Contaminants from dredged sediments alter the transcriptome of Manila clam and induce shifts in microbiota composition.

BACKGROUND: The reuse of dredged sediments in ports and lagoons is a big issue as it should not affect the quality and the equilibrium of ecosystems. In the lagoon of Venice, sediment management is of crucial importance as sediments are often utilized to built-up structures necessary to limit erosion. However, the impact of sediment reuse on organisms inhabiting this delicate area is poorly known. The Manila clam is a filter-feeding species of high economic and ecological value for the Venice lagoon experiencing a drastic decline in the last decades. In order to define the molecular mechanisms behind sediment toxicity, we exposed clams to sediments sampled from different sites within one of the Venice lagoon navigable canals close to the industrial area. Moreover, we investigated the impacts of dredged sediments on clam's microbial communities. RESULTS: Concentrations of the trace elements and organic chemicals showed increasing concentrations from the city of Venice to sites close to the industrial area of Porto Marghera, where PCDD/Fs and PCBs concentrations were up to 120 times higher than the southern lagoon. While bioaccumulation of organic contaminants of industrial origin reflected sediments' chemical concentrations, metal bioaccumulation was not consistent with metal concentrations measured in sediments probably due to the activation of ABC transporters. At the transcriptional level, we found a persistent activation of the mTORC1 signalling pathway, which is central in the coordination of cellular responses to chemical stress. Microbiota characterization showed the over-representation of potential opportunistic pathogens following exposure to the most contaminated sediments, leading to host immune response activation. Despite the limited acquisition of new microbial species from sediments, the latter play an important role in shaping Manila clam microbial communities. CONCLUSIONS: Sediment management in the Venice lagoon will increase in the next years to maintain and create new canals as well as to allow the operation of the new mobile gates at the three Venice lagoon inlets. Our data reveal important transcriptional and microbial changes of Manila clams after exposure to sediments, therefore reuse of dredged sediments represents a potential risk for the conservation of this species and possibly for other organisms inhabiting the Venice lagoon.

Viral nervous necrosis resistance in gilthead sea bream (Sparus aurata) at the larval stage: heritability and accuracy of genomic prediction with different training and testing settings.

BACKGROUND: The gilthead sea bream (Sparus aurata) has long been considered resistant to viral nervous necrosis (VNN), until recently, when significant mortalities caused by a reassortant nervous necrosis virus (NNV) strain were reported. Selective breeding to enhance resistance against NNV might be a preventive action. In this study, 972 sea bream larvae were subjected to a NNV challenge test and the symptomatology was recorded. All the experimental fish and their parents were genotyped using a genome-wide single nucleotide polymorphism (SNP) array consisting of over 26,000 markers. RESULTS: Estimates of pedigree-based and genomic heritabilities of VNN symptomatology were consistent with each other (0.21, highest posterior density interval at 95% (HPD95%): 0.1-0.4; 0.19, HPD95%: 0.1-0.3, respectively). The genome-wide association study suggested one genomic region, i.e., in linkage group (LG) 23 that might be involved in sea bream VNN resistance, although it was far from the genome-wide significance threshold. The accuracies (r) of the predicted estimated breeding values (EBV) provided by three Bayesian genomic regression models (Bayes B, Bayes C, and Ridge Regression) were consistent and on average were equal to 0.90 when assessed in a set of cross-validation (CV) procedures. When genomic relationships between training and testing sets were minimized, accuracy decreased greatly (r = 0.53 for a validation based on genomic clustering, r = 0.12 for a validation based on a leave-one-family-out approach focused on the parents of the challenged fish). Classification of the phenotype using the genomic predictions of the phenotype or using the genomic predictions of the pedigree-based, all data included, EBV as classifiers was moderately accurate (area under the ROC curve 0.60 and 0.66, respectively). CONCLUSIONS: The estimate of the heritability for VNN symptomatology indicates that it is feasible to implement selective breeding programs for increased resistance to VNN of sea bream larvae/juveniles. Exploiting genomic information offers the opportunity of developing prediction tools for VNN resistance, and genomic models can be trained on EBV using all data or phenotypes, with minimal differences in classification performance of the trait phenotype. In a long-term view, the weakening of the genomic ties between animals in the training and test sets leads to decreased genomic prediction accuracies, thus periodical update of the reference population with new data is mandatory.

