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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.

As filter-feeders, bivalve molluscs accumulate Vibrio into edible tissues. Consequently, an accurate assessment of depuration procedures and the characterization of the persistent Vibrio community in depurated shellfish represent a key issue to guarantee food safety in shellfish products. The present study investigated changes in the natural Vibrio community composition of the Ruditapes philippinarum microbiota with specific focus on human pathogenic species. For this purpose, the study proposed a MLSA-NGS approach (rRNA 16S, recA and pyrH) for the detection and identification of Vibrio species. Clam microbiota were analysed before and after depuration procedures performed in four depuration plants, using culture-dependent and independent approaches. Microbiological counts and NGS data revealed differences in terms of both contamination load and Vibrio community between depuration plants. The novel MLSA-NGS approach allowed for a clear definition of the Vibrio species specific to each depuration plant. Specifically, depurated clam microbiota showed presence of human pathogenic species. Ozone treatments and the density of clams in the depuration tank probably influenced the level of contamination and the Vibrio community composition. The composition of Vibrio community specific to each plant should be carefully evaluated during the risk assessment to guarantee a food-safe shellfish-product for the consumer.

The molecular factors and gene regulation involved in sex determination and gonad differentiation in bivalve molluscs are unknown. It has been suggested that doubly uniparental inheritance (DUI) of mitochondria may be involved in these processes in species such as the ubiquitous and commercially relevant Manila clam, Ruditapes philippinarum. We present the first long-read-based de novo genome assembly of a Manila clam, and a RNA-Seq multi-tissue analysis of 15 females and 15 males. The highly contiguous genome assembly was used as reference to investigate gene expression, alternative splicing, sequence evolution, tissue-specific co-expression networks, and sexual contrasting SNPs. Differential expression (DE) and differential splicing (DS) analyses revealed sex-specific transcriptional regulation in gonads, but not in somatic tissues. Co-expression networks revealed complex gene regulation in gonads, and genes in gonad-associated modules showed high tissue specificity. However, male gonad-associated modules showed contrasting patterns of sequence evolution and tissue specificity. One gene set was related to the structural organization of male gametes and presented slow sequence evolution but high pleiotropy, whereas another gene set was enriched in reproduction-related processes and characterized by fast sequence evolution and tissue specificity. Sexual contrasting SNPs were found in genes overrepresented in mitochondrial-related functions, providing new candidates for investigating the relationship between mitochondria and sex in DUI species. Together, these results increase our understanding of the role of DE, DS, and sequence evolution of sex-specific genes in an understudied taxon. We also provide resourceful genomic data for studies regarding sex diagnosis and breeding in bivalves.

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.

Vibrios represent a natural contaminant of seafood products. V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus are the most hazardous species to human health. Given the worldwide consumption of mollusc products, reliable detection of Vibrio species is recommended to prevent human vibriosis. In this study, culture-dependent and -independent methods were compared and integrated to implement knowledge of the Manila clam Vibrio community composition. Here, 16S and recA-pyrH metabarcoding were applied to compare the microbial communities of homogenate clam samples (culture-independent method) and their culture-derived samples plated on three different media (culture-dependent method). In addition, a subset of plated clam samples was investigated using shotgun metagenomics. Homogenate metabarcoding characterized the most abundant taxa (16S) and Vibrio species (recA-pyrH). Culture-dependent metabarcoding detected the cultivable taxa, including rare species. Moreover, marine agar medium was found to be a useful substrate for the recovery of several Vibrio species, including the main human pathogenic ones. The culture-dependent shotgun metagenomics detected all the main human pathogenic Vibrio species and a higher number of vibrios with respect to the recA-pyrH metabarcoding. The study revealed that integration of culture-dependent and culture-independent methods might be a valid approach for the characterization of Vibrio biodiversity.

