Phylogenomics investigation of sparids (Teleostei: Spariformes) using high-quality proteomes highlights the importance of taxon sampling

Sparidae (Teleostei: Spariformes) are a family of fish constituted by approximately 150 species with high popularity and commercial value, such as porgies and seabreams. Although the phylogeny of this family has been investigated multiple times, its position among other teleost groups remains ambiguous. Most studies have used a single or few genes to decipher the phylogenetic relationships of sparids. Here, we conducted a thorough phylogenomic analysis using five recently available Sparidae gene-sets and 26 high-quality, genome-predicted teleost proteomes. Our analysis suggested that Tetraodontiformes (puffer fish, sunfish) are the closest relatives to sparids than all other groups used. By analytically comparing this result to our own previous contradicting finding, we show that this discordance is not due to different orthology assignment algorithms; on the contrary, we prove that it is caused by the increased taxon sampling of the present study, outlining the great importance of this aspect in phylogenomic analyses in general

Muscle and liver transcriptome characterization and genetic marker discovery in the farmed meagre, Argyrosomus regius

Meagre (Argyrosomus regius), a teleost fish of the family Sciaenidae, is part of a group of marine fish species considered new for Mediterranean aquaculture representing the larger fish cultured in the region. Meagre aquaculture started ~ 25 years ago in West Mediterranean, and the supply of juveniles has been dominated by few hatcheries. This fact has raised concerns on possible inbreeding, urging the need for genetic information on the species and for an assessment of the polymorphisms found in the genome. To that end we characterized the muscle and liver transcriptome of a pool of meagre individuals, from different families and phenotypic size, to obtain a backbone that can support future studies regarding physiology, immunology and genetics of the species. The assembled transcripts were assigned to a wide range of biological processes including growth, reproduction, metabolism, development, stress and behavior. Then, to infer its genetic diversity and provide a catalogue of markers for future use, we scanned the reconstructed transcripts for polymorphic genetic markers. Our search revealed a total of 42,933 high quality SNP and 20,581 STR markers. We found a relatively low rate of polymorphism in the transcriptome that may indicate that inbreeding has taken place. This study has led to a catalogue of genetic markers at the expressed part of the genome and has set the ground for understanding growth and other traits of interest in meagre.info:eu-repo/semantics/acceptedVersio

Linkage mapping, comparative genome analysis, and QTL detection for growth in a non-model teleost, the meagre Argyrosomus regius, using ddRAD sequencing

Meagre (Argyrosomus regius), is a benthopelagic species rapidly emerging in aquaculture, due to its low food to biomass conversion rate, good fillet yield and ease of production. Tracing a species genomic background along with describing the genetic basis of important traits can greatly influence both conservation strategies and production perspectives. In this study, we employed ddRAD sequencing of 266 fish from six F1 meagre families, to construct a high-density genetic map comprising 4529 polymorphic SNP markers. The QTL mapping analysis provided a genomic appreciation for the weight trait identifying a statistically significant QTL on linkage group 15 (LG15). The comparative genomics analysis with six teleost species revealed an evolutionarily conserved karyotype structure. The synteny observed, verified the already well-known fusion events of the three-spine stickleback genome, reinforced the evidence of reduced evolutionary distance of Sciaenids with the Sparidae family, reflected the evolutionary proximity with Dicentrarchus labrax, traced several putative chromosomal rearrangements and a prominent putative fusion event in meagre’s LG17. This study presents novel elements concerning the genome evolutionary history of a non-model teleost species recently adopted in aquaculture, starts to unravel the genetic basis of the species growth-related traits, and provides a high-density genetic map as a tool that can help to further establish meagre as a valuable resource for research and production.info:eu-repo/semantics/publishedVersio

Development and validation of a combined species SNP array for the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata)