Resistance to viral nervous necrosis in European sea bass (Dicentrarchus labrax L.): heritability and relationships with body weight, cortisol concentration, and antibody titer.

BACKGROUND: Susceptibility of European sea bass (Dicentrarchus labrax L.) to viral nervous necrosis (VNN) is well-known. Interest towards selective breeding as a tool to enhance genetic resistance in this species has increased sharply due to the major threat represented by VNN for farmed sea bass and limitations concerning specific therapeutical measures. A sea bass experimental population (N = 650) was challenged with nervous necrosis virus (NNV) to investigate genetic variation in VNN mortality. In addition, relationships of this trait with serum cortisol concentration after stress exposure, antibody titer against NNV antigens, and body weight at a fixed age were studied to identify potential indicator traits of VNN resistance. RESULTS: The estimate of heritability for VNN mortality was moderate and ranged from 0.15 (HPD95%, 95% highest posterior density interval: 0.02, 0.31) to 0.23 (HPD95%: 0.06, 0.47). Heritability estimates for cortisol concentration, antibody titer, and body weight were 0.19 (HPD95%: 0.07, 0.34), 0.36 (HPD95%: 0.16, 0.59) and 0.57 (HPD95%: 0.33, 0.84), respectively. Phenotypic relationships between traits were trivial and not statistically significant, except for the estimated correlation between antibody titer and body weight (0.24). Genetic correlations of mortality with body weight or antibody titer (- 0.39) exhibited a 0.89 probability of being negative. A negligible genetic correlation between mortality and cortisol concentration was detected. Antibody titer was estimated to be positively correlated with body weight (0.49). CONCLUSIONS: Antibody titer against NNV offers the opportunity to use indirect selection to enhance resistance, while the use of cortisol concentration as an indicator trait in breeding programs for VNN resistance is questionable. The estimate of heritability for VNN mortality indicates the feasibility of selective breeding to enhance resistance to NNV and raises attention to the development of genomic prediction tools to simplify testing procedures for selection candidates.

Host-microbiota interactions shed light on mortality events in the striped venus clam Chamelea gallina.

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.

Multidisciplinary long-term survey of Manila clam grown in farming sites subjected to different environmental conditions.

In recent years recurrent bivalve mass mortalities considerably increased around the world, causing the collapse of natural and farmed populations. Venice Lagoon has historically represented one of the major production areas of the Manila clam Ruditapes philippinarum in Europe. However, in the last 20 years a 75 % decrease in the annual production has been experienced. While climate change and anthropogenic interventions may have played a key role in natural and farmed stocks reductions, no studies investigated at multiple levels the environmental stressors affecting farmed Manila clam to date. In this work we carried out a long-term monitoring campaign on Manila clam reared in four farming sites located at different distances from the southern Venice Lagoon inlet, integrating (meta)genomic approaches (i.e. RNA-seq; microbiota characterization), biometric measurements and chemical-physical parameters. Our study allowed to characterize the molecular mechanisms adopted by this species to cope with the different environmental conditions characterizing farming sites and to propose hypotheses to explain mortality events observed in recent years. Among the most important findings, the disruption of clam's immune response, the spread of Vibrio spp., and the up-regulation of molecular pathways involved in xenobiotic metabolism suggested major environmental stressors affecting clams farmed in sites placed close to Chioggia's inlet, where highest mortality was also observed. Overall, our study provides knowledge-based tools for managing Manila clam farming on-growing areas. In addition, the collected data is a snapshot of the time immediately before the commissioning of MoSE, a system of mobile barriers aimed at protecting Venice from high tides, and will represent a baseline for future studies on the effects of MoSE on clams farming and more in general on the ecology of the Venice Lagoon.