The vertebrate mitochondrial genomes generally present a typical gene order. Exceptions are uncommon and important to study the genetic mechanisms of gene order rearrangements and their consequences on phylogenetic output and mitochondrial function. Antarctic notothenioid fish carry some peculiar rearrangements of the mitochondrial gene order. In this first systematic study of 28 species, we analysed known and undescribed mitochondrial genome rearrangements for a total of eight different gene orders within the notothenioid fish. Our reconstructions suggest that transpositions, duplications and inversion of multiple genes are the most likely mechanisms of rearrangement in notothenioid mitochondrial genomes. In Trematominae, we documented an extremely rare inversion of a large genomic segment of 5300 bp that partially affected the gene compositional bias but not the phylogenetic output. The genomic region delimited by nad5 and trnF, close to the area of the Control Region, was identified as the hot spot of variation in Antarctic fish mitochondrial genomes. Analysing the sequence of several intergenic spacers and mapping the arrangements on a newly generated phylogeny showed that the entire history of the Antarctic notothenioids is characterized by multiple, relatively rapid, events of disruption of the gene order. We hypothesised that a pre-existing genomic flexibility of the ancestor of the Antarctic notothenioids may have generated a precondition for gene order rearrangement, and the pressure of purifying selection could have worked for a rapid restoration of the mitochondrial functionality and compactness after each event of rearrang

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.

Summary Whole-genome sequencing data were produced from a single flathead grey mullet female and assembled into a draft genome sequence, whereas publicly available sequence data were used to obtain a male draft sequence. Two pools, each consisting of 60 unrelated individuals, respectively, of male and female fish were analyzed using Pool-Sequencing. Mapping and analysis of Pool-Seq data against the draft genome(s) revealed >30 loci potentially associated with sex, the most promising locus of which, encoding the follicle-stimulating hormone receptor (fshr) and harboring two missense variants, was genotyped on 245 fish from four Mediterranean populations. Genotype data showed that fshr represents a previously unknown sex-determining locus, although the incomplete association pattern between fshr genotype and sex-phenotype, the variability of such pattern across different populations, and the presence of other candidate loci reveal that a greater complexity underlies sex determination in the flathead grey mullet., Graphical abstract, Highlights • Three SNPs with sex-specific allele frequencies found in the grey mullet fshr gene • Two missense variants at the fshr locus are significantly associated with male sex • fshr male genotypes show divergent distribution across geographical populations • Incomplete penetrance of fshr variants suggests that additional factors exist, Genetics; Evolutionary Biology; Genomics

The tight connection between immunity and reproduction has been studied for decades. However, basic knowledge at the molecular level of the effect of mating on immune function is still lacking in many taxa. Determining whether and how the immune system is engaged after mating is a crucial step in understanding post-mating mechanisms of reproduction and sexual selection. Here, we study the transcriptional changes in immunity-related genes caused by the ejaculate in the female reproductive tract using a model species for sexual selection studies, the guppy Poecilia reticulata. To study changes triggered by the ejaculate only, rather than caused by mating, we used artificial inseminations to transfer ejaculate into females. We then compared gene expression in the reproductive tract (gonoduct and ovary) of females artificially inseminated either with ejaculate or with a control solution, after 1 hr and after 6 hr. Overall, contact with ejaculate caused short-term changes in the expression of immune-related genes in the female reproductive tract, with a complex pattern of up- and down-regulation of immune-related pathways, but with clear indication of a marked down-regulation of the immune system shortly after ejaculate contact. This suggests a link between immune function and processes occurring between mating and fertilization in this species.

Whole-genome sequence data were produced from a single flathead grey mullet female and assembled into a draft genome sequence, whereas publicly available sequence data were used to obtain a male draft sequence. Two pools, each consisting of 60 unrelated individuals respectively of male and female fish were analysed using Pool-Sequencing. Mapping and analysis of Pool-Seq data against the draft genome(s) revealed >30 loci potentially associated with sex. The most promising of these loci encoding the follicle stimulating hormone receptor (FSHR) and harbouring two missense variants, was genotyped on 245 fish from four Mediterranean populations. Genotype data showed that FSHR represents a previously unknown sex-determining locus. However, the incomplete association pattern between FSHR genotype and sex-phenotype, the variability of such pattern across different natural populations and the presence of other candidate loci suggest a greater complexity underling sex determination in the flathead grey mullet.

