The toxic dinoflagellate Alexandrium catenella adversely affects early life stages of tehuelche scallop.

The effects of the toxic dinoflagellate Alexandrium catenella were investigated on growth, survival, and histopathology in larvae and spat of the Tehuelche scallop Aequipecten tehuelchus from Patagonia, Argentina. The study consisted of laboratory incubations of scallop larvae/spat with A. catenella, using environmentally realistic abundances of the dinoflagellate. Survival, growth, and histopathological effects were documented for scallop larvae/spat before, during, and after 7-day-long exposure to A. catenella. The scallops were grouped in flasks containing 0 (control), 20, 200, and 2000 cells mL-1 of A. catenella. The presence of A. catenella induced reduced larvae survival after 24 h, whereas a clear effect was observed after 3 days (survival of control larvae 95%, 72, and 79% for 20 and 200 cells mL-1, respectively, and 43% for 2000 cells mL-1). The growth rates of the control larvae and those exposed to 20 mL-1 cells were significantly different from zero. Histopathological effects (melanization, loss of connective tissue, necrosis, and inflammatory responses) were observed in spat exposed to A. catenella. These effects were more pronounced at the highest dinoflagellate concentration. Blooms of A. catenella frequently coincide with the reproductive season of A. tehuelchus, thus there is a need to further study the relationship between harmful algal blooms and the effect on scallops' natural populations in the region.

Collapse of scallop Nodipecten nodosus production in the tropical Southeast Brazil as a possible consequence of global warming and water pollution.

Mollusc rearing is a relevant global socioeconomic activity. However, this activity has faced severe problems in the last years in southeast Brazil. The mariculture scallop production dropped from 51,2 tons in 2016 to 10,2 tons in 2022 in the Baia da Ilha Grande (BIG; Rio de Janeiro). However, the possible causes of this collapse are unknown. This study aimed to analyze decadal trends of water quality in Nodipecten nodosus spat and adult production in BIG. We also performed physical-chemical and biological water quality analyses of three scallop farms and two nearby locations at BIG in 2022 to evaluate possible environmental stressors and risks. Scallop spat production dropped drastically in the last five years (2018-2022: mean ± stdev: 0.47 ± 0.45 million). Spat production was higher in colder waters and during peaks of Chlorophyll a in the last 13 years. Reduction of Chlorophyll a coincided with decreasing spat production in the last five years. Warmer periods (>27 °C) of the year may hamper scallop development. Counts of potentially pathogenic bacteria (Vibrios) and Escherichia coli were significantly higher in warmer periods which may further reduce scallop productivity. Shotgun metagenomics of seawater samples from the five studied corroborated these culture-based counts. Vibrios and fecal indicator bacteria metagenomic sequences were abundant across the entire study area throughout 2022. The results of this study suggest the collapse of scallop mariculture is the result of a synergistic negative effect of global warming and poor seawater quality.

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.

First Report, Characterization and Pathogenicity of Vibrio chagasii Isolated from Diseased Reared Larvae of Chilean Scallop, Argopecten purpuratus (Lamarck, 1819).

Two Vibrio strains (VPAP36 and VPAP40) were isolated from moribund-settled larvae of the Chilean scallop Argopecten purpuratus during vibriosis outbreaks that occurred in two commercial scallop larvae hatcheries located in the Inglesa and Tongoy bays in Northern Chile. The strains were identified as Vibrio chagasii using phenotypic characterization and whole genome sequence analysis. Both strains exhibited the phenotypic properties associated with virulence, gelatin hydrolysis and ß-hemolysis, whereas only VPAP36 produced phospholipase and only VPAP40 produced caseinase. The whole genome analysis showed that the strains harbored genes encoding for the virulence factors, the EPS type II secretion system, and Quorum Sensing (auto-inductor 1 and auto-inductor 2), whereas genes encoding a metalloproteinase and a capsular polysaccharide were detected only in the VPAP40 genome. When challenge bioassays using healthy 11-day-old scallop larvae were performed, the V. chagasii VPAP36 and VPAP40 strains exhibited significant (p < 0.05) differences in their larval lethal activity, producing, after 48 h, larval mortalities of 65.51 ± 4.40% and 28.56 ± 5.35%, respectively. Otherwise, the cell-free extracellular products of the VPAP36 and VPAP40 strains produced larval mortalities of 20.86 ± 2.40% and 18.37 ± 2.40%, respectively, after 48 h of exposure. This study reports for the first time the isolation of V. chagasii from the massive larval mortalities of the farmed scallop (Argopecten purpuratus) in Chile, and demonstrates the pathogenic activity of V. chagasii towards the Chilean scallop, the second most important species for Chilean mariculture.

