Evidence for Selection on Mitochondrial OXPHOS Genes in the Mediterranean Killifish Aphanius fasciatus Valenciennes, 1821.

Mitochondrial oxidative phosphorylation (OXPHOS) genes are a system subject to selection under determined environmental constraints despite a neutral evolution model that has long been hypothesized for the mitochondrial genome. In this study, the sequences of ND1, Cytb, and COI OXPHOS genes were analyzed in six populations of the eurythermal and euryhaline killifish A. fasciatus, to detect non-synonymous mutations leading to amino acid changes and to check whether selection acted on them using tests of recombination and selection. The results indicate a high COI and Cytb gene diversity and a high percentage of private haplotypes in all populations. In the Greek population, non-synonymous nucleotide substitutions were observed in the N-terminal region of COI and Cytb. Positively selected sites were also found. The information we obtained from the mitochondrial DNA sequences of A. fasciatus adds to the growing data on selective pressure acting on mitochondrial DNA in non-model species. These results should be explored from the perspective of the local adaptation of eurythermal and euryhaline species and supported using experimental evidence to better understand the interplay between historical climatic events and local adaptation and how each of them contributes to shaping the genetic structure of this species.

First Report of Mitochondrial DNA Copy Number Variation in Opsius heydeni (Insecta, Hemiptera, Cicadellidae) from Polluted and Control Sites.

Mitochondrial DNA easily undergoes alterations due to exposure to stress factors. In particular, mitochondrial DNA copy number (mtDNAcn) variation can be used as a biomarker of the effect of exposure to various environmental contaminants. In this study, a molecular investigation based on the evaluation of mtDNAcn variation was applied for the first time to individuals belonging to the species Opsius heydeni. A total of 20 samples were collected from two sites in eastern Sicily: Priolo Gargallo, a site with a strong anthropic impact, and the Simeto river Oasis, a control site. Specimens identified based on morphological traits were used to obtain COI gene sequences from this species that were not previously available in GenBank. After processing, the relative mtDNAcn was evaluated using real-time PCR of a portion of the COI and 18S genes. A decrease in the mtDNAcn in the specimens from the polluted site was observed. These results highlight how environmental contaminants can alter the mitochondrial genome and how Opsius heydeni can be considered a potential bioindicator species of environmental quality.

COIBar-RFLP Molecular Strategy Discriminates Species and Unveils Commercial Frauds in Fishery Products.

The DNA analysis is the best approach to authenticate species in seafood products and to unveil frauds based on species substitution. In this study, a molecular strategy coupling Cytochrome Oxidase I (COI) DNA barcoding with the consolidated methodology of Restriction Fragment Length Polymorphisms (RFLPs), named COIBar-RFLP, was applied for searching pattern of restriction enzyme digestion, useful to discriminate seven different fish species (juveniles of Engraulis encrasicolus and Sardina pilchardus sold in Italy as "bianchetto" and Aphia minuta sold as "rossetto"; icefish Neosalanx tangkahkeii; European perch, Perca fluviatilis and the Nile Perch, Lates niloticus; striped catfish, Pangasianodon hypophthalmus). A total of 30 fresh and frozen samples were processed for DNA barcoding, analyzed against a barcode library of COI sequences retrieved from GenBank, and validated for COIBar-RFLP analysis. Cases of misdescription were detected: 3 samples labeled as "bianchetto" were substituted by N. tangkahkeii (2 samples) and A. minuta (1 sample); 3 samples labeled as "persico reale" (P. fluviatilis) were substituted by L. niloticus and P. hypophthalmus. All species were simultaneously discriminated through the restriction pattern obtained with MspI enzyme. The results highlighted that the COIBar-RFLP could be an effective tool to authenticate fish in seafood products by responding to the emerging interest in molecular identification technologies.

Genetic hypervariability of a Northeastern Atlantic venomous rockfish.