The giant Antarctic toothfish Dissostichus mawsoni is one of the most economically valued fishery resources from the Southern Ocean. However, the distinctive biological characteristics and life history of this species, including large body size, long life span, late sexual maturity, and specialization to freezing polar waters, make it particularly vulnerable to overfishing and global climate change. It is therefore of fundamental importance to administer sound management and conservation actions to avoid depletion of this unique species. The crucial issue of whether this species consists of distinct biological units or stocks, which is needed to inform appropriate management and conservation, is currently unresolved. We report here the first RAD-seq analysis of Antarctic toothfish populations, a powerful approach of genome-wide discovery of SNP loci, to assess genetic differentiation between the geographically distant individuals from CCAMLAR Subarea 48.1 locations (South Shetland Islands and West Antarctic Peninsula coasts) and Subarea 88.1 (Ross Sea and McMurdo Sound). We recovered 19,611 SNPs belonging to 13,251 widely shared loci. Comprehensive population structure analyses unambiguously indicated absence of any significant level of population differentiation, thus the null hypothesis of panmixia cannot be rejected. Together with currently known life history traits of D. mawsoni, we suggest this striking lack of genomic differentiation likely reflects high degrees of contemporary and/or historical gene flow rather than shared spawning grounds. Complementary analyses, particularly otolith microchemistry, would contribute useful inference of natal origins.

A study was conducted on the habitat distribution of four sympatric species of Periophthalmus (the silver-lined mudskipper Periophthalmus argentilineatus, the slender mudskipper Periophthalmus gracilis, the kalolo mudskipper Periophthalmus kalolo and the Malacca mudskipper Periophthalmus malaccensis) from northern Sulawesi. Molecular phylogenetic reconstructions based on one mtDNA marker (16S) were used to validate the morphological taxa, identifying five molecular clades. Periophthalmus argentilineatus includes two molecular species, which are named Periophthalmus argentilineatus clades F and K. Multivariate direct gradient analysis show that these species form three distinct ecological guilds, with the two molecular species occurring in different guilds. Periophthalmus clade F is ecologically eurytypic; Periophthalmus clade K and P. kalolo are prevalent in ecosystems isolated by strong oceanic currents and at shorter distances from the sea; P. gracilis plus P. malaccensis are prevalent in ecosystems connected by shallow coastal waters, in vegetated habitats at larger distances from the sea. This indicates for the first time that mudskipper species exhibit a range of adaptations to semiterrestrialism not only within genera, but even within morphospecies, delineating a much more complex adaptive scenario than previously assumed.

Antarctic fish belonging to Notothenioidei represent an extraordinary example of radiation in the cold. In addition to the absence of hemoglobin, icefish show a number of other striking peculiarities including large-diameter blood vessels, high vascular densities, mitochondria-rich muscle cells, and unusual mitochondrial architecture. In order to investigate the bases of icefish adaptation to the extreme Southern Ocean conditions we sequenced the complete genome of the icefish Chionodraco myersi. Comparative analyses of the icefish genome with those of other teleost species, including two additional white-blooded and five red-blooded notothenioids, provided a new perspective on the evolutionary loss of globin genes. Muscle transcriptome comparative analyses against red-blooded notothenioids as well as temperate fish revealed the peculiar regulation of genes involved in mitochondrial function in icefish. Gene duplication and promoter sequence divergence were identified as genome-wide patterns that likely contributed to the broad transcriptional program underlying the unique features of icefish mitochondria.

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Antarctic fish belonging to Notothenioidei represent an extraordinary example of radiation in the cold. In addition to the absence of hemoglobin, icefish show a number of other striking peculiarities including large-diameter blood vessels, high vascular densities, mitochondria-rich muscle cells, and unusual mitochondrial architecture. In order to investigate the bases of icefish adaptation to the extreme Southern Ocean conditions we sequenced the complete genome of the icefish Chionodraco myersi. Comparative analyses of the icefish genome with those of other teleost species, including two additional white-blooded and five red-blooded notothenioids, provided a new perspective on the evolutionary loss of globin genes. Muscle transcriptome comparative analyses against red-blooded notothenioids as well as temperate fish revealed the peculiar regulation of genes involved in mitochondrial function in icefish. Gene duplication and promoter sequence divergence were identified as genome-wide patterns that likely contributed to the broad transcriptional program underlying the unique features of icefish mitochondria., Bargelloni et al. assemble the genome of the icefish Chionodraco myersi and use comparative analysis to examine the basis of adaptation to extreme Southern Ocean conditions. They show evidence for the evolutionary loss of globin genes and the peculiar regulation of genes involved in mitochondrial function.