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 phylogenetically unresolved apicomplexan (APXSc) causing swirl lesions in the Tehuelche scallop, Aequipecten tehuelchus, from the Southwest Atlantic coast.

The study reports a previously unknown apicomplexan (APXSc) parasite infecting wild scallops Aequipecten tehuelchus (d'Orbigny, 1842) from two separate areas (La Tapera and Punta Conos) of the San José gulf, in Patagonia Argentina. Histology, transmission electron microscope, molecular analyses and in situ hybridization were performed to describe the morphology of APXSc, and confirm its phylogenetic status. The prevalence of APXSc infection was 24% and 72% in scallops from La Tapera and Punta Conos, respectively. Seasonal variation was observed for scallops from La Tapera, recording highest prevalence in summer. A positive relationship between the presence of the APXSc and the size of the scallops was observed. A SSU rDNA consensus sequence of 1758 base pairs was generated which has a 94.8% identity to sequences obtained from a pathogenic apicomplexan parasite infecting Ostrea chilensis in New Zealand, but not closely related to other apicomplexans. The asexual reproduction, i.e. merogony, occurs in the Tehuelche scallop whilst the gamogonic and sporogonic stages were absent, suggesting a yet unknown definitive host. Severe host inflammation response involving fibroblast-like hemocytes surrounding the APXSc in the form of granuloma-like "swirls" is characteristic for this apicomplexan infection. Further studies are needed to reveal the life cycle, and presumable pathogenicity of APXSc.

Analysis and gonadal localization of Speedy A mRNA transcript, a novel gene associated with early germline cells in the scallop, Argopecten purpuratus.

The Speedy A (spdya) gene is a member of the Speedy/RINGO family, encoding a spdya protein associated with cellular cycle and meiosis in vertebrates. Results from genetic analyses indicated spdya conditional knockout mice are sterile, suggesting that this protein has essential functions in mammalian reproduction. There, however, are no published reports on the localization of spdya mRNA in the germline or in somatic cell lineages within the gonads from mollusks or other invertebrate species. Using a previously obtained transcriptome assembly from the scallop Argopecten purpuratus, an economically important hermaphroditic scallop species from Chile and Peru, there was identification of a complete coding sequence of the spdya mRNA. Phylogenetically spdya protein has sequence conservation homology with other scallops and mollusks. The relative mRNA transcript abundances at different gametogenic stages was assessed using quantitative PCR procedures. Results indicated there was an increase of spdya mRNA transcript abundance in testicular region samples at the late active stage, followed by a decrease in testis of reproductively mature individuals. To gain insight into the cellular localization of ap-spdya transcript within the gonads, specific RNA probes were synthesized for in situ hybridization analyses of gonad histological sections. Results indicated spdya mRNA is located exclusively in early germline (previtellogenic oocytes and spermatogonia) and somatic proliferative tissues of A. purpuratus ovarian and testicular regions. Overall, these results indicate there are putative functions of spdya in the early oogenesis and spermatogenesis of A. purpuratus and will contribute to furthering the understanding of gametogenesis in this species.

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.

First Report of Domoic Acid Production from Pseudo-nitzschia multistriata in Paracas Bay (Peru).

The Peruvian sea is one of the most productive ecosystems in the world. Phytoplankton production provides food for fish, mammals, mollusks and birds. This trophic network is affected by the presence of toxic phytoplankton species. In July 2017, samples of phytoplankton were obtained from Paracas Bay, an important zone for scallop (Argopecten purpuratus) aquaculture in Peru. Morphological analysis revealed the presence of the genus Pseudo-nitzschia, which was isolated and cultivated in laboratory conditions. Subsequently, the monoclonal cultures were observed by scanning electron microscopy (SEM), and identified as P. multistriata, based on both the morphological characteristics, and internal transcribed spacers region (ITS2) sequence phylogenetic analysis. Toxin analysis using liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) revealed the presence of domoic acid (DA) with an estimated amount of 0.004 to 0.010 pg cell-1. This is the first report of DA from the coastal waters of Peru and its detection in P. multistriata indicates that it is a potential risk. Based on our results, routine monitoring of this genus should be considered in order to ensure public health.

