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.

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.

Multiple-stressor effects of ocean acidification, warming and predation risk cues on the early ontogeny of a rocky-shore keystone gastropod.

To understand how climate change stressors might affect marine organisms and support adequate projections it is important to know how multiple stressors may be modulated by the presence of other species. We evaluated the direct effects of ocean warming (OW) and ocean acidification (OA) together with non-consumptive effects (NCEs) of the predatory crab Acanthocyclus hassleri on early ontogeny fitness-related traits of the commercially important rocky-shore keystone gastropod Concholepas concholepas. We measured the response of nine traits to these stressors at either the organismal level (survival, growth, feeding rates, tenacity, metabolic rate, calcification rate) or sub-organismal level (nutritional status, ATP-supplying capacity, stress condition). C. concholepas survival was not affected by any of the stressors. Feeding rates were not affected by OW or OA; however, they were reduced in the presence of crab NCEs compared with control conditions. Horizontal tenacity was affected by the OA × NCEs interaction; in the presence of NCEs, OA reduced tenacity. The routine metabolic rate, measured by oxygen consumption, increased significantly with OW. Nutritional status assessment determined that carbohydrate content was not affected by any of the stressors. However, protein content was affected by the OA × NCEs interaction; in the absence of NCEs, OA reduced protein levels. ATP-supplying capacity, measured by citrate synthase (CS) activity, and cellular stress condition (HSP70 expression) were reduced by OA, with reduction in CS activity found particularly at the high temperature. Our results indicate C. concholepas traits are affected by OA and OW and the effects are modulated by predator risk (NCEs). We conclude that some C. concholepas traits are resilient to climate stressors (survival, growth, horizontal tenacity and nutritional status) but others are affected by OW (metabolic rate), OA (ATP-supplying capacity, stress condition), and NCEs (feeding rate). The results suggest that these negative effects can adversely affect the associated community.

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.

The combined effects of climate change stressors and predatory cues on a mussel species.

In order to make adequate projections on the consequences of climate change stressors on marine organisms, it is important to know how impacts of these stressors are affected by the presence of other species. Here we assessed the direct effects of ocean warming (OW) and acidification (OA) along with non-consumptive effects (NCEs) of a predatory crab and/or a predatory snail on the habitat-forming mussel Perumytilus purpuratus. Mussels were exposed for 10-14 weeks to contrasting pCO2 (500 and 1400 µatm) and temperature (15 and 20 °C) levels, in the presence/absence of cues from one or two predator species. We compared mussel traits at sub-organismal (nutritional status, metabolic capacity-ATP production-, cell stress condition via HSP70 expression) and organismal (survival, oxygen consumption, growth, byssus biogenesis, clearance rates, aggregation) levels. OA increased the mussels' oxygen consumption; and OA combined with OW increased ATP demand and the use of carbohydrate reserves. Mussels at present-day pCO2 levels had the highest protein content. Under OW the predatory snail cues induced the highest cell stress condition on the mussels. Temperature, predator cues and the interaction between them affected mussel growth. Mussels grew larger at the control temperature (15 °C) when crab and snail cues were present. Mussel wet mass and calcification were affected by predator cues; with highest values recorded in crab cue presence (isolated or combined with snail cues). In the absence of predator cues in the trails, byssus biogenesis was affected by OA, OW and the OA × OW and OA × predator cues interactions. At present-day pCO2 levels, more byssus was recorded with snail than with crab cues. Clearance rates were affected by temperature, pCO2 and the interaction between them. The investigated stressors had no effects on mussel aggregation. We conclude that OA, OW and the NCEs may lead to neutral, positive or negative consequences for mussels.

The combined effects of ocean acidification and warming on a habitat-forming shell-crushing predatory crab.