Glyphosate, the most widely used herbicide worldwide, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. While the potential for glyphosate to induce a broad range of biological effects in exposed organisms has been demonstrated, the global molecular mechanisms of toxicity and potential effects in bacterial symbionts remain unclear, in particular for ecologically important marine species such as bivalve molluscs. Here, the effects of glyphosate (GLY), its degradation product aminomethylphosphonic acid (AMPA), and a mixture of both (MIX) on the mussel M. galloprovincialis were assessed in a controlled experiment. For the first time, next generation sequencing (RNA-seq and 16S rRNA amplicon sequencing) was used to evaluate such effects at the molecular level in both the host and its respective microbiota. The results suggest that the variable capacity of bacterial species to proliferate in the presence of these compounds and the impairment of host physiological homeostasis due to AMPA and GLY toxicity may cause significant perturbations to the digestive gland microbiota, as well as elicit the spread of potential opportunistic pathogens such as Vibrio spp.. The consequent host-immune system activation identified at the molecular and cellular level could be aimed at controlling changes occurring in the composition of symbiotic microbial communities. Overall, our data raise further concerns about the potential adverse effects of glyphosate and AMPA in marine species, suggesting that both the effects of direct toxicity and the ensuing changes occurring in the host-microbial community must be taken into consideration to determine the overall ecotoxicological hazard of these compounds.

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata, a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata, first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes. European Union KBBE.2013.1.2-10 European Community 311920 Fondazione Cassa di Risparmio Padova e Rovigo FCT - Foundation for Science and Technology research grant SPARCOMP under the Call ARISTEIA I of the National Strategic Reference Framework - by the EU 36 Hellenic Republic through the European Social Fund info:eu-repo/semantics/publishedVersion

Yellowfin tuna, Thunnus albacares, is one of the most important seafood commodities in the world. Despite its great biological and economic importance, conflicting evidence arises from classical genetic and tagging studies concerning the yellowfin tuna population structure at local and global oceanic scales. Access to more powerful and cost effective genetic tools would represent the first step towards resolving the population structure of yellowfin tuna across its distribution range. Using a panel of 939 neutral Single Nucleotide Polymorphisms (SNPs), and the most comprehensive data set of yellowfin samples available so far, we found genetic differentiation among the Atlantic, Indian and Pacific oceans. The genetic stock structure analysis carried out with 33 outlier SNPs, putatively under selection, identified discrete populations within the Pacific Ocean and, for the first time, also within the Atlantic Ocean. Stock assessment approaches that consider genetic differences at neutral and adaptive genomic loci should be routinely implemented to check the status of the yellowfin tuna, prevent illegal trade, and develop more sustainable management measures.

Insect mitochondrial genomes (mtDNA) are usually double helical and circular molecules containing 37 genes that are encoded on both strands. The arrangement of the genes is not constant for all species, and produces distinct gene orders (GOs) that have proven to be diagnostic in defining clades at different taxonomic levels. In general, it is believed that distinct taxa have a very low chance of sharing identically arranged GOs. However, examples of identical, homoplastic local rearrangements occurring in distinct taxa do exist. In this study, we sequenced the complete mtDNAs of the ants Formica fusca and Myrmica scabrinodis (Formicidae, Hymenoptera) and compared their GOs with those of other Insecta. The GO of F. fusca was found to be identical to the GO of Dytrisia (the largest clade of Lepidoptera). This finding is the first documented case of an identical GO shared by distinct groups of Insecta, and it is the oldest known event of GO convergent evolution in animals. Both Hymenoptera and Lepidoptera acquired this GO early in their evolution. Using a phylogenetic approach combined with new bioinformatic tools, the chronological order of the evolutionary events that produced the diversity of the hymenopteran GOs was determined. Additionally, new local homoplastic rearrangements shared by distinct groups of insects were identified. Our study showed that local and global homoplasies affecting the insect GOs are more widespread than previously thought. Homoplastic GOs can still be useful for characterizing the various clades, provided that they are appropriately considered in a phylogenetic and taxonomic context.

International audience; Global population genetic structure of yellowfin tuna (Thunnus albacares) is still poorly understood despite its relevance for the tuna fishery industry. Low levels of genetic differentiation among oceans speak in favour of the existence of a single panmictic population worldwide of this highly migratory fish. However, recent studies indicated genetic structuring at a much smaller geographic scales than previously considered, pointing out that YFT population genetic structure has not been properly assessed so far. In this study, we demonstrated for the first time, the utility of 2b-RAD genotyping technique for investigating population genetic diversity and differentiation in high gene-flow species. Running de novo pipeline in Stacks, a total of 6772 high-quality genome-wide SNPs were identified across Atlantic, Indian and Pacific population samples representing all major distribution areas. Preliminary analyses showed shallow but significant population structure among oceans (FST=0.0273; P-value