First description outside Europe of the emergent pathogen Vibrio europaeus in shellfish aquaculture.

Vibrio europaeus is an emergent pathogen affecting the most important bivalve species reared in Spanish and French hatcheries. Using a genomic approach, we identified V. europaeus outside Europe for the first time from massive larval mortalities of scallop (Argopecten purpuratus) in Chile and from seawater near a shellfish hatchery in the US West Coast. Results show the worldwide spreading and potential impact of V. europaeus for aquaculture; these four countries are among the 10 major producers of mollusks. Pathogenicity of V. europaeus was demonstrated for the first time towards scallop, the second most important species for Chilean mariculture.

Midterm Follow-up of Fenestrated and Scalloped Physician-Modified Endovascular Grafts for Zone 2 TEVAR.

Purpose: To investigate the midterm outcomes of scalloped or fenestrated physician-modified endovascular grafts (PMEGs) for zone 2 thoracic endovascular aortic repairs (TEVAR). Materials and Methods: Between November 2013 and May 2019, 54 consecutive patients (mean age 63 years; 41 men) were treated with thoracic PMEGs modified with 7 scallops or 47 fenestrations for the left subclavian artery (LSA). Indications for aortic repair were acute complicated type B aortic dissection (17, 31%), degenerative aneurysm (13, 24%), acute traumatic rupture of the aortic isthmus (9, 16%), post chronic dissection aneurysmal evolution (8, 15%), penetrating aortic ulcer (3, 6%), intramural hematoma (2, 4%), and floating thrombus (2, 4%). Results: Technical success was 94%; 3 (6%) LSAs were unintentionally covered. An intraoperative type Ia endoleak was treated during the index procedure. One (2%) patient suffered spinal cord ischemia, with irreversible bilateral paraplegia. Three (6%) patients experience postoperative minor strokes with full neurological recovery. Four (7%) patients died in the perioperative period; 2 (2%) were due to aneurysm rupture. Mean follow-up was 26±16 months; 15 (28%) patients had at least 3 years of follow-up. Two (4%) type II endoleaks were identified and successfully treated (4% reintervention rate); no other endoleaks were identified. All the LSAs remained clinically and radiologically patent. There were no conversions to open repair, ruptures, retrograde dissection, stent fracture, migrations, or other aortic complications. Conclusion: Scalloped or single-fenestrated PMEGs for the LSA appear to be durable and safe in the midterm. Combined with low periprocedural morbidity and mortality, these results suggest that this approach can be considered as an off-label alternative to extend proximal seal to zone 2 for TEVAR. Further studies with a larger number of patients and long-term outcomes are needed to fully validate this approach.

Efecto de medicamentos homeopáticos sobre crecimiento, supervivencia y microbiota gastrointestinal, en juveniles del pectínido Argopecten ventricosus / Effect of homeopathic medicines on growth, survival and gastrointestinal microbiota of juvenile scallop Argopecten ventricosus

RESUMEN Objetivo. Estudiar el efecto de medicamentos homeopáticos sobre el crecimiento, supervivencia y microbiota del tracto gastrointestinal (TGI) de almeja Catarina Argopecten ventricosus. Materiales y Métodos. Se aplicaron cinco tratamientos homeopáticos derivados de bacterias [(T1) ViP-ViA 1D, (T2) ViP-ViA 7C], minerales [(T3) AcF-MsS 1D, (T4) PhA-SiT 7C], o venenos [(T5) ViT 31C] y tres controles: (C1) etanol diluido 1:99, (C2) etanol dinamizado 1C y (C3) agua destilada. La microbiota se determinó secuenciando la región V3-V5 del gen 16S rRNA. Resultados. El mayor crecimiento en longitud de la concha correspondió a T1 (117 µm d-1) y T2 (108 µm d-1), la mayor supervivencia a T3 y T5 y el mejor resultado global a T3. Las curvas de rarefacción de los grupos tratados y controles mostraron una clara separación. Se encontraron diferencias significativas (p≤0.05) entre filos (Proteobacteria > Actinobacteria > Firmicutes > Bacterloidetes> Chloroflexi y para los Géneros: Symbiobacterium > Microbacterium > Methylobacillus > Bacillus > Paenibacillus > Burkholderia > Nostoc > Methylobacterium > Leucobacter). El género Symbiobacterium fue dominante (p≤0.05) para T5, respecto a todos los tratamientos y grupos controles. La especie Microbacterium maritypicum (Actinobacteria) mostró la mayor abundancia relativa (p≤0.05) en T1 y T3 y Symbiobacterium toebii (Firmicutes) en T5 y T2 (p≤0.05), ambas con respecto al inicio del estudio T0. Conclusiones. Se presenta por primera vez la composición de la microbiota del TGI de A. ventricosus y la aplicabilidad potencial de la homeopatía para mejorar el rendimiento productivo y modular la microbiota gastrointestinal de la especie.

