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.

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.

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.

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.

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.

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.

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.

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.

Metabolic Cost of the Immune Response During Early Ontogeny of the Scallop Argopecten purpuratus.

The scallop Argopecten purpuratus is an important resource for Chilean and Peruvian aquaculture. Seed availability from commercial hatcheries is critical due to recurrent massive mortalities associated with bacterial infections, especially during the veliger larval stage. The immune response plays a crucial role in counteracting the effects of such infections, but being energetically costly, it potentially competes with the physiological and morphological changes that occur during early development, which are equally expensive. Consequently, in this study, energy metabolism parameters at the individual and cellular levels, under routine-basal status and after the exposure to the pathogenic strain bacteria (Vibrio splendidus VPAP18), were evaluated during early ontogeny (trochophore, D-veliger, veliger, pediveliger, and early juveniles) of A. purpuratus. The parameters measured were as follows: (1) metabolic demand, determined as oxygen consumption rate and (2) ATP supplying capacity measured by key mitochondrial enzymes activities [citrate synthase (CS), electron transport system (ETS), and ETS/CS ratio, indicative of ATP supplying efficiency], mitochondrial membrane potential (ΔΨm), and mitochondrial density (ρ m) using an in vivo image analysis. Data revealed that metabolic demand/capacity varies significantly throughout early development, with trochophores being the most efficient in terms of energy supplying capacity under basal conditions. ATP supplying efficiency decreased linearly with larval development, attaining its lowest level at the pediveliger stage, and increasing markedly in early juveniles. Veliger larvae at basal conditions were inefficient in terms of energy production vs. energy demand (with low ρ m, ΔΨm, enzyme activities, and ETS:CS). Post-challenged results suggest that both trochophore and D-veliger would have the necessary energy to support the immune response. However, due to an immature immune system, the immunity of these stages would rely mainly on molecules of parental origin, as suggested by previous studies. On the other hand, post-challenged veliger maintained their metabolic demand but decreased their ATP supplying capacity, whereas pediveliger increased CS activity. Overall, results suggest that veliger larvae exhibit the lowest metabolic capacity to overcome a bacterial challenge, coinciding with previous works, showing a reduced capacity to express immune-related genes. This would result in a higher susceptibility to pathogen infection, potentially explaining the higher mortality rates occurring during A. purpuratus farming.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Induction of foxp3 during the Crosstalk between Antigen Presenting Like-Cells MHCII+CD83+ and Splenocytes CD4+IgM- in Rainbow Trout.

In fish, the spleen is one of the major immune organs in the animal, and the splenocytes could play a key role in the activation and modulation of the immune response, both innate and adaptive. However, the crosstalk between different types of immune cells in the spleen has been poorly understood. In this work, an in vitro strategy is carried out to obtain and characterize mononuclear splenocytes from rainbow trout, using biomarkers associated with lymphocytes (CD4 and IgM) and antigen-presenting cells (CD83 and MHC II). Using these splenocytes, co-cultures of 24 and 48 h are used to determine the gene expression of master transcriptional factors that coordinate the polarization of T cells (t-bet, gata3, and foxp3). The results show a proportional upregulation of foxp3 (compared to t-bet and gata3) in co-cultures (at 24 h) of IFNγ-induced splenocytes with and without stimulation of Piscirickettsia salmonis proteins. In addition, foxp3 upregulation was established in co-cultures with IFNγ-induced cells and in cells only stimulated previously with P. salmonis proteins at 48 h of co-culture. These results show a potential communication between antigen-presenting-like cells and lymphocyte in the spleen, which could be induced towards a Treg phenotype.

Host Defense Effectors Expressed by Hemocytes Shape the Bacterial Microbiota From the Scallop Hemolymph.

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.

Functional Insights From the Evolutionary Diversification of Big Defensins.

Big defensins are antimicrobial polypeptides believed to be the ancestors of ß-defensins, the most evolutionary conserved family of host defense peptides (HDPs) in vertebrates. Nevertheless, big defensins underwent several independent gene loss events during animal evolution, being only retained in a limited number of phylogenetically distant invertebrates. Here, we explore the evolutionary history of this fascinating HDP family and investigate its patchy distribution in extant metazoans. We highlight the presence of big defensins in various classes of lophotrochozoans, as well as in a few arthropods and basal chordates (amphioxus), mostly adapted to life in marine environments. Bivalve mollusks often display an expanded repertoire of big defensin sequences, which appear to be the product of independent lineage-specific gene tandem duplications, followed by a rapid molecular diversification of newly acquired gene copies. This ongoing evolutionary process could underpin the simultaneous presence of canonical big defensins and non-canonical (ß-defensin-like) sequences in some species. The big defensin genes of mussels and oysters, two species target of in-depth studies, are subjected to gene presence/absence variation (PAV), i.e., they can be present or absent in the genomes of different individuals. Moreover, big defensins follow different patterns of gene expression within a given species and respond differently to microbial challenges, suggesting functional divergence. Consistently, current structural data show that big defensin sequence diversity affects the 3D structure and biophysical properties of these polypeptides. We discuss here the role of the N-terminal hydrophobic domain, lost during evolution toward ß-defensins, in the big defensin stability to high salt concentrations and its mechanism of action. Finally, we discuss the potential of big defensins as markers for animal health and for the nature-based design of novel therapeutics active at high salt concentrations.

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.

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.

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.

Reproduction Immunity Trade-Off in a Mollusk: Hemocyte Energy Metabolism Underlies Cellular and Molecular Immune Responses.

Immune responses, as well as reproduction, are energy-hungry processes, particularly in broadcast spawners such as scallops. Thus, we aimed to explore the potential reproduction-immunity trade-off in Argopecten purpuratus, a species with great economic importance for Chile and Peru. Hemocytes, key immunological cells in mollusks, were the center of this study, where we addressed for the first time the relation between reproductive stage, hemocyte metabolic energetics and their capacity to support immune responses at cellular and molecular levels. Hemocyte metabolic capacity was assessed by their respiration rates, mitochondrial membrane potential and citrate synthase (CS) activity. Cellular immune parameters such as the number of circulating and tissue-infiltrating hemocytes and their reactive oxygen species (ROS) production capacity were considered. Molecular immune responses were examined through the transcriptional levels of two pattern recognition receptors (ApCLec and ApTLR) and two anti-microbial effectors (ferritin and big defensin). Their expressions were measured in hemocytes from immature, matured and spawned scallops under basal, and one of the following challenges: (i) in vitro, where hemocytes were challenged with the ß glucan zymosan, to determine the immune potentiality under standardized conditions; or (ii) in vivo challenge, using hemocytes from scallops injected with the pathogenic bacteria Vibrio splendidus. Results indicate a post-spawning decrease in the structural components of the immune system (hemocyte number/quality) and their potential capacity of performing immune functions (with reduced ATP-producing machinery and exhaustion of energy reserves). Both in vitro and in vivo challenges demonstrate that hemocytes from immature scallops have, in most cases, the best metabolic potential (increased CS activity) and immune performances, with for example, over threefold higher ROS production and tissue-infiltration capacity than those from mature and spawned scallops after the bacterial challenge. Agreeing with cellular responses, hemocytes from immature individuals induced the highest levels of immune receptors and antimicrobial effectors after the bacterial challenge, while spawned scallops presented the lowest values. Overall, results suggest a trade-off between resource allocation in reproduction and the immune responses in A. purpuratus, with hemocyte energy metabolic capacity potentially underlying cellular and molecular immune responses. Further research would be necessary to explore regulatory mechanisms such as signaling pleiotropy which may potentially be underlying this trade-off.

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.

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.