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.

Transcriptome analysis of Catarina scallop (Argopecten ventricosus) juveniles treated with highly-diluted immunomodulatory compounds reveals activation of non-self-recognition system.

Marine bivalve hatchery productivity is continuously challenged by apparition and propagation of new diseases, mainly those related to vibriosis. Disinfectants and antibiotics are frequently overused to prevent pathogen presence, generating a potential negative impact on the environment. Recently, the use of highly diluted compounds with immunostimulant properties in marine organisms has been trailed successfully to activate the self-protection mechanisms of marine bivalves. Despite their potential as immunostimulants, little is known about their way of action. To understand their effect, a comparative transcriptomic analysis was performed with Argopecten ventricosus juveniles. The experimental design consisted of four treatments formulated from pathogenic Vibrio lysates at two dilutions: [(T1) Vibrio parahaemolyticus and Vibrio alginolyticus 1D; (T2) V. parahaemolyticus and V. alginolyticus 7C]; minerals [(T3) PhA+SiT 7C], scorpion venom [(T4) ViT 31C]; and one control (C1) hydro-alcoholic solution (ethanol 1%). The RNA sequencing (RNAseq) analysis showed a higher modulation of differentially expressed genes (DEG) in mantle tissue compared to gill tissue. The scallops that showed a higher number of DEG related to immune response in mantle tissue corresponded to T1 (V. parahaemolyticus and V. alginolyticus lysate) and T3 (Silicea terra® - Phosphoric acid®). The transcriptome analysis allowed understanding some interactions between A. ventricosus juveniles and highly-diluted treatments.

The immune response of the scallop Argopecten purpuratus is associated with changes in the host microbiota structure and diversity.

All organisms live in close association with a variety of microorganisms called microbiota. Furthermore, several studies support a fundamental role of the microbiota on the host health and homeostasis. In this context, the aim of this work was to determine the structure and diversity of the microbiota associated with the scallop Argopecten purpuratus, and to assess changes in community composition and diversity during the host immune response. To do this, adult scallops were immune challenged and sampled after 24 and 48 h. Activation of the immune response was established by transcript overexpression of several scallop immune response genes in hemocytes and gills, and confirmed by protein detection of the antimicrobial peptide big defensin in gills of Vibrio-injected scallops at 24 h post-challenge. Then, the major bacterial community profile present in individual scallops was assessed by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA genes and dendrogram analyses, which indicated a clear clade differentiation of the bacterial communities noticeable at 48 h post-challenge. Finally, the microbiota structure and diversity from pools of scallops were characterized using 16S deep amplicon sequencing. The results revealed an overall modulation of the microbiota abundance and diversity according to scallop immune status, allowing for prediction of some changes in the functional potential of the microbial community. Overall, the present study showed that changes in the structure and diversity of bacterial communities associated with the scallop A. purpuratus are detected after the activation of the host immune response. Now, the relevance of microbial balance disruption in the immune capacity of the scallop remains to be elucidated.

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.

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.

Homeopathy outperforms antibiotics treatment in juvenile scallop Argopecten ventricosus: effects on growth, survival, and immune response.

BACKGROUND: Mortality from vibriosis in mollusk production is attributed to pathogenic bacteria, particularly Vibrio alginolyticus. Use of increasingly potent antibiotics has led to bacterial resistance and increased pathogenicity. Alternatives in sanitation, safety, and environmental sustainability are currently under analysis. To-date, homeopathy has been investigated in aquaculture of freshwater fish, but not in marine mollusks. The effect of the homeopathic complexes in the growth, survival, and immune response of the Catarina scallop Argopecten ventricosus were assessed. METHODS: A bioassay to assess the potential of homeopathy in improving cultivation of juvenile A. ventricosus was conducted for 21 days, with a final challenge of 120 h with V. alginolyticus. The experimental design included two homeopathic formulas The homeopathic complex Passival, consisting of Passiflora incarnata 30 CH, Valeriana officinalis 30 CH, Ignatia amara 30 CH and Zincum valerianicum 30 CH plus Phosphoricum acid 30 CH (treatment TH1) or Silicea terra 30 CH (TH2), two antibiotics (ampicillin = AMP, oxytetracycline = OXY), and two reference treatments (without homeopathic or antibiotic treatment = CTRL, ethanol 30° GL = ETH). Additionally, a negative control CTRL- (untreated/uninfected) is included in the challenge test. Juvenile scallops (4.14 ± 0.06 mm, 13.33 mg wet weight) were cultivated in 4 L tanks provided with aerated, filtered (1 µm), and UV-sterilized seawater that was changed every third day. They were fed a blend of the microalgae Isochrysis galbana and Chaetoceros calcitrans (150,000 cells mL-1 twice a day). All treatments were directly added to the tank water and then 500 mL challenge units were inoculated with 1 × 107 CFU/mL (LD50) of V. alginolyticus. RESULTS: Juveniles grew significantly larger and faster in height and weight with TH2 compared to the ETH and CTRL (P < 0.05, ANOVA). Higher concentrations of proteins occurred in scallops exposed to TH2 (160.57 ± 7.79 mg g-1), compared to other treatments and reference treatments. Higher survival rate during the challenge bioassay occurred with TH1 (85%), compared to AMP (53%), OXY (30%), and CTRL (0%), and superoxide dismutase (P < 0.05) was significantly higher in scallops treated with TH1, compared to other treatments and reference treatments. CONCLUSIONS: Homeopathic treatments improved growth and survival and enhanced survival against V. alginolyticus in juvenile A. ventricosus. This suggests that homeopathy is a viable treatment for this mollusk to reduce use of antibiotics in scallops and its progressive increase in pathogenicity in mollusk hatcheries.

