of ApBD1 in the mucosal immune response of A. purpuratus.

Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas.

Although antimicrobial histones have been isolated from multiple metazoan species, their role in host defense has long remained unanswered. We found here that the hemocytes of the oyster Crassostrea gigas release antimicrobial H1-like and H5-like histones in response to tissue damage and infection. These antimicrobial histones were shown to be associated with extracellular DNA networks released by hemocytes, the circulating immune cells of invertebrates, in response to immune challenge. The hemocyte-released DNA was found to surround and entangle vibrios. This defense mechanism is reminiscent of the neutrophil extracellular traps (ETs) recently described in vertebrates. Importantly, oyster ETs were evidenced in vivo in hemocyte-infiltrated interstitial tissues surrounding wounds, whereas they were absent from tissues of unchallenged oysters. Consistently, antimicrobial histones were found to accumulate in oyster tissues following injury or infection with vibrios. Finally, oyster ET formation was highly dependent on the production of reactive oxygen species by hemocytes. This shows that ET formation relies on common cellular and molecular mechanisms from vertebrates to invertebrates. Altogether, our data reveal that ET formation is a defense mechanism triggered by infection and tissue damage, which is shared by relatively distant species suggesting either evolutionary conservation or convergent evolution within Bilateria.

The new insights into the oyster antimicrobial defense: Cellular, molecular and genetic view.

Oysters are sessile filter feeders that live in close association with abundant and diverse communities of microorganisms that form the oyster microbiota. In such an association, cellular and molecular mechanisms have evolved to maintain oyster homeostasis upon stressful conditions including infection and changing environments. We give here cellular and molecular insights into the Crassostrea gigas antimicrobial defense system with focus on antimicrobial peptides and proteins (AMPs). This review highlights the central role of the hemocytes in the modulation and control of oyster antimicrobial response. As vehicles for AMPs and other antimicrobial effectors, including reactive oxygen species (ROS), and together with epithelia, hemocytes provide the oyster with local defense reactions instead of systemic humoral ones. These reactions are largely based on phagocytosis but also, as recently described, on the extracellular release of antimicrobial histones (ETosis) which is triggered by ROS. Thus, ROS can signal danger and activate cellular responses in the oyster. From the current literature, AMP production/release could serve similar functions. We provide also new lights on the oyster genetic background that underlies a great diversity of AMP sequences but also an extraordinary individual polymorphism of AMP gene expression. We discuss here how this polymorphism could generate new immune functions, new pathogen resistances or support individual adaptation to environmental stresses.

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus.

OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for ß-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase Cg-EcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD.

Silencing of the Vasa gene by RNA Interference Affects Embryonic Development and Reproductive Output in the Sea Louse Caligus rogercresseyi.

The sea louse Caligus rogercresseyi is a major ectoparasitic copepod that causes significant economic losses in the salmon farming industry. Despite recent advancements, the mechanisms underlying germline and embryo development in this species remain poorly understood. The Vasa gene encodes a highly conserved DEAD box helicase that is required for germ cell formation and function in many species. In this study, the Vasa gene was characterized in C. rogercresseyi, and its expression and function were analyzed. Phylogenetic analysis showed that the Cr-Vasa gene product formed clusters in clades with Vasa proteins from closely related species of crustaceans. Cr-Vasa gene expression patterns were assessed by qPCR, and the results showed a significantly higher relative expression level in adult females compared to copepodid, chalimus, and adult male stages. Tissue-specific localization of Cr-Vasa mRNA in C. rogercresseyi was determined using chromogenic in situ hybridization, and strong positive signal was observed in male testes, but also in the intestine and cuticle, while in females, it was observed in the ovaries, oocytes, cuticle, intestine, and egg strings. RNAi-mediated gene silencing of Cr-Vasa impacted embryonic development and reproductive output in adult female lice. Females from the dsVasa-treated group displayed unusual phenotypes, including shorter egg strings with numerous extra-embryonic inclusions, irregularly shaped abnormal embryos, and aborted egg strings. This study provides insights into the role of the Vasa gene in C. rogercresseyi embryonic development and reproductive output, which may have implications for the control of this parasitic copepod in the salmon farming industry.

Evidence of the Autophagic Process during the Fish Immune Response of Skeletal Muscle Cells against Piscirickettsia salmonis.

Autophagy is a fundamental cellular process implicated in the health of the cell, acting as a cytoplasmatic quality control machinery by self-eating unfunctional organelles and protein aggregates. In mammals, autophagy can participate in the clearance of intracellular pathogens from the cell, and the activity of the toll-like receptors mediates its activation. However, in fish, the modulation of autophagy by these receptors in the muscle is unknown. This study describes and characterizes autophagic modulation during the immune response of fish muscle cells after a challenge with intracellular pathogen Piscirickettsia salmonis. For this, primary cultures of muscle cells were challenged with P. salmonis, and the expressions of immune markers il-1ß, tnfα, il-8, hepcidin, tlr3, tlr9, mhc-I and mhc-II were analyzed through RT-qPCR. The expressions of several genes involved in autophagy (becn1, atg9, atg5, atg12, lc3, gabarap and atg4) were also evaluated with RT-qPCR to understand the autophagic modulation during an immune response. In addition, LC3-II protein content was measured via Western blot. The challenge of trout muscle cells with P. salmonis triggered a concomitant immune response to the activation of the autophagic process, suggesting a close relationship between these two processes.