In mid rocky intertidal habitats the mussel Perumytilus purpurarus monopolizes the substratum to the detriment of many other species. However, the consumption of mussels by the shell-crushing crab Acanthocyclus hassleri creates within the mussel beds space and habitat for several other species. This crab uses its disproportionately large claw to crush its shelled prey and plays an important role in maintaining species biodiversity. This study evaluated the consequences of projected near-future ocean acidification (OA) and warming (OW) on traits of A. hassleri linked with their predatory performance. Individual A. hassleri were maintained for 10-16 weeks under contrasting pCO2 (~500 and 1400 µatm) and temperature (~15 and 20 °C) levels. We compared traits at the organismal (oxygen consumption rate, survival, calcification rate, feeding rates, crusher claw pinching strength, self-righting speed, sarcomere length of the crusher claw muscles) and cellular (nutritional status ATP provisioning capacity through citrate synthase activity, expression of HSP70) level. Survival, calcification rate and sarcomere length were not affected by OA and OW. However, OW increased significantly feeding and oxygen consumption. Pinching strength was reduced by OA; meanwhile self-righting was increased by OA and OW. At 20 °C, carbohydrate content was reduced significantly by OA. Regardless of temperature, a significant reduction in energy reserves in terms of protein content by OA was found. The ATP provisioning capacity was significantly affected by the interaction between temperature and pCO2 and was highest at 15 °C and present day pCO2 levels. The HSP70 levels of crabs exposed to OW were higher than in the control crabs. We conclude that OA and OW might affect the amount and size of prey consumed by this crab. Therefore, by reducing the crab feeding performance these stressors might pose limits on their role in generating microhabitat for other rocky intertidal species inhabiting within mussel beds.

Are we neglecting earth while conquering space? Effects of aluminized solid rocket fuel combustion on the physiology of a tropical freshwater invertebrate.

Space launchers often use aluminized-solid fuel ("propergol") as propellant and its combustion releases tons of Al2O3 and HCl that sink in terrestrial and aquatic environments, polluting and decreasing water pH. We studied the impact of these events on the biochemical/physiological performance of the freshwater shrimp Macrobrachium jelskii, with wild specimens collected from a non-impacted site in French Guiana. In the laboratory, shrimps were exposed for one week to: i) undisturbed conditions; ii) Al2O3 exposure (0.5 mg L-1) at normal pH (6.6); iii) decreased pH (4.5) (mimicking HCl release in the environment) with no Al2O3; or iv) Al2O3 0.5 mg L-1 and pH 4.5, representing the average conditions found in the water bodies around the Ariane 5 launch pad. Results showed that shrimps bioaccumulated aluminium (Al) regardless of water pH. The combined effect of Al2O3 and low pH caused the most impact: acetylcholinesterase and carboxylesterase activities decreased, indicating neurotoxicity and reduced detoxification capacity, respectively. Animal respiration was enhanced with Al2O3 and pH variations alone, but the synergic interaction of both stressors caused respiration to decrease, suggesting metabolic depression. Oxidative damage followed a similar pattern to respiration rates across conditions, suggesting free radical-mediation in Al toxicity. Antioxidant activities varied among enzymes, with glutathione reductase being the most impacted by Al2O3 exposure. This study shows the importance of addressing space ports' impact on the environment, setting the bases for selecting the most appropriate biomarkers for future monitoring programs using a widespread and sensitive crustacean in the context of an increasing space-oriented activity across the world.

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.

Effect of predation risk and ectoparasitic louse flies on physiological stress condition of the red-tailed tropicbird (Phaethon rubricauda) from Rapa Nui and Salas & Gómez islands.

BACKGROUND: Introduced predators at seabird colonies and parasites may have lethal and/or sub-lethal consequences for bird populations. We assessed the potential sub-lethal negative effects of these stressors in a native seabird listed as vulnerable in its south-eastern pacific distribution. This study was conducted in two red-tailed tropicbird (Phaethon rubricauda) colonies, one located in Rapa Nui Island, which is threatened by the presence of introduced predators, and the other located in Salas & Gómez Island, which has no introduced predators, but birds are infested by ectoparasitic louse flies. METHODS: The effects on physiological stress traits of both, predation risk on different nest types (protected and exposed) on Rapa Nui, and different levels of louse flies' parasitic loads on Salas & Gómez were studied. Three variables were analyzed: the heterophil/lymphocyte (H/L) ratio, the transcriptional levels of mRNA HSP70 in blood, and the body condition. These stress indicators and leukocyte counts were compared between colonies. RESULTS: No significant differences were found in any stress indicator between different nest types within Rapa Nui, showing that the effect of predator's presence was the same for adults nesting in both, protected and exposed nests. No significant correlation was found between louse flies' parasitic loads and any stress indicators in the birds of Salas & Gómez. Also, there was no difference in any stress indicator between islands. However, a significant opposite trend between islands was found in the eosinophil, lymphocyte, and monocyte counts when related to body condition. CONCLUSIONS: We found a lack of significant differentiation in all the stress level indicators assessed within and between islands. The presence of louse flies in Salas & Gómez vs. the absence of this parasite at Rapa Nui may be the cause for the significant difference in the trend of eosinophil, lymphocyte and monocyte counts between the islands. However, further studies are necessary to elucidate the reason for this difference and to better investigate the lethal effects of introduced predators on the Rapa Nui colony to evaluate appropriate conservation measures for this native seabird.