BACKGROUND: Understanding the interplay between climate and current and historical factors shaping genetic diversity is pivotal to infer changes in marine species range and communities' composition. A phylogeographical break between the Atlantic and the Mediterranean has been documented for several marine organisms, translating into limited dispersal between the two basins. METHODS: In this study, we screened the intraspecific diversity of 150 individuals of the Madeira rockfish (Scorpaena maderensis) across its distributional range (seven sampling locations in the Atlantic and Mediterranean basins) using the mitochondrial control region and the nuclear S7 first intron. RESULTS: The present work is the most comprehensive study done for this species, yielding no genetic structure across sampled locations and no detectable Atlantic-Mediterranean break in connectivity. Our results reveal deep and hyper-diverse bush-like genealogies with large numbers of singletons and very few shared haplotypes. The genetic hyper-diversity found for the Madeira rockfish is relatively uncommon in rocky coastal species, whose dispersal capability is limited by local oceanographic patterns. The effect of climate warming on the distribution of the species is discussed.

Signals from the deep-sea: Genetic structure, morphometric analysis, and ecological implications of Cyclothone braueri (Pisces, Gonostomatidae) early life stages in the Central Mediterranean Sea.

Cyclothone braueri (Stomiiformes, Gonostomatidae) is a widely distributed fish inhabiting the mesopelagic zone of marine tropical and temperate waters. Constituting one of the largest biomasses of the ocean, C. braueri is a key element in most of the ecological processes occurring in the twilight layer. We focused on the ecological processes linked to early life stages in relation to marine pelagic environmental drivers (temperature, salinity, food availability and geostrophic currents) considering different regions of the Central Mediterranean Sea. A multivariate morphometric analysis was carried out using six parameters with the aim of discerning different larval morphotypes, while a fragment of 367 bp representing the 12S ribosomal RNA gene was used to perform molecular analyses aimed at determining the intraspecific genetic variability. Analysis highlighted two geographically distinct morphotypes not genetically discernible and related to the different nutritional conditions due to spatial heterogeneities in terms of temperature and food availability. The body depth (BD) emerged as an appropriate morphometric parameter to detect the larval condition in this species. Molecular analysis highlighted a moderate genetic divergence in the fish population, showing the recurrence of two phylogroups not geographically separated.

Geographic Pattern of Sushi Product Misdescription in Italy-A Crosstalk between Citizen Science and DNA Barcoding.

The food safety of sushi and the health of consumers are currently of high concern for food safety agencies across the world due to the globally widespread consumption of these products. The microbiological and toxicological risks derived from the consumption of raw fish and seafood have been highlighted worldwide, while the practice of species substitution in sushi products has attracted the interest of researchers more than food safety agencies. In this study, samples of sushi were processed for species authentication using the Cytochrome Oxidase I (COI) gene as a DNA barcode. The approach of Citizen Science was used to obtain the sushi samples by involving people from eighteen different Italian cities (Northern, Central and Southern Italy). The results indicate that a considerable rate of species substitution exists with a percentage of misdescription ranging from 31.8% in Northern Italy to 40% in Central Italy. The species most affected by replacement was Thunnus thynnus followed by the flying fish roe substituted by eggs of Mallotus villosus. These results indicate that a standardization of fish market names should be realized at the international level and that the indication of the scientific names of species should be mandatory for all products of the seafood supply chain.

Transposable Elements and Stress in Vertebrates: An Overview.

Since their identification as genomic regulatory elements, Transposable Elements (TEs) were considered, at first, molecular parasites and later as an important source of genetic diversity and regulatory innovations. In vertebrates in particular, TEs have been recognized as playing an important role in major evolutionary transitions and biodiversity. Moreover, in the last decade, a significant number of papers has been published highlighting a correlation between TE activity and exposition to environmental stresses and dietary factors. In this review we present an overview of the impact of TEs in vertebrate genomes, report the silencing mechanisms adopted by host genomes to regulate TE activity, and finally we explore the effects of environmental and dietary factor exposures on TE activity in mammals, which is the most studied group among vertebrates. The studies here reported evidence that several factors can induce changes in the epigenetic status of TEs and silencing mechanisms leading to their activation with consequent effects on the host genome. The study of TE can represent a future challenge for research for developing effective markers able to detect precocious epigenetic changes and prevent human diseases.

Fish-Based Baby Food Concern-From Species Authentication to Exposure Risk Assessment.