ABSTRACT Objective. To study the effect of homeopathic medicines on growth, survival and gastrointestinal (GIT) microbiota of Catarina scallop Argopecten ventricosus. Materials and methods. Five homeopathic (HOM) treatments derived from bacteria [(T1) ViP-ViA 1D, (T2) ViP-ViA 7C], minerals [(T3) AcF-MsS 1D, (T4) PhA-SiT 7C] or venoms [T5) ViT 31C] and three controls: [(C1) diluted ethanol 1:99, (C2) diluted/succussed ethanol 1C and (C3) distilled water] were evaluated (21 days) in triplicate. Microbiota was analysed by sequencing the V3-V5 region of the 16S rRNA genes. Results. The best growth in shell-length corresponded to T1 (117 µm d-1) and T2 (108 µm d-1) and the highest survival to T3 and T5, stating T3 as the best HOM-treatment. A clear separation was found in rarefaction curves of HOM-treated against un-treated control scallops. Significant differences (p≤0.05) were found for Phyla (Proteobacteria> Actinobacteria> Firmicutes> Bacterloidetes>Chloroflexi and for Genera: Symbiobacterium> Microbacterium> Methylobacillus> Bacillus> Paenibacillus> Burkholderia> Nostoc> Methylobacterium> Leucobacter). The genus Symbiobacterium was dominant in T5, finding significant differences (p≤0.05) with respect to all treatments. At species level, Microbacterium maritypicum (Actinobacteria) showed a greater relative abundance (p≤0.05) in T1 and T3 and Symbiobacterium toebii (Firmicutes) was also significantly higher (p≤0.05) in abundance in T5 and T2, both against initial T0. Conclusions. This study showed for a first time, the composition of GIT microbiota in A. ventricosus and focused on the potential applicability of homeopathy to improve overall performance and modulate the GIT microbiota of the species.

Chaetopterus antarcticus (Polychaeta: Chaetopteridae) in Argentinian shelf scallop beds: from infaunal to epifaunal life habits

Introduction: The shelf-break frontal area in the Argentine Sea, between 37ｼ S and 40ｼ S, is characterized by high frequency and abundance of the parchment worm Chaetopterus antarcticus Kinberg, 1866 associated to Zygochlamys patagonica scallop beds. This polychaete was usually collected within its U tubes, typical of infaunal habit. During 2007, a surprising massive settlement of worms on scallops was observed. Objective: Our objectives is to report the variability of life habits of C. antarcticus in scallop grounds distributed between 37º S and 40º S in Argentinean waters. Methods: The presence of the parchment tube worms on Z. patagonica was analyzed in 892 scallops coming from three samples collected in September (previously obtained for other purposes) and November 2007 and April 2008, between 99-106 m depth in Management Unit B (16.186 km2, 38º S - 39º 52' S). Polychaetes were identified and quantified on both valves. In addition, both epibiont and infaunal worms were registered through time. The settlement of C. antarcticus on scallops was monitored during the period 2007-2015 and the presence of free tubes, both empty and inhabited by worms, were registered during the period 2008-2015 from a total of 374 representative subsamples (10 l) collected with trawling tools between 37º S and 40º S. Results: Scallops collected in 2007 and 2008 showed that 468 (> 50 %) specimens were encrusted; in 66 % (311 specimens) of them C. antarcticus was settled. The temporal analysis showed that the spatial distribution of epibiotic worms decreased in 2009, 2010 and 2012, with its total absence in 2013, 2014 and 2015 in the study area. During the period 2007 - 2015, infaunal worms were registered throughout the area, collected in part of the locations monitored. Conclusions: The variability of life habits of C. antarcticus was not commonly registered previously in the study area; epibiotic behavior could be the result of intensive soft sediment disturbance due to trawling and hence, the selection of other available primary settlement substrate.