Human pharmaceuticals such as Acetaminophen, Atenolol and Carbamazepine are pseudo persistent aquatic pollutants with yet unknown sub-lethal effects at environmentally relevant concentrations. Gilthead seabream (Sparus aurata) were exposed to Acetaminophen: 31.90 ± 11.07 μg L−1; Atenolol: 0.95 ± 0.38 μg L−1 and Carbamazepine: 6.95 ± 0.13 μg L−1 in a 28 day flow through experiment to (1) determine whether exposure to low concentrations in the μg·L−1 range of the pharmaceuticals alters the brain transcriptome and, (2) identify different expression profiles and treatment specific modes of action and pathways. Despite low exposure concentrations, 411, 7 and 612 differently expressed transcripts were identified in the individual treatments with Acetaminophen, Atenolol and Carbamazepine, respectively. Functional analyses of differentially expressed genes revealed a significant over representation of several biological processes, cellular compartment features and molecular functions for both Acetaminophen and Carbamazepine treatments. Overall, the results obtained in seabream brain suggest similar physiological responses to those observed in humans also at environmental concentrations, as well as the existence of treatment specific processes that may be useful for the development of biomarkers of contamination., This study was funded by a Marie Curie European Reintegration Grant (Proposal N° 239325-ERA4PHARM, FP7-PEOPLE-ERG-2008), the project SCARCE, funded by the Spanish Ministry for Sciences and Innovation (Consolider-Ingenio 2010 CSD2009-00065) and the project I2TEP for Cross Border Research and Technology Transfer between Spain and Portugal funded by the European Commission (0432_I2TEP_5_E Interreg EU). M.H. is supported by a Ramón y Cajal contract (RYC-2012-12217) from the Spanish Ministry of Economy and Competitiveness (MINECO).

We sequenced the mitochondrial genomes of the spider crabs Maja crispata and Maja squinado (Majidae, Brachyura). Both genomes contain the whole set of 37 genes characteristic of Bilaterian genomes, encoded on both α- and β-strands. Both species exhibit the same gene order, which is unique among known animal genomes. In particular, all the genes located on the β-strand form a single block. This gene order was analysed together with the other nine gene orders known for the Brachyura. Our study confirms that the most widespread gene order (BraGO) represents the plesiomorphic condition for Brachyura and was established at the onset of this clade. All other gene orders are the result of transformational pathways originating from BraGO. The different gene orders exhibit variable levels of genes rearrangements, which involve only tRNAs or all types of genes. Local homoplastic arrangements were identified, while complete gene orders remain unique and represent signatures that can have a diagnostic value. Brachyura appear to be a hot-spot of gene order diversity within the phylum Arthropoda. Our analysis, allowed to track, for the first time, the fully evolutionary pathways producing the Brachyuran gene orders. This goal was achieved by coupling sophisticated bioinformatic tools with phylogenetic analysis.

The aim of the present study was to identify transcriptional modifications and regulatory networks accounting for physiological and metabolic responses to specific nutrients in the liver of young Belgian Blue × Holstein bulls using RNA-sequencing. A larger trial has been carried out in which animals were fed with different diets: 1] a conventional diet; 2] a low-protein/low-mineral diet (low-impact diet) and 3] a diet enriched in n-3 fatty acids (FAs), conjugated linoleic acid (CLA) and vitamin E (nutraceutical diet). The initial hypothesis was that the administration of low-impact and nutraceutical diets might influence the transcriptional profiles in bovine liver and the resultant nutrient fluxes, which are essential for optimal liver function and nutrient interconversion. Results showed that the nutraceutical diet significantly reduced subcutaneous fat covering in vivo and liver pH. Dietary treatments did not affect overall liver fat content, but significantly modified the liver profile of 33 FA traits (out of the total 89 identified by gas-chromatography). In bulls fed nutraceutical diet, the percentage of n-3 and CLA FAs increased around 2.5-fold compared with the other diets, whereas the ratio of n6/n3 decreased 2.5-fold. Liver transcriptomic analyses revealed a total of 198 differentially expressed genes (DEGs) when comparing low-impact, nutraceutical and conventional diets, with the nutraceutical diet showing the greatest effects on liver transcriptome. Functional analyses using ClueGo and Ingenuity Pathway Analysis evidenced that DEGs in bovine liver were variously involved in energy reserve metabolic process, glutathione metabolism, and carbohydrate and lipid metabolism. Modifications in feeding strategies affected key transcription factors regulating the expression of several genes involved in fatty acid metabolism, e.g. insulin-induced gene 1, insulin receptor substrate 2, and RAR-related orphan receptor C. This study provides noteworthy insights into the molecular changes occurring as a result of nutrient variation in diets (aimed at reducing the environmental impact and improving human health) and broadens our understanding of the relationship between nutrients variation and phenotypic effects.