In this work, two different but complementary approaches were used to evaluate the reliability of fish-based baby foods as a source of safe nourishment for babies. More specifically, barcoding analysis based on the Cytochrome Oxidase I sequences was used for fish species authentication and an analysis of metal/metalloid levels was performed to estimate the exposure risk assessment derived from consumption of selected fish-based baby food in infants and toddlers. COI DNA barcoding revealed that in three samples the species detected did not match the common name of the species shown on the label. In particular, G. chalcogrammus and M. australis were found in place of M. merluccius and O. mykiss was found in place of S. salar. The analysis of exposure risk assessment indicated a low risk for developing chronic systemic and carcinogenic effects in infants and toddler, under an exposure scenario based on daily consumption of a single box of fish-based baby food. However, it is important to highlight that in order to provide a comprehensive risk assessment it would be important to supplement the levels of exposure resulting from the total diet. Overall, our results suggest that more attention should be paid by authorities to ensure the safety of food for infants and toddlers.

Swordfish or Shark Slice? A Rapid Response by COIBar-RFLP.

Market transparency is in strong demand by consumers, and the authentication of species is an important step for seafood traceability. In this study, a simple molecular strategy, COIBar-RFLP (cytochrome oxidase I barcode-restriction fragment length polymorphism), is proposed to unveil commercial fraud based on the practice of species substitution in the swordfish trade. In particular, COI barcoding allowed the identification of the species Prionace glauca, Mustelus mustelus, and Oxynotus centrina in slices labeled as Xiphias gladius. Furthermore, the enzymatic digestion of COI amplicons using the MboI restriction endonuclease allowed the simultaneous discrimination of the four species. Interestingly, an intraspecific differential MboI pattern was obtained for the swordfish samples. This pattern was useful to differentiate the two different clades revealed in this species by phylogenetic analyses using several molecular markers. These results indicate the need to strengthen regulations and define molecular tools for combating the occurrence of fraud along the seafood supply chain and show that COIBar-RFLP could become a standardized molecular tool to assess seafood authenticity.

From Fish Eggs to Fish Name: Caviar Species Discrimination by COIBar-RFLP, an Efficient Molecular Approach to Detect Fraud in the Caviar Trade.

The demand for caviar is growing as is its price on the market. Due to the decline of true caviar production from sturgeons, eggs from other fish species and other animals have been used as substitutes for caviar. The labels on these products should indicate the species from which the eggs were derived, but the label can be misleading in some cases. In this context, species identification using DNA analysis is crucial for traceability and authentication of caviar products. In this work, we applied the COIBar-RFLP procedure to obtain species-specific endonuclease restriction patterns useful to discriminate "caviar" species. The tested caviar products were identified as originating from eight species: Acipenser transmontanus, A. gueldenstaedtii, A. stellatus, A. baerii, Mallotus villosus, Huso huso, Cyclopterus lumpus and Eumicrotremus orbis. The results demonstrated that 14% of the caviar products examined have a label that does not indicate the species from which the eggs were originated. The MboI restriction enzyme produced specific profiles discriminating the eight species, confirming that the COIBar-RFLP is a useful approach for routine screening of seafood products due to its ease and rapid execution, as the results of screening can be obtained within 7 h, by-passing the need for sequencing.

How soil granulometry, temperature, and water predict genetic differentiation in Namibian spiders (Ariadna: Segestriidae) and explain their behavior.

The Namib Desert is a biodiversity hotspot for many invertebrates, including spiders. Tube-dwelling spiders belonging to the Ariadna genus are widespread in gravel plains. These sit-and-wait predators share a particular behavior, as they spend their life in tunnels in the soil, surrounding the entrance of their burrow with stone rings. We investigated five spider populations taking into account environmental parameters, functional traits, and molecular data. We have chosen the temperature at the soil surface and at the bottom of the burrow, the air humidity, and the soil granulometry to define the environment. The chosen functional traits were the diameter and depth of the burrows, the ratio between weight and length, the thermal properties of their silks, and the number of ring elements. The molecular branch lengths and the evolutionary distance emerging from cytochrome oxidase I gene sequences summarized the molecular analysis. Our study highlights a strong coherence between the resulting evolutionary lineages and the respective geographical distribution. Multivariate analyses of both environmental and molecular data provide the same phylogenetic interpretation. Low intrapopulation sequence divergence and the high values between population sequence divergence (between 4.9% and 26.1%) might even suggest novel taxa which deserve further investigation. We conclude that both the Kimura distance and the branch lengths are strengthening the environmental clustering of these peculiar sites in Namibia.

