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.

Histopathological survey of parasites harboured by the clam Tawera elliptica (Lamarck, 1818) from Chiloé Archipelago, southeastern Pacific.

Tawera elliptica (commonly known as "Almeja Juliana", is a venerid clam that inhabits sandy bottoms and is distributed from Valparaíso on the Pacific coast up to the Mar del Plata area along the Atlantic coast. Harvests of this clam have declined substantially over the last decade. Therefore, an analysis of common parasites and pathological conditions of this clam was undertaken along with histopathology. Monthly samples were prepared for routine histology for examination under light and electron microscopy. T. elliptica has a sex ratio of 1:1 and the relationship between the shell length and the wet tissue weight is not significantly different between females and males. The maximum values for de condition index and meat yield were found during the austral winter. The following parasites (and their overall prevalence) were detected: intracellular microcolonies of bacteria in digestive gland (22.9%), intestinal epithelium (9.3%) and gills (3.17%), an unidentified cyst in gills (59,3%), a Steinhausia-like intraoocytic microsporidian (5.2%), Gregarine spores (41.3%), ciliated protozoa (16.7%), two metazoa, a Paravortex like flatworm (4.3%), and a digenean trematode (8%). The monthly mean intensity of the most relevant parasites was between 2.3 and 35.6 for digestive gland intracellular microcolonies of bacteria (IMC), 0-5.1 for intestinal epithelium IMC, 0-2 for branchial IMC and 0 - 48 for intraoocytic microsporidium. The prevalence and the infection intensity were low-to very low, and no World Organisation for Animal Health OIE listed parasite was detected. It is concluded that this is a healthy clam, and no disease risks for the cultivation are visualized at present. However, IMC at high prevalence and intensities of infection could be potentially impactful, and the intraoocytic microsporidian could jeopardize reproduction if present in high intensities of infection.

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.

Minimal impact at current environmental concentrations of microplastics on energy balance and physiological rates of the giant mussel Choromytilus chorus.

Microplastic particles (MP) uptake by marine organisms is a phenomenon of global concern. Nevertheless, there is scarce evidence about the impacts of MP on the energy balance of marine invertebrates. We evaluated the mid-term effect of the microplastic ingestion at the current higher environmental concentrations in the ocean on the energy balance of the giant mussel Choromytilus chorus. We exposed juvenile mussels to three concentrations of microplastics (0, 100, and 1000 particles L-1) and evaluated the effect on physiology after 40 days. The impacts of MP on the ecophysiological traits of the mussels were minimum at all the studied concentrations. At intermediate concentrations of MP, Scope for Growth (SFG) had little impact. Other relevant key life-history and physiological processes, such as size and metabolism, were not affected by microplastics. However, individuals treated with MP presented histopathological differences compared to control group, which could result in adverse health effects for mussels.

Cosmopolitan Distribution of Endozoicomonas-Like Organisms and Other Intracellular Microcolonies of Bacteria Causing Infection in Marine Mollusks.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia-like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae, the genera Mycoplasma and Francisella, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens.

Microbiota of the Digestive Gland of Red Abalone (Haliotis rufescens) Is Affected by Withering Syndrome.

Withering syndrome (WS), an infectious disease caused by intracellular bacteria Candidatus Xenohaliotis californiensis, has provoked significant economic losses in abalone aquaculture. The pathogen infects gastroenteric epithelia, including digestive gland, disrupting the digestive system and causing a progressive wilting in abalone. Nonetheless, our knowledge about WS implications in digestive gland microbiota, and its role in diseases progress remains largely unknown. This study aims to determine whether digestive gland-associated microbiota differs between healthy red abalone (Haliotis rufescens) and red abalone affected with WS. Using high-throughput sequencing of the V4 region of the 16S rRNA gene, our results revealed differences in microbiota between groups. Bacterial genera, including Mycoplasma, Lactobacillus, Cocleimonas and Tateyamaria were significantly more abundant in healthy abalones, whilst Candidatus Xenohaliotis californiensis and Marinomonas were more abundant in WS-affected abalones. Whilst Mycoplasma was the dominant genus in the healthy group, Candidatus Xenohaliotis californiensis was dominant in the WS group. However, Candidatus Xenohaliotis californiensis was present in two healthy specimens, and thus the Mycoplasma/Candidatus Xenohaliotis californiensis ratio appears to be more determinant in specimens affected with WS. Further research to elucidate the role of digestive gland microbiota ecology in WS pathogenesis is required.