Information on the population dynamics of a reservoir species have been increasingly adopted to understand and eventually predict the dispersal patterns of infectious diseases throughout an area. Although potentially relevant, to date there are no studies which have investigated the genetic structure of the red fox population in relation to infectious disease dynamics. Therefore, we genetically and spatially characterised the red fox population in the area stretching between the Eastern and Dinaric Alps, which has been affected by both distemper and rabies at different time intervals. Red foxes collected from north-eastern Italy, Austria, Slovenia and Croatia between 2006-2012, were studied using a set of 21 microsatellite markers. We confirmed a weak genetic differentiation within the fox population using Bayesian clustering analyses, and we were able to differentiate the fox population into geographically segregated groups. Our finding might be due to the presence of geographical barriers that have likely influenced the distribution of the fox population, limiting in turn gene flow and spread of infectious diseases. Focusing on the Italian red fox population, we observed interesting variations in the prevalence of both diseases among distinct fox clusters, with the previously identified Italy 1 and Italy 2 rabies as well as distemper viruses preferentially affecting different sub-groups identified in the study. Knowledge of the regional-scale population structure can improve understanding of the epidemiology and spread of diseases. Our study paves the way for an integrated approach for disease control coupling pathogen, host and environmental data to inform targeted control programs in the future.

Skeletal anomalies in farmed fish are a relevant issue affecting animal welfare and health and causing significant economic losses. Here, a high-density genetic map of European seabass for QTL mapping of jaw deformity was constructed and a genome-wide association study (GWAS) was carried out on a total of 298 juveniles, 148 of which belonged to four full-sib families. Out of 298 fish, 107 were affected by mandibular prognathism (MP). Three significant QTLs and two candidate SNPs associated with MP were identified. The two GWAS candidate markers were located on ChrX and Chr17, both in close proximity with the peaks of the two most significant QTLs. Notably, the SNP marker on Chr17 was positioned within the Sobp gene coding region, which plays a pivotal role in craniofacial development. The analysis of differentially expressed genes in jaw-deformed animals highlighted the “nervous system development” as a crucial pathway in MP. In particular, Zic2, a key gene for craniofacial morphogenesis in model species, was significantly down-regulated in MP-affected animals. Gene expression data revealed also a significant down-regulation of Sobp in deformed larvae. Our analyses, integrating transcriptomic and GWA methods, provide evidence for putative mechanisms underlying seabass jaw deformity.

The advent of next-generation sequencing (NGS) has dramatically changed bacterial typing technologies, increasing our ability to differentiate bacterial isolates. Despite it is now possible to sequence a bacterial genome in a few days and at reasonable costs, most genetic analyses do not require whole-genome sequencing, which also remains impractical for large population samples due to the cost of individual library preparation and bioinformatics. More traditional sequencing approaches, however, such as MultiLocus Sequence Typing (mlst) are quite laborious and time-consuming, especially for large-scale analyses. In this study, a genotyping approach based on restriction site-associated (RAD) tag sequencing, 2b-RAD, was applied to characterize Listeria monocytogenes strains. To verify the feasibility of the method, an in silico analysis was performed on 30 available complete genomes. For the same set of strains, in silico mlst analysis was conducted as well. Subsequently, 2b-RAD and mlst analyses were experimentally carried out on 58 isolates collected from food samples or food-processing sites. The obtained results demonstrate that 2b-RAD predicts mlst types and often provides more detailed information on population structure than mlst. Moreover, the majority of variants differentiating identical sequence type isolates mapped against accessory fragments, thus providing additional information to characterize strains. Although mlst still represents a reliable typing method, large-scale studies on molecular epidemiology and public health, as well as bacterial phylogenetics, population genetics and biosafety could benefit of a low cost and fast turnaround time approach such as the 2b-RAD analysis proposed here.