Impaired reproduction, energy reserves and dysbiosis: The overlooked consequences of heatwaves in a bivalve mollusc.

Extreme events like Marine Heatwaves (MHWs) are becoming more intense, severe, and frequent, threatening benthic communities, specifically bivalves. However, the consequences of non-lethal MHWs on animals are still poorly understood. Here, we exposed the Manila clam Ruditapes philippinarum to non-lethal MHW for 30 days and provided an integrative view of its effects. Our result indicated that albeit non-lethal, MHW reduced clam's energy reserves (by reducing their hepato-somatic index), triggered antioxidant defenses (particularly in males), impaired reproduction (via the production of smaller oocytes in females), triggered dysbiosis in the digestive gland microbiota and altered animals' behaviour (by impacting their burying capacity) and filtration rate. Such effects were seen also at RNA-seq (i.e. many down-regulated genes belonged to reproduction) and metabolome level. Interestingly, negative effects were more pronounced in males than in females. Our results show that MHWs influence animal physiology at multiple levels, likely impacting its fitness and its ecosystem services.

Molecular Monitoring of SARS-CoV-2 in Different Sewage Plants in Venice and the Implications for Genetic Surveillance.

Wastewater-based epidemiology is now widely used as an indirect tool to monitor the spread of SARS-CoV-2. In this study, five different sample matrices representing diverse phases of the wastewater treatment process were collected during the second wave of SARS-CoV-2 from two wastewater treatment plants (WWTPs) serving the Civil Hospital and Sacca Fisola island in Venice, Italy. Positive SARS-CoV-2 detections occurred at both WWTPs, and data on viral genome detection rate and quantification suggest that the pellet (i.e., the particulate resulting from the influent) is a sensitive matrix that permits reliable assessment of infection prevalence while reducing time to results. On the contrary, analysis of post-treatment matrices provides evidence of the decontamination efficacy of both WWTPs. Finally, direct sequencing of wastewater samples enabled us to identify B.1.177 and B.1.160 as the prevalent SARS-CoV-2 lineages circulating in Venice at the time of sampling. This study confirmed the suitability of wastewater testing for studying SARS-CoV-2 circulation and established a simplified workflow for the prompt detection and characterization of the virus.

Long-lasting effects of chronic exposure to chemical pollution on the hologenome of the Manila clam.

Chronic exposure to pollutants affects natural populations, creating specific molecular and biochemical signatures. In the present study, we tested the hypothesis that chronic exposure to pollutants might have substantial effects on the Manila clam hologenome long after removal from contaminated sites. To reach this goal, a highly integrative approach was implemented, combining transcriptome, genetic and microbiota analyses with the evaluation of biochemical and histological profiles of the edible Manila clam Ruditapes philippinarum, as it was transplanted for 6 months from the polluted area of Porto Marghera (PM) to the clean area of Chioggia (Venice lagoon, Italy). One month post-transplantation, PM clams showed several modifications to its resident microbiota, including an overrepresentation of the opportunistic pathogen Arcobacter spp. This may be related to the upregulation of several immune genes in the PM clams, potentially representing a host response to the increased abundance of deleterious bacteria. Six months after transplantation, PM clams demonstrated a lower ability to respond to environmental/physiological stressors related to the summer season, and the hepatopancreas-associated microbiota still showed different compositions among PM and CH clams. This study confirms that different stressors have predictable effects in clams at different biological levels and demonstrates that chronic exposure to pollutants leads to long-lasting effects on the animal hologenome. In addition, no genetic differentiation between samples from the two areas was detected, confirming that PM and CH clams belong to a single population. Overall, the obtained responses were largely reversible and potentially related to phenotypic plasticity rather than genetic adaptation. The results here presented will be functional for the assessment of the environmental risk imposed by chemicals on an economically important bivalve species.

Potential for Genetic Improvement of Resistance to Perkinsus olseni in the Manila Clam, Ruditapes philippinarum, Using DNA Parentage Assignment and Mass Spawning.