SNP arrays are powerful tools for high-resolution studies of the genetic basis of complex traits, facilitating both population genomic and selective breeding research. The European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata) are the two most important fish species for Mediterranean aquaculture. While selective breeding programmes increasingly underpin stocky supply for this industry, genomic selection is not yet widespread. Genomic selection has major potential to expedite genetic gain, in particular for traits practically impossible to measure on selection candidates, such as disease resistance and fillet yield. The aim of our study was to design a combined-species 60K SNP array for both European seabass and gilthead seabream, and to validate its performance on farmed and wild populations from numerous locations throughout the species range. To achieve this, high coverage Illumina whole genome sequencing of pooled samples was performed for 24 populations of European seabass and 27 populations of gilthead seabream. This resulted in a database of ~20 million SNPs per species, which were then filtered to identify high-quality variants and create the final set for the development of the ‘MedFish’ SNP array. The array was then tested by genotyping a subset of the discovery populations and demonstrated a high conversion rate to functioning polymorphic assays on the array (92% in seabass: 89% in seabream) and repeatability (99.4 - 99.7%). The platform interrogates ~30K markers in each fish species, includes features such as SNPs previously shown to be associated with performance traits, and is enriched for SNPs predicted to alter protein function. The array was demonstrated to be effective at detecting population structure across a wide range of fish populations from diverse geographical origins, and to examine the extent of haplotype sharing among Mediterranean fish farms. Therefore, the MedFish array enables efficient and accurate high-throughput genotyping for genome-wide distributed SNPs on each fish species, and will facilitate stock management, population genomics approaches, and acceleration of selective breeding through genomic selection.submittedVersio

Development of a new spectrophotometric assay for rapid detection and differentiation of KPC, MBL and OXA-48 carbapenemase-producing Klebsiella pneumoniae clinical isolates

The increased prevalence of carbapenemase-producing Enterobacteriaceae (CPE) has made essential the design of quicker tests for CPE detection. In the present study, a simple and rapid assay was developed based on measurement of the hydrolytic activity of imipenem at a final concentration of 65 µg/mL (100 µM) through ultraviolet–visible (UV-Vis) spectrophotometry. All measurements were conducted at 297 nm. A total of 83 carbapenem-non-susceptible CPE, consisting of Klebsiella pneumoniae clinical isolates and genotypically characterised as KPC-, VIM-, NDM- or OXA-48-producers, were tested. For comparison, 30 carbapenem-non-susceptible clinical isolates, consisting of Escherichia coli and K. pneumoniae and genotypically confirmed as non-CPE, were also examined. The spectrophotometric assay enabled efficient discrimination of CPE from non-CPE isolates even in 45 min (P < 0.0001). Moreover, the presence of phenylboronic acid (PBA) or ethylene diamine tetra-acetic acid (EDTA) in the reaction mixture was able to inhibit the hydrolytic capacity of KPC- or metallo-β-lactamase (MBL)-producers, respectively, while the hydrolytic activity of OXA-48-producing strains was not affected by the presence of these inhibitors (P < 0.001). The newly developed assay presented 100% sensitivity and specificity to detect and differentiate KPC-, MBL- and OXA-48-producers compared with genotypic characterisation. Thus, the proposed spectrophotometric method can be considered as an easy, fast, accurate and cost-effective diagnostic tool for screening carbapenem-non-susceptible K. pneumoniae isolates in the clinical laboratory. © 2020 Elsevier Ltd and International Society of Antimicrobial Chemotherap

Development of a multiplex RT-PCR assay for the identification of recombination types at different genomic regions of vaccine-derived polioviruses

Polioviruses (PVs) are the causal agents of acute paralytic poliomyelitis. Since the 1960s, poliomyelitis has been effectively controlled by the use of two vaccines containing all three serotypes of PVs, the inactivated poliovirus vaccine and the live attenuated oral poliovirus vaccine (OPV). Despite the success of OPV in polio eradication programme, a significant disadvantage was revealed: the emergence of vaccine-associated paralytic poliomyelitis (VAPP). VAPP is the result of accumulated mutations and putative recombination events located at the genome of attenuated vaccine Sabin strains. In the present study, ten Sabin isolates derived from OPV vaccinees and environmental samples were studied in order to identify recombination types located from VP1 to 3D genomic regions of virus genome. The experimental procedure that was followed was virus RNA extraction, reverse transcription to convert the virus genome into cDNA, PCR and multiplex-PCR using specific designed primers able to localize and identify each recombination following agarose gel electrophoresis. This multiplex RT-PCR assay allows for the immediate detection and identification of multiple recombination types located at the viral genome of OPV derivatives. After the eradication of wild PVs, the remaining sources of poliovirus infection worldwide would be the OPV derivatives. As a consequence, the immediate detection and molecular characterization of recombinant derivatives are important to avoid epidemics due to the circulation of neurovirulent viral strains. © 2016, Springer Science+Business Media New York

