A Weight of Evidence approach to support the assessment of the quality of Manila clam farming sites in a coastal lagoon.

Aquaculture productivity in coastal lagoons is endangered by a complex interplay of anthropogenic and environmental factors, amplified by the effects of climate change in these sensitive areas. To reach a more comprehensive assessment of farming sites quality, a quantitative Weight of Evidence approach (QWoE) is applied for the first time to data collected at four Manila clam (R. philippinarum) farming sites in the Venice lagoon (Italy). This included sediment quality, chemical bioaccumulation, and biological responses. Results revealed a greater hazard for sites closer to the open sea. In these areas, the combination of sediment characteristics and a higher frequency of salinity and temperature stress could explain the alterations measured at a transcriptional and biomarker level. The findings demonstrate that a QWoE approach that integrates multiple sources of evidence should also include physicochemical conditions in order to better understand the impacts of human activities and other stressors on clam aquaculture productivity.

Effects of environmental concentrations of the fragrance amyl salicylate on the mediterranean mussel Mytilus galloprovincialis.

Amyl salicylate (AS) is a fragrance massively used as a personal care product and following the discharged in wastewaters may end up in the aquatic environment representing a potential threat for the ecosystem and living organisms. AS was recently detected in water of the Venice Lagoon, a vulnerable area continuously subjected to the income of anthropogenic chemicals. The lagoon is a relevant area for mollusc farming, including the Mediterranean mussels (Mytilus galloprovincialis) having an important economic and ecological role. Despite high levels of AS occurred in water of the Lagoon of Venice, no studies investigated the possible consequences of AS exposures on species inhabiting this ecosystem to date. For the first time, we applied a multidisciplinary approach to investigate the potential effects of the fragrance AS on Mediterranean mussels. To reach such a goal, bioaccumulation, cellular, biochemical, and molecular analyses (RNA-seq and microbiota characterization) were measured in mussels treated for 7 and 14 days with different AS Venice lagoon environmental levels (0.1 and 0.5 µg L-1). Despite chemical investigations suggested low AS bioaccumulation capability, cellular and molecular analyses highlighted the disruption of several key cellular processes after the prolonged exposures to the high AS concentration. Among them, potential immunotoxicity and changes in transcriptional regulation of pathways involved in energy metabolism, stress response, apoptosis and cell death regulations have been observed. Conversely, exposure to the low AS concentration demonstrated weak transcriptional changes and transient increased representation of opportunistic pathogens, as Arcobacter genus and Vibrio aestuarianus. Summarizing, this study provides the first overview on the effects of AS on one of the most widely farmed mollusk species.

Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae.

The gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom "reactomes" (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response.

A comparative synthesis of transcriptomic analyses reveals major differences between WSSV-susceptible Litopenaeus vannamei and WSSV-refractory Macrobrachium rosenbergii.

Since the 1990s White Spot Syndrome Virus (WSSV) has severely affected shrimp aquaculture worldwide causing a global pandemic of White Spot Disease (WSD) in penaeid culture. However, not all decapod species that can be infected by WSSV show the same susceptibility to the virus, thus raising interesting questions regarding the potential genetic traits that might confer resistance to WSSV. In order to shed light into the genetic markers of WSSV resistance, we employed a dual approach: i) we initially analysed the transcriptomes derived from the hepatopancreas of two species, the susceptible white shrimp Litopenaeus vannamei and the refractory fresh water prawn Macrobrachium rosenbergii, both infected with WSSV. We found a large number of differentially expressed genes (DEGs) belonging to the immune system (mostly anti-microbial peptides and haemolymph clotting components) that were generally up-regulated in M. rosenbergii and down-regulated in L. vannamei. Further, in both species we identified many up-regulated DEGs that were related to metabolism (suggesting a metabolic shift during the infection) and, interestingly, in L. vannamei only, we found several DEGs that were related to moult and suggested an inhibition of the moult cycle in this species following WSSV infection. ii) we then identified a limited number of genetic markers putatively linked with WSD tolerance by employing an ecological genomics approach in which we compared published reports with our own RNA-seq datasets for different decapod species infected with WSSV. Using this second comparative approach, we found nine candidate genes which are consistently down-regulated in susceptible species and up-regulated in refractory species and which have a role in immune response. Together our data offer novel insights into gene expression differences that can be found in susceptible and refractory decapod species infected with WSSV and provide a valuable resource towards our understanding of the potential genetic basis of tolerance to WSSV.

Comparative analysis of shrimp (Penaeus vannamei) miRNAs expression profiles during WSSV infection under experimental conditions and in pond culture.

In recent years, the importance of viral and host microRNAs (miRNAs) in mediating viral replication and control of host cellular machinery, has been realised and increasing efforts have been taken in order to understand the interactions of miRNAs from host and pathogen during infection. However, all existing studies has thus far been conducted in controlled experimental conditions and the veracity of these data for field conditions are yet to be established. In this framework, small RNA sequencing was performed to identify the miRNAs involved in shrimp (Penaeus vannamei) immune responses under two different WSSV infection conditions of natural infection and experimentally challenged conditions. The expression profiles of miRNAs of shrimp infected with WSSV under two contrasting conditions were compared and as a result, 23365 known miRNAs and 481 novel miRNAs were identified. Amongst the most abundantly expressed miRNAs, the hypoxia related miR-210 and immune pathway related miR-29b were expressed only in infected shrimps of both conditions. miR-8-5p, having a functional role in modulation of chitin biosynthesis was exclusively represented in higher numbers in the WSSV -infected shrimps under natural conditions whilst four of the miRNAs (mja-miR-6493-5p, mja-miR-6492, mmu-miR-3968, tcf-miR-9b-5p) identified from shrimps collected from pond culture targeted chitinase, an important enzyme involved in growth and moulting in shrimps, indicating an interaction between WSSV infection and moult cycle under culture conditions. Some of the miRNAs (tca-miR-87b-3p, cte-miR-277a) and miRNAs belonging to class miR-9, miR-981 that were identified only in WSSV infected shrimps under experimental conditions, are known to respond against WSSV infection in shrimps. Moreover, the miRNA target prediction revealed several immune-related gene targets such as cathepsin, c-type lectin, haemocyanin and ubiquitin protein ligase were commonly identified under both the conditions. However, the miRNAs identified from challenge experiment had wide number of gene targets as compared to the miRNAs of natural infection. The shrimp miRNA mja-miR-6489-3p, was also found to target early virus gene wsv001 of WSSV. Our study, therefore, provides the comparative analysis of miRNA expression from shrimp during WSSV infection in two different conditions.