Resistance of Argopecten purpuratus scallop larvae to vibriosis is associated with the front-loading of immune genes and enhanced antimicrobial response.

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.

A Diet Rich in HUFAs Enhances the Energetic and Immune Response Capacities of Larvae of the Scallop Argopecten purpuratus.

Massive mortalities in farmed larvae of the scallop Argopecten purpuratus have been associated with pathogenic Vibrio outbreaks. An energetic trade-off between development-associated demands and immune capacity has been observed. Given that highly unsaturated fatty acids (HUFAs) are essential nutrients for larval development, we evaluated the effect of diets based on microalgae low and high in HUFAs (LH and HH, respectively) on the energetic condition and the immune response of scallop larvae. The results showed that the HH diet increased cellular membrane fluidity in veliger larvae. The routine respiration rate was 64% higher in the HH-fed veligers than in the LH-fed veligers. Additionally, the metabolic capacity tended to be higher in the HH-fed veligers than in the LH-fed veligers after the Vibrio challenge. After the challenge, the HH-fed veligers presented higher transcript induction of ApTLR (immune receptor) and ApGlys (immune effector) genes, and the HH-fed pediveligers presented higher induction of ApLBP/BPI1 (antimicrobial immune effector) gene, than the LH-fed larvae. Furthermore, the HH-fed veligers controlled total Vibrio proliferation (maintaining near basal levels) after the bacterial challenge, while the LH-fed veligers were not able to control this proliferation, which increased three-fold. Finally, the HH-fed larvae showed 20-25% higher growth and survival rates than the LH-fed veligers. Overall, the results indicated that the administration of a HH diet increases cell membrane fluidity and energy metabolic capacity, which in turn enhances immunity and the ability to control Vibrio proliferation. The administration of microalgae high in HUFAs would be a promising strategy for improving scallop larval production efficiency.

Antimicrobial Spectrum of Activity and Mechanism of Action of Linear Alpha-Helical Peptides Inspired by Shrimp Anti-Lipopolysaccharide Factors.

Shrimp antilipopolysaccharide factors (ALFs) form a multifunctional and diverse family of antimicrobial host defense peptides (AMPs) composed of seven members (groups A to G), which differ in terms of their primary structure and biochemical properties. They are amphipathic peptides with two conserved cysteine residues stabilizing a central ß-hairpin that is understood to be the core region for their biological activities. In this study, we synthetized three linear (cysteine-free) peptides based on the amino acid sequence of the central ß-hairpin of the newly identified shrimp (Litopenaeus vannamei) ALFs from groups E to G. Unlike whole mature ALFs, the ALF-derived peptides exhibited an α-helix secondary structure. In vitro assays revealed that the synthetic peptides display a broad spectrum of activity against both Gram-positive and Gram-negative bacteria and fungi but not against the protozoan parasites Trypanosoma cruzi and Leishmania (L.) infantum. Remarkably, they displayed synergistic effects and showed the ability to permeabilize bacterial membranes, a mechanism of action of classical AMPs. Having shown low cytotoxicity to THP-1 human cells and being active against clinical multiresistant bacterial isolates, these nature-inspired peptides represent an interesting class of bioactive molecules with biotechnological potential for the development of novel therapeutics in medical sciences.

The Gill Microbiota of Argopecten purpuratus Scallop Is Dominated by Symbiotic Campylobacterota and Upwelling Intensification Differentially Affects Their Abundance.

Despite the great importance of gills for bivalve mollusks (respiration, feeding, immunity), the microbiota associated with this tissue has barely been characterized in scallops. The scallop Argopecten purpuratus is an important economic resource that is cultivated in areas where coastal upwelling is intensifying by climate change, potentially affecting host-microbiota interactions. Thus, we first characterized the bacterial community present in gills from cultivated scallops (by 16S rRNA gene amplicon sequencing) and assessed their stability and functional potential in animals under farm and laboratory conditions. Results showed that under both conditions the gill bacterial community is dominated by the phylum Campylobacterota (57%), which displays a chemoautotrophic potential that could contribute to scallop nutrition. Within this phylum, two phylotypes, namely symbionts A and B, were the most abundant; being, respectively, taxonomically affiliated to symbionts with nutritional functions in mussel gills, and to uncultured bacteria present in coral mucus. Additionally, in situ hybridization and scanning electron microscopy analyses allowed us to detect these symbionts in the gills of A. purpuratus. Given that shifts in upwelling phenology can cause disturbances to ecosystems, affecting bacteria that provide beneficial functions to the host, we further assessed the changes in the abundance of the two symbionts (via qPCR) in response to a simulated upwelling intensification. The exposure to combined decreasing values in the temperature, pH, and oxygen levels (upwelling conditions) favored the dominance of symbiont B over symbiont A; suggesting that symbiont abundances are modulated by these environmental changes. Overall, results showed that changes in the main Campylobacterota phylotypes in response to upwelling intensification could affect its symbiotic function in A. purpuratus under future climate change scenarios. These results provide the first insight into understanding how scallop gill-microbial systems adapt and respond to climate change stressors, which could be critical for managing health, nutrition, and scallop aquaculture productivity.