Insight into the messenger role of reactive oxygen intermediates in immunostimulated hemocytes from the scallop Argopecten purpuratus.

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.

Molecular characterization of an inhibitor of NF-κB in the scallop Argopecten purpuratus: First insights into its role on antimicrobial peptide regulation in a mollusk.

Inhibitors of nuclear factor kappa B (IκBs) are major control components of the Rel/NF-κB signaling pathway, a key regulator in the modulation of the expression of immune-related genes in vertebrates and invertebrates. The activation of the Rel/NF-κB signaling pathway depends largely in the degradation of IκB proteins and thus, IκBs are a main target for the identification of genes whose expression is controlled by Rel/NF-κB pathway. In order to identify such regulation in bivalve mollusks, the cDNA sequence encoding an IκB protein was characterized in the scallop Argopecten purpuratus, ApIκB. The cDNA sequence of ApIκB is comprised of 1480 nucleotides with a 1086 bp open reading frame encoding for 362 amino acids. Bioinformatics analysis showed that ApIκB displays the conserved features of IκB proteins. The deduced amino acid sequence consists of a 39.7 kDa protein, which has an N-terminal degradation motif, six ankyrin repeats and a C-terminal phosphorylation site motif. Phylogenetic analysis revealed a high degree of identity between ApIκB and other IκBs from mollusks, but also to arthropod cactus proteins and vertebrate IκBs. Tissue expression analysis indicated that ApIκB is expressed in all examined tissues and it is upregulated in circulating hemocytes from scallops challenged with the pathogenic Gram-negative bacterium Vibrio splendidus. After inhibiting ApIκB gene expression using the RNA interference technology, the gene expression of the antimicrobial peptide big defensin was upregulated in hemocytes from non-challenged scallops. Results suggest that ApIκB may control the expression of antimicrobial effectors such as big defensin via a putative Rel/NF-κB signaling pathway. This first evidence will help to deepen the knowledge of the Rel/NF-κB conserved pathway in scallops.

Morphofunctional study of hemocytes from lions-paw scallop Nodipecten subnodosus.

Hemocytes play an important role in internal defense in mollusk bivalves; they are generally divided into granular and hyaline types, where the granules possess molecules to combat foreign particles. We investigated the morphology, staining reactions, and immune-related activities of hemocytes from the pectinid lions-paw scallop Nodipecten subnodosus. We showed that, in contrast to the conditions in most bivalves and similar to other pectinids, no granular cells were observed; only very few semi-granular cells were present and the large majority of cells were hyalinocytes and blast-like cells. The cytoplasm in many hyalinocytes showed vesicles of various sizes, but none with the staining characteristics of typical granules. When hemolymph was removed from these scallops, the plasma did not clot and the hemocytes rapidly adhered to one another in suspension. When living hemocytes were placed on a glass surface, they clustered into groups of various sizes; within minutes, pseudopods extended from the cells and outward migration began. Many cells spread to form extensive networks of flat cells, and other undifferentiated cells did not spread and kept a rounded morphology. Some hemocytes were immunologically active because they phagocytize Escherichia coli bacteria. Hemocytes expressed the carbohydrates residues N-acetyl-D-glucosamine, α-D-glucose, α-D-mannose, and α-L-fucose moieties, and cytochemical assays showed activity of acid phosphatase, specific esterase, and non-specific esterase, enzymes commonly associated with lysosomes and immunologic activity. These observations serve as a useful tool for further investigation of morphofunctional studies and physiological status of scallops.