The euryhaline European sea bass Dicentrarchus labrax L., inhabiting the coasts of the eastern Atlantic Ocean and Mediterranean Sea, has had many opportunities for differentiation throughout its large natural range. However, evidence for this has been incompletely documented geographically and with an insufficient number of markers. Therefore, its full range was sampled at 22 sites and individuals were genotyped with a suite of mapped markers, including 14 microsatellite loci (N = 536) and 46 neutral or gene-linked single nucleotide polymorphisms (SNPs; N = 644). We confirm that the Atlantic and Mediterranean basins harbour two distinct lineages. Within the Atlantic Ocean no pattern was obvious based on the microsatellite and SNP genotypes, except for a subtle difference between South-eastern and North-eastern Atlantic sea bass attributed to limited introgression of alleles of Mediterranean origin. SNP genotypes of the Mediterranean lineage differentiated into three groups, probably under the influence of geographical isolation. The Western Mediterranean group showed genetic homogeneity without evidence for outlier loci. The Adriatic group appeared as a distinct unit. The Eastern Mediterranean group showed a longitudinal gradient of genotypes and most interestingly an outlier locus linked to the somatolactin gene. Overall, the spatial pattern fits those observed with other taxa of between-basin segregation and within-basin connectivity, which concurs well with the swimming capabilities of European sea bass. Evidence from a few outlier loci in this and other studies encourages further exploration of its regional connectivity and adaptive evolution. ispartof: Biological Journal of the Linnean Society vol:116 issue:1 pages:86-105 status: published

Next-generation sequencing (NGS) technologies now offer the opportunity to move from population genetics to population genomics, even in non-model species. In the present study, two NGS platforms, the Roche 454 FLX and the Illumina Genome Analyzer were used to discover Single Nucleotide Polymorphisms (SNPs) in the muscle transcriptome of the European hake (Merluccius merluccius), which represents one of the most important demersal fisheries in Europe. Transcriptome sequencing and de novo assembly into unique contigs, in silico SNP detection, and validation by high-throughput genotyping yielded a set of 395 polymorphic SNP loci. Of these, 381 nuclear SNPs were used to scan genomic variation in 19 Atlantic and Mediterranean populations of M. merluccius. As all SNPs reside in the functional (transcribed) portion of the hake genome, it is expected that a subset of sequence variants might be located in genomic regions potentially involved in local adaptation, conveying greater resolving power for the detection of spatial structuring on a large and/or local scale. Combining genome scans and correlation analysis between SNP allele frequencies and environmental factors, several candidate loci that are potentially under divergent selection were identified, mainly genes involved in muscles structural organization/function and protein synthesis regulation, or implicated in energy metabolism. Such “outlier” loci showed high performance in resolving population structure, unveiling distinct genetic clusters at multiple spatial scales, which possibly correspond to locally adapted populations. Following a precautionary approach, these results suggest the need to revise the current management strategies of European hake stocks, particularly within basins.

[Background] The Manila clam (Ruditapes philippinarum) is a cultured bivalve with worldwide commercial importance, and diseases cause high economic losses. For this reason, interest in the immune genes in this species has recently increased. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and its application to study the gene transcription profiles of hemocytes from clams infected with V. alginolyticus through a time course., [Results] The complete set of sequences from R. philippinarum available in the public databases and the hemocyte sequences enriched in immune transcripts were assembled successfully. A total of 12,156 annotated sequences were used to construct the 8 ×15 k oligo-microarray. The microarray experiments yielded a total of 579 differentially expressed transcripts. Using the gene expression results, the associated Gene Ontology terms and the enrichment analysis, we found different response mechanisms throughout the experiment. Genes related to signaling, transcription and apoptosis, such as IL-17D, NF-κB or calmodulin, were typically expressed as early as 3 hours post-challenge (hpc), while characteristic immune genes, such as PGRPs, FREPs and defense proteins appeared later at 8 hpc. This immune-triggering response could have affected a high number of processes that seemed to be activated 24 hpc to overcome the Vibrio challenge, including the expression of many cytoskeleton molecules, which is indicative of the active movement of hemocytes. In fact functional studies showed an increment in apoptosis, necrosis or cell migration after the infection. Finally, 72 hpc, activity returned to normal levels, and more than 50% of the genes were downregulated in a negative feedback of all of the previously active processes., [Conclusions] Using a new version of the R. philippinarum oligo-microarray, a putative timing for the response against a Vibrio infection was established. The key point to overcome the challenge seemed to be 8 hours after the challenge, when we detected immune functions that could lead to the destruction of the pathogen and the activation of a wide variety of processes related to homeostasis and defense. These results highlight the importance of a fast response in bivalves and the effectiveness of their innate immune system., This work has been partially funded by the EU Project REPROSEED (245119). RM wishes to acknowledge the Spanish MICINN for her FPI Spanish research grant (BES-2009-029765).

