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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. [ABSTRACT FROM AUTHOR]

Abstract Background The insect order Neuroptera encompasses more than 5,700 described species. To date, only three neuropteran mitochondrial genomes have been fully and one partly sequenced. Current knowledge on neuropteran mitochondrial genomes is limited, and new data are strongly required. In the present work, the mitochondrial genome of the ascalaphid owlfly Libelloides macaronius is described and compared with the known neuropterid mitochondrial genomes: Megaloptera, Neuroptera and Raphidioptera. These analyses are further extended to other endopterygotan orders. Results The mitochondrial genome of L. macaronius is a circular molecule 15,890 bp long. It includes the entire set of 37 genes usually present in animal mitochondrial genomes. The gene order of this newly sequenced genome is unique among Neuroptera and differs from the ancestral type of insects in the translocation of trnC. The L. macaronius genome shows the lowest A+T content (74.50%) among known neuropterid genomes. Protein-coding genes possess the typical mitochondrial start codons, except for cox1, which has an unusual ACG. Comparisons among endopterygotan mitochondrial genomes showed that A+T content and AT/GC-skews exhibit a broad range of variation among 84 analyzed taxa. Comparative analyses showed that neuropterid mitochondrial protein-coding genes experienced complex evolutionary histories, involving features ranging from codon usage to rate of substitution, that make them potential markers for population genetics/phylogenetics studies at different taxonomic ranks. The 22 tRNAs show variable substitution patterns in Neuropterida, with higher sequence conservation in genes located on the α strand. Inferred secondary structures for neuropterid rrnS and rrnL genes largely agree with those known for other insects. For the first time, a model is provided for domain I of an insect rrnL. The control region in Neuropterida, as in other insects, is fast-evolving genomic region, characterized by AT-rich motifs. Conclusions The new genome shares many features with known neuropteran genomes but differs in its low A+T content. Comparative analysis of neuropterid mitochondrial genes showed that they experienced distinct evolutionary patterns. Both tRNA families and ribosomal RNAs show composite substitution pathways. The neuropterid mitochondrial genome is characterized by a complex evolutionary history.

Populations of marine top predators have been sharply declining during the past decades, and one-third of chondrichthyans are currently threatened with extinction. Sustainable management measures and conservation plans of large pelagic sharks require knowledge on population genetic differentiation and demographic connectivity. Here, we present the case of the Mediterranean blue shark ( Prionace glauca , L. 1758), commonly found as bycatch in longline fisheries and classified by the IUCN as critically endangered. The management of this species suffers from a scarcity of data about population structure and connectivity within the Mediterranean Sea and between this basin and the adjacent Northeast Atlantic. Here, we assessed the genetic diversity and spatial structure of blue shark from different areas of the Mediterranean Sea and the Northeast Atlantic through genome scan analyses. Pairwise genetic differentiation estimates ( F ST ) on 203 specimens genotyped at 14,713 ddRAD-derived SNPs revealed subtle, yet significant, genetic differences within the Mediterranean sampling locations, and between the Mediterranean Sea and the Northeast Atlantic Ocean. Genetic differentiation suggests some degree of demographic independence between the Western and Eastern Mediterranean blue shark populations. Furthermore, results show limited genetic connectivity between the Mediterranean and the Atlantic basins, supporting the hypothesis of two distinct populations of blue shark separated by the Strait of Gibraltar. Although reproductive interactions may be limited, the faint genetic signal of differentiation suggests a recent common history between these units. Therefore, Mediterranean blue sharks may function akin to a metapopulation relying upon local demographic processes and connectivity dynamics, whereby the limited contemporary gene flow replenishment from the Atlantic may interplay with currently poorly regulated commercial catches and large-scale ecosystem changes. Altogether, these results emphasise the need for revising management delineations applied to these critically endangered sharks., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Evolutionary Applications published by John Wiley & Sons Ltd.)