Introducción: El área del frente de talud en el Mar Argentino, entre 37° S y 40° S, se caracteriza por alta frecuencia y abundancia del poliqueto Chaetopterus antarcticus Kinberg, 1866 asociado a bancos de vieira Zygochlamys patagonica (King, 1832). Este poliqueto era colectado usualmente habitando los tubos en U, típicos de hábito infaunal. Durante 2007, un sorprendente asentamiento masivo de gusanos sobre vieiras fue observado. Objetivos: Los objetivos del trabajo son reportar la variabilidad del hábito de vida de C. antarcticus en fondos de vieiras entre 37° S y 40° S en aguas de Argentina. Métodos: La presencia de gusanos tubícolas sobre Z. patagonica se analizó en 892 vieiras colectadas en tres muestras durante Septiembre (muestra tomada con otros propósitos) y Noviembre 2007 y Abril 2008, entre 99-106 m de profundidad en la Unidad de Manejo B (16.186 km2, 38º S - 39º 52' S). Los poliquetos fueron identificados y cuantificados en ambas valvas. Además, los gusanos tanto epibiontes como infaunales se registraron en un período de tiempo. El asentamiento de C. antarcticus sobre vieiras se monitoreó durante 2007-2015 y la presencia de tubos libres, tanto vacíos como habitados por gusanos, durante 2008-2015 a partir de 374 submuestras representativas (10 l) colectadas con artes de arrastre entre 37° S y 40° S. Resultados: Las vieiras colectadas en 2007 y 2008 mostraron que 468 (> 50 %) especímenes estaban incrustados; C. antarcticus estaba asentado en 66 % (311 especímenes) de los mismos. El análisis temporal mostró que la distribución espacial de gusanos epibiontes decreció en 2009, 20101 y 2012, y su ausencia total en 2013, 2014 y 2015 en el área de estudio. Durante el período 2007 - 2015 los gusanos infaunales se registraron en toda el área, en parte de las localidades relevadas. Conclusiones: La variabilidad del hábito de vida de C. antarcticus no había sido registrada previamente en el área de estudio; el comportamiento epibiótico podría ser el resultado del disturbio intensivo de los sedimentos blandos debido al arrastre y en consecuencia, la selección de otro sustrato de asentamiento primario disponible.

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.

Isolation and Pathogenic Characterization of Vibrio bivalvicida Associated With a Massive Larval Mortality Event in a Commercial Hatchery of Scallop Argopecten purpuratus in Chile.

The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods, to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of the pathology. The pathogenic strain was identified as Vibrio bivalvicida based on its phenotypic properties, the multilocus sequence analysis (MLSA) of eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) and different in silico genome-to-genome comparisons. When triplicate cultures of healthy 10 days old scallop larvae were challenged with 1 × 105 colony forming units (CFU) mL-1 of the VPAP30 strain, percentages of larval survival of 78.9 ± 3.3%, 34.3 ± 4.9%, and 0% were observed at 12, 2,4 and 36 h, respectively, whereas uninfected larval cultures showed survival rates of 97.4 ± 1.2% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.7%, significantly (P < 0.001) lower than the control group (97.4 ± 1.2%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.8% after 48 h of exposure. V. bivalvicida VPAP30 exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds. This report constitutes the first isolation of V. bivalvicida out of Europe and extends the host range of this species, having demonstrated its pathogenic activity on the Chilean scallop larvae (A. purpuratus). These results supporting the pathogenic potential of V. bivalvicida to kill the larvae of a broad range of bivalve species reared in hatcheries located in the Atlantic and the Pacific coasts.

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.

Sex determination and differentiation genes in a functional hermaphrodite scallop, Nodipecten subnodosus.