Rapid molecular identification of necrophagous diptera by means of variable-length intron sequences in the wingless gene.

The arrival of arthropods at a corpse exhibits specific temporal patterns, and Diptera play a key role in the initial stages of the decomposition process. Thus, the correct species assignment of the insect larvae found on a decomposing body is an important step in forensic investigations. Here, we describe a molecular procedure to define the species at larval age found on a corpse more quickly and easily than current systems. Our method involves a unique PCR amplification of a DNA segment within the evolutionarily conserved wingless gene, involved in embryo development. The amplified DNA segment contains the fourth intron of wingless, which we found to be variable in length, from about 800 to 3000 bp, among species of necrophagous Diptera. The identification of the amplified segment size in species from Lucilia, Calliphora and Sarcophaga genera, allowed us to determine the species at larval age collected in the early stages of a decomposing body, with a simple PCR amplification and subsequent electrophoresis. This procedure may help in forensic investigations to estimate the minimum Post Mortem Interval (PMI-min) of a body colonized by these larvae, avoiding the use of time-consuming and/or more expensive procedures.

Genomic properties of chromosomal bands are linked to evolutionary rearrangements and new centromere formation in primates.

Chromosomal rearrangements in humans are largely related to pathological conditions, and phenotypic effects are also linked to alterations in the expression profile following nuclear relocation of genes between functionally different compartments, generally occupying the periphery or the inner part of the cell nuclei. On the other hand, during evolution, chromosomal rearrangements may occur apparently without damaging phenotypic effects and are visible in currently phylogenetically related species. To increase our insight into chromosomal reorganisation in the cell nucleus, we analysed 18 chromosomal regions endowed with different genomic properties in cell lines derived from eight primate species covering the entire evolutionary tree. We show that homologous loci, in spite of their evolutionary relocation along the chromosomes, generally remain localised to the same functional compartment of the cell nuclei. We conclude that evolutionarily successful chromosomal rearrangements are those that leave the nuclear position of the regions involved unchanged. On the contrary, in pathological situations, the effect typically observed is on gene structure alteration or gene nuclear reposition. Moreover, our data indicate that new centromere formation could potentially occur everywhere in the chromosomes, but only those emerging in very GC-poor/gene-poor regions, generally located in the nuclear periphery, have a high probability of being retained through evolution. This suggests that, in the cell nucleus of related species, evolutionary chromosomal reshufflings or new centromere formation does not alter the functionality of the regions involved or the interactions between different loci, thus preserving the expression pattern of orthologous genes.

Bioaccumulation of metals and biomarkers of environmental stress in Parablennius sanguinolentus (Pallas, 1814) sampled along the Italian coast.

Heavy metal pollution is one of the greatest threats to the ecosystems because it degrades the habitat and is potentially toxic to wildlife and human populations. In the last few decades, bioaccumulation studies performed with a multimarker approach have been a valuable tool for the investigation of environmental and animal safety. We perform an analysis of a benthic teleost fish species - Parablennius sanguinolentus - sampled at several Italian coastal sites with different degrees of anthropogenic pressure. Our integrative analysis encompasses bioaccumulation of 10 metals, biomarkers of environmental stress (micronuclei and nuclear abnormalities) and neutral genetic variation (using sequences of the mtDNA control region). We find a clear and significant correlation of metal bioaccumulation with micronuclei and nuclear abnormalities, especially with undisputed genotoxic metals, such as Cd, Cr, Hg and Pb. Furthermore, the molecular genetic analysis revealed a decrease of genetic variability in the populations more subjected to anthropic pressure.

Heavy metal content and molecular species identification in canned tuna: Insights into human food safety.