12 pages, 5 figures, 1 table, Pecten maximus, the king scallop, is a bivalve species with important commercial value for both fisheries and aquaculture, traditionally consumed in several European countries. Major problems in larval rearing, however, still limit hatchery-based seed production. High mortalities during early larval stages, likely related to bacterial pathogens, represent the most relevant bottleneck. To address this issue, understanding host defense mechanisms against microbes is extremely important. In this study next-generation RNA-sequencing was carried on scallop hemocytes. To enrich for immune-related transcripts, cDNA libraries from hemocytes challenged in vivo with inactivated-Vibrio anguillarum and in vitro with pathogen-associated molecular patterns, as well as unchallenged controls, were sequenced yielding 216,444,674 sequence reads. De novo assembly of the scallop hemocyte transcriptome consisted of 73,732 contigs (31% annotated). A total of 934 contigs encoded proteins with a known immune function, grouped into several functional categories. Particular attention was reserved to Toll-like receptors (TLRs), a family of pattern recognition receptors (PRRs) involved in non-self recognition. Through mining the scallop hemocyte transcriptome, at least four TLRs could be identified. The organization of canonical TLR domains demonstrated that single cysteine cluster and multiple cysteine cluster TLRs co-exist in this species. In addition, preliminary data concerning their mRNA level following bacterial challenge suggested that different members of this family could exhibit opposite responses to pathogenic stimuli. Finally, a global analysis of differential expression comparing gene-expression levels in in vitro and in vivo stimulated hemocytes against controls provided evidence on a large set of transcripts involved in the great scallop immune response., This work was funded by the EU Project REPROSEED (FP7- KBBE-2009-1-2-11). M.P had a PhD fellowship from the University of Padova.

The first set of eight polymorphic microsatellites markers was successfully optimized from a partial genomic library enriched for an AC motif of the killifish Aphanius fasciatus Nardo, 1827 (Teleostei, Cyprinodontidae), a brackish-water fish widely distributed along the coasts of the Mediterranean Sea. The developed loci proved to be polymorphic, displaying from two to six alleles per locus with expected heterozygosity ranging from 0.277 to 0.752. All loci were under Hardy–Weinberg equilibrium, except Af8 that showed significant heterozygotes deficiency, and there was no evidence of linkage associations between any pair of loci. Markers reported here will be potentially useful in monitoring of genetic population structure in this species.

The Mediterranean yellowtail Seriola dumerilii (Risso, 1810) is a semi-pelagic fish economically important to fisheries and aquaculture. We developed nine microsatellite primer pairs for this species. Microsatellites were isolated from a partial genomic library enriched for an AC motif. The developed loci proved to be polymorphic showing three to 11 alleles per locus. They can be employed for population studies as well as for parentage assessment. Cross-species amplification was successfully tested in Seriola fasciata and Seriola rivoliana.

This study provides a first description of the phylogeographic patterns and evolutionary history of two species of the mudskipper genus Periophthalmus. These amphibious gobies are distributed throughout the whole Indo-Pacific region and Atlantic coast of Africa, in peritidal habitats of soft-bottom coastal ecosystems. Three sequence datasets of two widely distributed species, Periophthalmus argentilineatus and P. kalolo, were obtained by amplifying and sequencing two mtDNA markers (D-loop and 16S rDNA) and the nDNA rag1 region. The three datasets were then used to perform phylogeographic, demographic and population genetic analyses. Our results indicate that tectonic events and past climatic oscillations strongly contributed to shape present genetic differentiation, phylogeographic and demographic patterns. We found support for the monophyly of P. kalolo, and only shallow genetic differentiation between East-African and Indo-Malayan populations of this species. However, our collections of the morphospecies P. argentilineatus include three molecularly distinct lineages, one of them more closely related to P. kalolo. The presence of Miocenic timings for the most recent common ancestors of some of these morphologically similar clades, suggests the presence of strong stabilising selection in mudskippers’ habitats. At population level, demographic analyses and palaeoecological records of mangrove ecosystems suggest that Pleistocene bottlenecks and expansion plus secondary contact events of the studied species were associated with recurrent sea transgressions during interglacials, and sea regressions or stable regimes during glacials, respectively.