Combined effect of pCO2 and temperature levels on the thermal niche in the early benthic ontogeny of a keystone species.

We evaluated the effects of projected, near future ocean acidification (OA) and extreme events of temperature (warming or cooling) on the thermal tolerance of Concholepas concholepas, a coastal benthic keystone species. Three separate trials of an experiment were conducted by exposing juvenile C. concholepas for 1 month to one of two contrasting pCO2 levels (~500 and ~1200 µatm). In addition, each pCO2 level was combined with one of four temperature treatments. The control was 15 °C, whilst the other temperatures were 10 °C (Trial 1), 20 °C (Trial 2) and 25 °C (Trial 3). At the end of each trial, we assessed Critical Thermal maximum (CTmax) and minimum (CTmin) via self-righting success, calculated partial thermal tolerance polygons, measured somatic growth, determined transcription of Heat Shock Proteins 70 (HSP70) and measured oxygen consumption rates. Regardless of pCO2 level, HSP70 transcript levels were significantly higher in juveniles after exposure to extreme temperatures (10 °C and 25 °C) indicating physiological stress. Oxygen consumption rates increased with increasing temperature from 10 °C to 20 °C though showed a decrease at 25 °C. This rate was not affected by pCO2 or the interaction between temperature and pCO2. Juveniles exposed to present-day and near future pCO2 levels at 20 °C showed similar thermal tolerance polygonal areas; whilst changes in both CTmin and CTmax at 25 °C and 10 °C caused narrower and broader areas, respectively. Temperature affected growth, oxygen consumption and HSP70 transcription in small juvenile C. concholepas. Exposure to elevated pCO2 did not affect thermal tolerance, growth or oxygen consumption at temperatures within the thermal range normally experienced by this species in northern Chile (15-20 °C). At elevated pCO2 conditions, however, exposure to warmer (25 °C) or colder (10 °C) temperatures reduced or increased the thermal area, respectively. This study demonstrates the importance of examining the thermal-tolerance edges to better understand how OA and temperature will combine to physiologically challenge inter-tidal organisms.

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.

Ocean warming and acidification pose synergistic limits to the thermal niche of an economically important echinoderm.

To make robust projectios of the impacts of climate change, it is critical to understand how abiotic factors may interact to constrain the distribution and productivity of marine flora and fauna. We evaluated the effects of projected end of the century ocean acidification (OA) and warming (OW) on the thermal tolerance of an important living marine resource, the sea urchin Loxechinus albus, a benthic shallow water coastal herbivore inhabiting part of the Pacific coast of South America. After exposing young juveniles for a 1-month period to contrasting pCO2 (~500 and 1400 µatm) and temperature (~15 °C and 20 °C) levels, critical thermal maximum (CTmax) and minimum (CTmin) as well as thermal tolerance polygons were assessed based on self-righting success as an end point. Transcription of heat shock protein 70 (HSP70), a chaperone protecting cellular proteins from environmental stress, was also measured. Exposure to elevated pCO2 significantly reduced thermal tolerance by increasing CTmin at both experimental temperatures and decreasing CTmax at 20 °C. There was also a strong synergistic effect of OA × OW on HSP70 transcription levels which were 75 times higher than in control conditions. If this species is unable to adapt to elevated pCO2 in the future, the reduction in thermal tolerance and HSP response suggests that near-future warming and OA will disrupt their performance and reduce their distribution with ecological and economic consequences. Given the wider latitudinal range (6 to 56°S) and environmental tolerance of L. albus compared to other members of this region's benthic invertebrate community, OW and OA may cause substantial changes to the coastal fauna along this geographical range.

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.