The Manila clam Ruditapes philippinarum, a major cultured shellfish species, is threatened by infection with the microparasite Perkinsus olseni, whose prevalence increases with high water temperatures. Under the current trend of climate change, the already severe effects of this parasitic infection might rapidly increase the frequency of mass mortality events. Treating infectious diseases in bivalves is notoriously problematic, therefore selective breeding for resistance represents a key strategy for mitigating the negative impact of pathogens. A crucial step in initiating selective breeding is the estimation of genetic parameters for traits of interest, which relies on the ability to record parentage and accurate phenotypes in a large number of individuals. Here, to estimate the heritability of resistance against P. olseni, a field experiment mirroring conditions in industrial clam production was set up, a genomic tool was developed for parentage assignment, and parasite load was determined through quantitative PCR. A mixed-family cohort of potentially 1,479 clam families was produced in a hatchery by mass spawning of 53 dams and 57 sires. The progenies were seeded in a commercial clam production area in the Venice lagoon, Italy, where high prevalence of P. olseni had previously been reported. Growth and parasite load were monitored every month and, after 1 year, more than 1,000 individuals were collected for DNA samples and phenotype recording. A pooled sequencing approach was carried out using DNA samples from the hatchery broodstock and from a Venice lagoon clam population, providing candidate markers used to develop a 245-SNP panel. Parentage assignment for 246 F1 individuals showed sire and dam representation were high (75 and 85%, respectively), indicating a very limited risk of inbreeding. Moderate heritability (0.23 ± 0.11-0.35 ± 0.13) was estimated for growth traits (shell length, shell weight, total weight), while parasite load showed high heritability, estimated at 0.51 ± 0.20. No significant genetic correlations were found between growth-associated traits and parasite load. Overall, the preliminary results provided by this study show high potential for selecting clams resistant to parasite load. Breeding for resistance may help limit the negative effects of climate change on clam production, as the prevalence of the parasite is predicted to increase under a future scenario of higher temperatures. Finally, the limited genetic correlation between resistance and growth suggests that breeding programs could incorporate dual selection without negative interactions.

Tracing seafood at high spatial resolution using NGS-generated data and machine learning: Comparing microbiome versus SNPs.

Developing reliable tools to trace food origin represents a major goal for producers and control authorities. Here, we test the hypothesis whether NGS-generated data could provide a reliable tool to ensure seafood traceability. As a test case, we used the Manila clam Ruditapes philippinarum, a bivalve mollusk of high commercial interest with worldwide distribution, collected in the Venice lagoon sites subjected to prohibition of clam harvesting because of chemical contamination as well as in authorized clam harvesting areas. The results obtained demonstrated that the geographic origin of Manila clam may be more accurately determined basing on microbiome data than single nucleotide polymorphisms. In particular, combining microbiome data with machine-learning techniques, we provide the experimental evidence that it is possible to trace the clam place of origin at high spatial resolution. Considering its low cost and portability, NGS-analysis of microbiome data might represent a cost-effective, high-resolution tool for reliable food traceability.

RAD SNP markers as a tool for conservation of dolphinfish Coryphaena hippurus in the Mediterranean Sea: Identification of subtle genetic structure and assessment of populations sex-ratios.

Dolphinfish is an important fish species for both commercial and sport fishing, but so far limited information is available on genetic variability and pattern of differentiation of dolphinfish populations in the Mediterranean basin. Recently developed techniques allow genome-wide identification of genetic markers for better understanding of population structure in species with limited genome information. Using restriction-site associated DNA analysis we successfully genotyped 140 individuals of dolphinfish from eight locations in the Mediterranean Sea at 3324 SNP loci. We identified 311 sex-related loci that were used to assess sex-ratio in dolphinfish populations. In addition, we identified a weak signature of genetic differentiation of the population closer to Gibraltar Strait in comparison to other Mediterranean populations, which might be related to introgression of individuals from Atlantic. No further genetic differentiation could be detected in the other populations sampled, as expected considering the known highly mobility of the species. The results obtained improve our knowledge of the species and can help managing dolphinfish stock in the future.