Characterizing the Bioluminescence of the Humboldt Squid, Dosidicus gigas (d'Orbigny, 1835): One of the Largest Luminescent Animals in the World.

Bioluminescence is found in a number of cephalopods, such as Watasenia scintillans and Sthenoteuthis oualaniensis; however, many species remain poorly studied, including the Humboldt squid, Dosidicus gigas. This is the largest member of the Ommastrephidae family and grows to 2 m in length, making it one of the largest luminescent animals ever observed. Humboldt squid have small photophores all over their body that emit a brilliant blue luminescence. Using lyophilized photophores from squid caught off the coast of Chile, experiments were conducted to isolate the luciferin and protein involved in its bioluminescence. Methanolic extracts of the photophores were shown to contain dehydrocoelenterazine, and a membrane-bound photoprotein was shown to be involved. This photoprotein was purified using ion exchange chromatography, and SDS-PAGE showed a clean band of approximately 60 kDa. The excised band was analyzed by LC/MS, and the obtained data were compared against the transcriptome data of D. gigas, allowing us to find two gene products which displayed high coverage (>80%), the enzymes symplectin and vanin-2, which potentially associate with light emission process in this organism. Finally, the purified photoprotein was shown to emit a blue light (470 nm) in the presence of dehydrocoelenterazine.

Assessment of oxytetracycline baths as therapeutic treatment for the control of the agent of withering syndrome (WS) in red abalone (Haliotis rufescens).

Withering Syndrome (WS) is a lethal disease that affects abalone species in both wild and farmed populations. This infection, caused by the rickettsial-like intracellular organism (RLO) Candidatus Xenohaliotis californiensis, can severely impair the normal development of affected animals, and ultimately, their survival. The most common line of action against the WS has been the use of antibiotics, specifically oxytetracycline (OTC), administered via intramuscular injection and per os via medicated feed. In the present study, we have assessed the effectiveness of OTC baths as therapeutic treatment for the control of the WS agent in H. rufescens. Clinical signs of infection were monitored for 11 months in treated juveniles, in addition to feed consumption rate, growth patterns and gonad development. Abalones were asymptomatic until the end of the experiment, when a small number of non-treated animals exhibited clinical signs of infection. Gonad maturity was not observed. OTC treated animals grew significantly less than their non-treated counterparts, being 4.3% shorter and 13.6% lighter at the end of the experiment. They also displayed negative allometry, i.e. for the same shell length, they were lighter than non-treated groups. Furthermore, the weight of muscle and soft tissues in OTC treated animals was lighter than in the other groups, while no differences were found in shell weight. The feed consumption rate was the same for all groups, thus the observed growth patterns cannot be attributed to a decreased feed intake. One possible explanation is that antibiotic treatment may have impacted gut microflora, thus preventing efficient nutrient digestion and absorption and, indirectly, reducing growth. Prevalence of RLOs causing WS (WS-RLO) and the variant form (RLOv), infected with a bacteriophague and non virulent, were significantly lower in the OTC-treated group than in the other groups. Similar results were observed for the mean intensity of RLOv, while for WS-RLO, the intensity in the OTC-treated group was higher, although not statistically significant, than the rest of the groups. These observations may be the consequence of an increased bacterial sensitivity to OTC effects associated with the phage infection or faster reproduction of WS-RLOs than RLOv after OTC treatment. Our results let us infer that the prophylactic use of OTC in abalone to avoid the negative effects of WS on abalone farms could have an undesired negative effect on the biological control exerted by the phage on the bacteria after OTC treatment.

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.