Cloning and molecular characterization of two ferritins from red abalone Haliotis rufescens and their expressions in response to bacterial challenge at juvenile and adult life stages.

Ferritins are ubiquitous proteins with a pivotal role in iron storage and homeostasis, and in host defense responses during infection by pathogens in several organisms, including mollusks. In this study, we characterized two ferritin homologues in the red abalone Haliotis rufescens, a species of economic importance for Chile, USA and Mexico. Two ferritin subunits (Hrfer1 and Hrfer2) were cloned. Hrfer1 cDNA is an 807 bp clone containing a 516 bp open reading frame (ORF) that corresponds to a novel ferritin subunit in H. rufescens. Hrfer2 cDNA is an 868 bp clone containing a 516 bp ORF that corresponds to a previously reported ferritin subunit, but in this study 5'- and 3'-UTR sequences were additionally found. We detected a putative Iron Responsive Element (IRE) in the 5'-UTR sequence, suggesting a posttranscriptional regulation of Hrfer2 translation by iron. The deduced protein sequences of both cDNAs possessed the motifs and domains required in functional ferritin subunits. Expression patterns of both ferritins in different tissues, during different developmental stages, and in response to bacterial (Vibrio splendidus) exposure were examined. Both Hrfer1 and Hrfer2 are most expressed in digestive gland and gonad. Hrfer1 mRNA levels increased about 34-fold along with larval developmental process, attaining the highest level in the creeping post-larvae. Exogenous feeding is initiated at the creeping larva stage; thus, the increase of Hrfer1 may suggest and immunity-related role upon exposure to bacteria. Highest Hrfer2 expression levels were detected at trochophore stage; which may be related with early shell formation. Upon challenge with, the bacteria an early mild induction of Hrfer2 (2 h post-challenge), followed by a stronger induction of Hrfer1 at 15 h post-challenge, was observed in haemocytes from adult abalones. While maximal upregulation of both genes in the whole individual occurred at 24 h post-challenge, in juveniles. A significant increase in ferritin protein levels from 6 h to 24 h post-challenge was also detected. Our results suggest an involvement of Hrfer1 and Hrfer2, and of ferritin proteins in the immune response of H. rufescens to bacterial infection.

First insight into the heritable variation of the resistance to infection with the bacteria causing the withering syndrome disease in Haliotis rufescens abalone.

Withering syndrome disease has experienced worldwide spread in the last decade. This fatal disease for abalone is produced by a rickettsia-like organism (WS-RLO), the bacterium "Candidatus Xenohaliotis californiensis". To evaluate the potential of the red abalone (Haliotis rufescens) to improve its resistance to infection by WS-RLO, the additive genetic component in the variation of this trait was estimated. For this, the variation in infection intensity with WS-RLOs and WS-RLOv (phage-infected RLOs) was analyzed in 56 families of full-sibs maintained for three years in a host-parasite cohabitation aquaculture system. A WS-RLO prevalence of 65% was observed in the analysed population; and from the total WS-RLO inclusions 60% were hyperparasited with the phage (WS-RLOv). The decrease in the food ingestion rate was the sole negative effect associated with increasing WS-RLO intensity of infection, suggesting that the high level of WS-RLOv load may have diminished the symptoms of WS disease in the analyzed abalones. The estimated heritabilities were moderate to mid, but significant, varying from 0.21 to 0.23 and 0.36 for WS-RLO and WS-RLOv infections, respectively. This suggests that variation in resistance to infection with WS-RLO may respond to selection in the evaluated red abalone population. Estimated response to selection (G) for the level of infection by WS-RLO indicated that if the 10% of red abalone with the lowest infection level is selected as broodstock, a 90% reduction in the intensity of infection in the progeny can be expected, even with the lowest estimation of heritability (h2=0.21). This strong response would be also due to the large phenotypic variance of this trait. Strong positive correlations, both phenotypic and genotypic, were observed between infection intensities with WS-RLO and WS-RLOv, indicating that selection to increase resistance to one of the types of RLOs will affect resistance in the other in the same direction. This is the first study that demonstrates the existence of additive genetic variation for resistance to WS-RLO in abalone. Consequently, it is possible to increase the resistance to WS-RLO in H. rufescens by selective breeding, which can be an economically attractive and environmentally friendly manner to reduce mortalities and growth effects caused by WS in abalone farms.

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.