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., (© 2023. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The mitogenome of the species belonging to the Papilionodea (Lepidoptera) is a double stranded circular molecule containing the 37 genes shared by Metazoa. Eight mitochondrial gene orders are known in the Papilionoidea. MIQGO is the plesiomorphic gene order for this superfamily, while other mitochondrial arrangements have a very limited distribution. 2S1GO gene order is an exception and is present in several Lycaenidae and one species of Hesperiidae. We studied the macrostructural changes generating the gene orders of butterflies by analysing a large data set (611 taxa) containing 5 new mitochondrial sequences/assemblies and 87 de novo annotated mitogenomes. Our analysis supports a possible origin of the intergenic spacer trnQ - nad2 , characterising MIQGO, from trnM . We showed that the homoplasious gene order IMQGO, shared by butterflies, species of ants, beetles and aphids, evolved through different transformational pathways. We identify a complicated evolutionary scenario for 2S1GO in Lycaenidae, characterised by multiple events of duplication/loss and change in anticodon of trnS1 . We show that the gene orders ES1GO and S1NGO originated through a tandem duplication random loss mechanism. We describe two novel gene orders. Ampittia subvittatus (Hesperiidae) exhibits the gene order 2FFGO, characterised by two copies of trnF , one located in the canonical position and a second placed in the opposite strand between trnR and trnN . Bhutanitis thaidina (Papilionidae) exhibits the gene order 4QGO, characterised by the quadruplication of trnQ .

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., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

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 rec A -pyr H 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 ( rec A -pyr H). 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 rec A -pyr H metabarcoding. The study revealed that integration of culture-dependent and culture-independent methods might be a valid approach for the characterization of Vibrio biodiversity.

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.

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 analyzed 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 5,300 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. Analyzing 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 hypothesized 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 rearrangement., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

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., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., (© 2020 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2020 European Society For Evolutionary Biology.)

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., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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., Competing Interests: The authors declare no competing interests.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

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 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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2015 John Wiley & Sons Ltd.)

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<0.01). Discriminant Analysis of Principal Components endorsed the presence of genetically discrete yellowfin tuna populations among three oceanic pools. Although such evidence needs to be corroborated by increasing sample size, these results showed the efficiency of this genotyping technique in assessing genetic divergence in a marine fish with high dispersal potential., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome-wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large- and fine-scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F(CT) = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F(CT) range 0.275-0.705) and fine-scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries., (© 2013 John Wiley & Sons Ltd.)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.

Background: The insect order Neuroptera encompasses more than 5,700 described species. To date, only three neuropteran mitochondrial genomes have been fully and one partly sequenced. Current knowledge on neuropteran mitochondrial genomes is limited, and new data are strongly required. In the present work, the mitochondrial genome of the ascalaphid owlfly Libelloides macaronius is described and compared with the known neuropterid mitochondrial genomes: Megaloptera, Neuroptera and Raphidioptera. These analyses are further extended to other endopterygotan orders., Results: The mitochondrial genome of L. macaronius is a circular molecule 15,890 bp long. It includes the entire set of 37 genes usually present in animal mitochondrial genomes. The gene order of this newly sequenced genome is unique among Neuroptera and differs from the ancestral type of insects in the translocation of trnC. The L. macaronius genome shows the lowest A+T content (74.50%) among known neuropterid genomes. Protein-coding genes possess the typical mitochondrial start codons, except for cox1, which has an unusual ACG. Comparisons among endopterygotan mitochondrial genomes showed that A+T content and AT/GC-skews exhibit a broad range of variation among 84 analyzed taxa. Comparative analyses showed that neuropterid mitochondrial protein-coding genes experienced complex evolutionary histories, involving features ranging from codon usage to rate of substitution, that make them potential markers for population genetics/phylogenetics studies at different taxonomic ranks. The 22 tRNAs show variable substitution patterns in Neuropterida, with higher sequence conservation in genes located on the α strand. Inferred secondary structures for neuropterid rrnS and rrnL genes largely agree with those known for other insects. For the first time, a model is provided for domain I of an insect rrnL. The control region in Neuropterida, as in other insects, is fast-evolving genomic region, characterized by AT-rich motifs., Conclusions: The new genome shares many features with known neuropteran genomes but differs in its low A+T content. Comparative analysis of neuropterid mitochondrial genes showed that they experienced distinct evolutionary patterns. Both tRNA families and ribosomal RNAs show composite substitution pathways. The neuropterid mitochondrial genome is characterized by a complex evolutionary history.