Canned tuna in olive oil and in brine of the most popular brands sold in Italian markets were analyzed to verify the authentication of transformed products, with the aim to unveil commercial frauds due to the substitutions of high value species with species of low commercial value, and to assess the health risk of consumers related to cadmium (Cd), lead (Pb) and mercury (Hg) contents. Species authentication was evaluated with amplification of COI DNA barcode and confirmed the declared species. Among tested metals, Hg had the highest concentrations, followed by Cd and Pb. None of the tested samples surpassed the European regulatory limits no. 1881/2006 fixed for Hg and Pb, whereas one batch of canned tuna in olive oil exceeded standard for Cd. Risk for human health was evaluated by the metals daily intake and target hazard quotient (THQ). As a result, Cd and Pb did not exceed the toxicological reference values established by World Health Organization (WHO) and the Environmental Protection Agency (EPA). Conversely, Hg content suggests a consumption no more than once a week and a continuous surveillance of this fishery products for consumer protection.

Mutagenic properties of linuron and chlorbromuron evaluated by means of cytogenetic biomarkers in mammalian cell lines.

Agricultural practices are usually supported by several chemical substances, such as herbicides. Linuron and chlorbromuron are phenylurea herbicides largely used to protect crops from weeds, blocking photosynthesis by inhibition of the photosystem II complex. The former, also commercially known as lorox or afalon, is selectively used to protect bean and French bean plants, fennels, and celeriacs; the second, commercially known as maloran, is selectively used for carrots, peas, potatoes, soy sprouts, and sunflowers. Considering the widespread use of herbicides and, more generally, pesticides, it is important to clarify their involvement on human health, one of them concerning the possible direct or indirect effect on the genome of exposed populations. Here, we show that these herbicides are endowed by mutagenic properties, as demonstrated by an increased number of chromosomal aberrations (CAs) in two exposed Chinese hamster cell lines derived from ovary and epithelial liver, respectively. This was also confirmed by sister chromatid exchange (SCE) and micronucleus (MN) assays. Our present and previously obtained data clearly indicate that phenylurea herbicides must be used with great caution, especially for agricultural workers who use large amounts of herbicides during their work, and particular attention should be given to residues of these herbicides and their involvement in environmental pollution.

A COI Nonsynonymous Mutation as Diagnostic Tool for Intraspecific Discrimination in the European Anchovy Engraulis encrasicolus (Linnaeus).

The European anchovy, Engraulis encrasicolus, is currently one of the principal target species for commercial fisheries in Europe. In this study, the mitochondrial Control Region (CR) and the Cytochrome Oxidase I (COI) mitochondrial gene were analyzed in 74 specimens of E. encrasicolus from four localities in the central Mediterranean. In both populations, the two markers revealed the presence of two main haplogroups, A and B, already detected in previous investigations of different classes of molecular markers. Both CR and COI markers consistently identified two haplogroups. The COI sequence analysis identified a non-synonymous transversion (T to G) at position 116 of the translated sequence, resulting in an amino acid change. All COI sequences of haplogroup A had an amino acid sequence with alanine in this position, while serine was present in the same position in haplogroup B. The two haplogroups A and B were also discriminated by the variable number of TACA elements at the 5'-end of the mitochondrial CR. The selection tests applied to the COI dataset revealed that codon 116 was not under positive selection, that seven amino acid changes were under purifying selection, and that two amino acids were under episodic positive selection.

Geographically widespread swordfish barcode stock identification: a case study of its application.

BACKGROUND: The swordfish (Xiphias gladius) is a cosmopolitan large pelagic fish inhabiting tempered and tropical waters and it is a target species for fisheries all around the world. The present study investigated the ability of COI barcoding to reliably identify swordfish and particularly specific stocks of this commercially important species. METHODOLOGY: We applied the classical DNA barcoding technology, upon a 682 bp segment of COI, and compared swordfish sequences from different geographical sources (Atlantic, Indian Oceans and Mediterranean Sea). The sequences of the 5' hyper-variable fragment of the control region (5'dloop), were also used to validate the efficacy of COI as a stock-specific marker. CASE REPORT: This information was successfully applied to the discrimination of unknown samples from the market, detecting in some cases mislabeled seafood products. CONCLUSIONS: The NJ distance-based phenogram (K2P model) obtained with COI sequences allowed us to correlate the swordfish haplotypes to the different geographical stocks. Similar results were obtained with 5'dloop. Our preliminary data in swordfish Xiphias gladius confirm that Cytochrome Oxidase I can be proposed as an efficient species-specific marker that has also the potential to assign geographical provenance. This information might speed the samples analysis in commercial application of barcoding.