Differential susceptibility to the Withering Syndrome agent and renal coccidia in juvenile Haliotis rufescens, Haliotis discus hannai and the interspecific hybrid.

Withering Syndrome (WS) is a pathogenic chronic disease caused by the intracellular rickettsial-like bacterium "Candidatus Xenohaliotis californiensis" (WS-RLOs), which affects many abalone species. The renal coccidium (Margolisiella haliotis) has often been observed concurrent with WS infection. The red abalone Haliotis rufescens is a very susceptible species to WS and is also infected by the coccidium M. haliotis. In contrast, the Japanese abalone Haliotis discus hannai is not infected by these parasites. Interspecific hybridization is a method for improving important traits in animal husbandry. The objective of this study was to determine susceptibility to WS-RLO and M. haliotis infection in the hybrid generated from a cross between red and Japanese abalones. Juveniles from both species and the interspecific hybrid were challenged by exposure to effluent from red abalone adults infected with both parasites. The animals were analyzed by histology at 130days post-challenge. A 33% prevalence WS-RLOs was observed in the red abalone H. rufescens, whereas a 20% prevalence was observed in the hybrid. Infections were graded on a scale of 0-3. Of these red abalones infected, 53% presented grade 1 infection intensity, 10% had grade 2 infections, and 50% had grade 3 infections. However, the hybrids only presented intensities at the extremes of the scale; of those infected 33% showed grade 1 infections and 66% had grade 3 infections. The coccidium prevalence was 7% in red abalone individuals and 13% in the hybrid abalone. In contrast, the Japanese abalone did not present infections with either parasite. As with the prevalence, the infection intensities for the coccidium were higher in the hybrid abalone; of those infected 25% had grade 2 infections, and 75% had grade 3 infections, but the red abalone presented only grade 2 infection intensities. Therefore, the hybrid did not inherited non-susceptibility or resistance characteristics of the parental H. discus hannai and possessed biological conditions that could foster development of both parasites. Development of a culture based on this hybrid abalone should consider its susceptibility to infection by coccidian, WS-RLOs and the potential for developing the WS disease.

Physiological performance of juvenile Haliotis rufescens and Haliotis discus hannai abalone exposed to the withering syndrome agent.

Withering syndrome (WS) is a serious chronic disease caused by infection with the bacterium Candidatus Xenohaliotis californiensis, a Rickettsiales-like organism (WS-RLO) that affects multiple abalone species in both natural and farmed populations. However, there is no available information regarding the effects of this disease on the physiological performance of infected abalone. We studied the effect of different levels of infection on components of energy balance and physiological indices (rates of absorption and assimilation, O/N ratio, and scope for growth) in the abalone species Haliotis rufescens and Haliotis discus hannai. Juveniles were exposed to C. X. californiensis transmission for 130 days, during which time the presence/absence of WS-RLOs was evaluated by PCR (following DNA sequencing-based confirmation of 100% identity with the sequence of C. X. californiensis from California), and the prevalence and intensity of infection were evaluated via histological analysis. Among H. rufescens juveniles exposed to the bacterium, 92% became infected (positive by histology), and the intensity of infection ranged from low (degree 1) to moderate (degree 2). In contrast, no H. discus hannai juveniles were positive for WS-RLO by histology, although 23% were positive by PCR, possibly indicating incipient WS-RLO infection that did not develop during the experimental period or to mere presence of WS-RLO DNA in the sample. Infection of H. rufescens juveniles by WS-RLOs negatively affected all components of the energy balance and physiological indices, such as scope for growth and the O/N ratio, in direct relation to the degree of infection. The most strongly affected functions were the rate of ingestion, standard metabolism, and production of feces, which were reduced by 60-80% in the most highly infected individuals. The reduced energy intake in the organisms produced a strong energy imbalance such that the energy available for growth was reduced by 49% in infected organisms. In contrast, juveniles of H. discus hannai carrying the bacterium developed no infection and showed no alterations of physiological function. Our results indicate that the level of early infection by WS-RLOs may exert a negative effect on physiological activity in H. rufescens, even when the disease is not evident.