The transcriptomic signature of different sexes in two protogynous hermaphrodites: Insights into the molecular network underlying sex phenotype in fish

Sex differentiation is a puzzling problem in fish due to the variety of reproductive systems and the flexibility of their sex determination mechanisms. The Sparidae, a teleost family, reflects this remarkable diversity of sexual mechanisms found in fish. Our aim was to capture the transcriptomic signature of different sexes in two protogynous hermaphrodite sparids, the common pandora Pagellus erythrinus and the red porgy Pagrus pagrus in order to shed light on the molecular network contributing to either the female or the male phenotype in these organisms. Through RNA sequencing, we investigated sex-specific differences in gene expression in both species’ brains and gonads. The analysis revealed common male and female specific genes/pathways between these protogynous fish. Whereas limited sex differences found in the brain indicate a sexually plastic tissue, in contrast, the great amount of sex-biased genes observed in gonads reflects the functional divergence of the transformed tissue to either its male or female character. Α common “crew” of well-known molecular players is acting to preserve either sex identity of the gonad in these fish. Lastly, this study lays the ground for a deeper understanding of the complex process of sex differentiation in two species with an evolutionary significant reproductive system

Molecular characterization of a new intergenotype Norovirus GII recombinant

Human noroviruses (NoVs) of the Caliciviridae family are a major cause of epidemic gastroenteritis. The NoV genus is genetically diverse and recombination of viral RNA is known to depend upon various immunological and intracellular constraints that may allow the emergence of viable recombinants. In the present study, we report the development of a broadly reactive RT-PCR assay, which allowed the characterization of strain A6 at molecular level, established its genetic relationship at the sub-genogroup level and classified A6 strain at the sub-genotype level. The detection was carried out initially by enzyme-linked immunosorbent assay (ELISA) and the subsequent detection and molecular characterization of NoV strain was achieved by reverse transcription-PCR and sequencing. Based on the sequence analysis, A6 strain was revealed to belong to the GII genogroup of NoVs. Partial ORF1 gene sequencing analysis and complete ORF2 gene sequencing revealed that ORF1 and ORF2 belonged to two distinct genotypes GII/9 and GII/6, respectively, making obvious that A6 strain is a rare intergenotypic recombinant within the genogroup GII between GII.9 and GII.6 genotypes. A6 strain represents the first human NoV from Greece, whose genome has been partially (ORF1&ORF3) and completed (ORF2) sequenced. To our knowledge the recombination event GII.9/GII.6 in RdRp and capsid gene, respectively, that was revealed in the present study is reported for the first time

A new RT-PCR assay for the identification of the predominant recombination types in 2C and 3D genomic regions of vaccine-derived poliovirus strains

In the post-eradication era of wild polioviruses, the only remaining sources of poliovirus infection worldwide would be the vaccine-derived polioviruses (VDPVs). As the preponderance of countries certified to be polio-free has switched from OPV (oral poliovirus vaccine) to IPV (inactivated poliovirus vaccine), importation of recombinant evolved derivatives of vaccinal strains would have serious implication for public health. To test the robustness of the proposed RT-PCR screening analysis, eleven recombinant vaccine-derived polioviruses that were characterized previously by sequencing by our group, in addition to three recently identified recombinant environmental isolates were assayed. Although the most definitive characterization of VDPVs is by genomic sequencing, in this study we describe a new, inexpensive and broadly applicable RT-PCR assay for the identification of the predominant recombination types S3/Sx in 2C and S2/Sx in 3D genomic regions respectively of VDPVs, that can be readily implemented in laboratories lacking sequencing facilities as a first approach for the early detection of vaccine-derived poliovirus (VDPVs). (C) 2009 Elsevier Ltd. All rights reserved