The lion-paw, Nodipecten subnodosus is one of three scallop species commercially exploited on the west coast of the Peninsula of Baja California. Because nothing is known about sex determination and sexual differentiation in hermaphrodite scallops, in the present work, a global transcriptomic analysis was performed in two early developmental stages, settling eyed-larvae and spat, as well as in three tissues (undifferentiated gonad, digestive gland, and adductor muscle). Over 27 million Illumina paired-end reads were obtained through the MiSeq platform. After processing the reads a total of 243,774 transcripts were assembled with an N50 of 980 and an average length of 775nt. A total of 43,252 proteins were inferred and 36,103 transcripts had at least one homolog in the SwissProt database according to a blastx search. After differential expression analyses and GO annotations it was possible to identify several sex-related genes in the scallop, including one known to be involved in the sex determination pathway of the hermaphrodite model organism Caenorhabditis elegans, N. subnodosus-sex1 (Ns-sex1). Other interesting sex determination and differentiation genes were Ns-dmrta2, Ns-sox9, Ns-wnt4, Ns-doa, Ns-ovo, Ns-vir, among others. Most of these genes were mainly expressed in the testis region, suggesting their participation in male gonad region sex differentiation. These results represent the first available information on the genetics of sex determination and differentiation in a functional hermaphrodite scallop.

Embryo-larvae and juvenile toxicity of Pb and Cd in Northern Chilean scallop Argopecten purpuratus.

This study aimed to investigate the effects of Cd and Pb on earlier stage of the commercially important scallop (Argopecten purpuratus) in the contamination context of Northern Chile where this specie is farmed, through acute exposure bioassays in embryo-larvae measuring cumulative abnormality (EC50) and juvenile cumulative mortality (LC50) as endpoints, based on environmentally detected concentrations and available toxicological data from similar species. Embryo-larvae exposure indicates 48 h EC50 of 1.55 mg/L Cd, and 0.044 mg/L Pb. On the contrary, 96 h LC50 in juvenile scallops was 0.48 mg/L Cd and 1.47 mg/L Pb. Our results demonstrated differential toxicity between embryo and juvenile scallops that might relate to different primary defense mechanisms or effect in morphological development of individuals in each ontogenetic stage. Compared to similar bivalve metal toxicity tests, this study demonstrated that A. purpuratus embryos are more sensitive to Pb than most other bivalve species. Our results indicate that maximum permitted levels of Pb in marine waters and estuaries (according to Chilean regulation) could pose a risk for scallops' first stage of life (embryo-larvae) development, and needs to be reviewed. Furthermore, Chilean environmental regulations do not have quality standards for marine sediments (currently under discussion), where high levels of metals have been continuously reported.

Potentially toxic filamentous fungi associated to the economically important Nodipecten nodosus (Linnaeus, 1758) scallop farmed in southeastern Rio de Janeiro, Brazil.

Numerous countries have been confronted with infectious diseases in mariculture activities, including fungi infections, although reports in scallops are scarce. Thus, this study aimed to investigate the occurrence of filamentous fungi in Nodipecten nodosus specimens from three marine farms in Southeastern Brazil. Eight fungi genera were observed in the branchial arches, intestine and muscle tissue of the scallop specimens. These include potentially toxin-producing species, such as Aspergillus, Penicillium and Fusarium. Their presence may lead to potential public health concerns, since all sampling sites showed the presence of fungi in all scallop organs, with special concern regarding edible muscle tissue. A significant number of species was observed at one of the study areas, which could indicate a previously unknown source of contamination, since increases in fungi species richness in polluted coastal waters have been reported. This is also, to the best of our knowledge, the first report of Pestalotiopsis in shellfish.

First Report of Vibrio tubiashii Associated with a Massive Larval Mortality Event in a Commercial Hatchery of Scallop Argopecten purpuratus in Chile.

The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of this pathology. The pathogenic strain was identified as Vibrio tubiashii based on its phenotypic properties and the sequence analysis of its 16S rRNA and housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA). When triplicate cultures of healthy 10-day-old scallop larvae were challenged with 1 × 105 colony forming units (CFU) mL-1 of the VPAP30 strain, percentages of larval survival of 78.87 ± 3.33%, 34.32 ± 4.94%, and 0% were observed at 12, 24, and 36 h, respectively; whereas uninfected larval cultures showed survival rates of 97.4 ± 1.24% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.66%, significantly (P < 0.001) lower than the control group (97.4 ± 1.20%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.84% after 48 h of exposure. This is the first report of the isolation of V. tubiashii from the diseased larvae of the scallop A. purpuratus, occurring in a commercial culture in Chile, and it was demonstrated that the VPAP30 strain exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds.