Anna Maria Pappalardo

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.

DNA barcoding and COI sequence variation in Mediterranean lanternfishes larvae

Abstract The potential use of Cytochrome c Oxidase I (COI)-DNA barcode sequences for the molecular identification of lanternfish larvae from the Sicilian Channel was investigated at two levels: at an interspecific level by confirming species identification based on morphological characters; and at an intraspecific level to test for the presence of geographical variation of COI-DNA sequences. A reference library of COI sequences was constructed starting from unambiguously identified specimens. Neighbor-joining analysis based on K2P genetic distances formed non-overlapping clusters for all species (Myctophum punctatum, Ceratoscopelus maderensis, Hygophum benoiti, Electrona risso and Lobianchia dofleini) with a 100% bootstrap support for each. Additional COI sequences of lanternfishes from Atlantic Ocean and Balearic Sea samples deposited in BOLD system database were included in the dataset. The present analysis allowed the identification of unknown fish larvae and indicated that there is a relative congruence between morphological and molecular identification approaches. Our preliminary data in Myctophidae species confirm that COI gene can be used as an efficient species-specific marker that is also useful for assessing the geographical provenance of larvae. This information will likely be applicable to the investigation of the population structure in these poorly studied species.

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.

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.

Comparative pattern of genetic structure in two Mediterranean killifishes Aphanius fasciatus and Aphanius iberus inferred from both mitochondrial and nuclear data

In this study, genetic variation was assessed in Aphanius fasciatus and Aphanius iberus characterized by similar ecological traits but with very different distribution ranges in the Mediterranean area. Five populations of A. iberus and five of A. fasciatus were analysed using five polymorphic microsatellite loci and partial mitochondrial control region (D-loop) sequences. Congruent results were found with both nuclear and mitochondrial molecular markers. The results showed that similar levels of genetic divergence, based on mitochondrial control region sequences, are present among populations of A. iberus and among populations of A. fasciatus despite the very different geographic distance existing among the examined populations of the two species (low geographic distance in A. iberus and high in A. fasciatus). A possible explanation could be that the populations of A. iberus were isolated for a longer time than the populations of A. fasciatus supporting the hypothesis that the split in the lineage leading to A. iberus is older than the split in the lineage leading to A. fasciatus. The possibility that the wide circum-Mediterranean distribution of A. fasciatus ensures the high connectivity of its populations, preventing, in some cases, local differentiation, however, cannot be ruled out.

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.

Mitochondrial and nuclear intraspecific variation in the rusty blenny (Parablennius sanguinolentus, Blenniidae)

The genetic structure of Parablennius sanguinolentus from the northeastern Atlantic and Mediterranean was assessed using sequences from the mitochondrial control region (CR) and the first intron of the nuclear S7 ribosomal protein gene (S7). Our data show high genetic diversity for the central Mediterranean populations, contrasting with very low diversity in the Atlantic populations and in the westernmost Mediterranean population of Cabo de Gata. Both CR mismatch analysis and neutrality tests provide evidence of demographic and spatial expansion for the Atlantic and central Mediterranean populations. The significant correlation between genetic and geographic distances supported isolation-by-distance for the S7, but not for the CR, suggesting a complex scenario for the history of the rusty blenny in the Mediterranean Sea. The pattern of genetic structure displayed by P. sanguinolentus could be explained as the result of a post-glacial colonization of the Atlantic coast of Europe from the Mediterranean Sea.

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.

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.

Differential flatfish species detection by COIBar-RFLP in processed seafood products

Common sole (Solea solea) is often the subject of fraudulent species substitutions in processed products because of their excellent organoleptic characteristics and high commercial interest. COIBar-RFLP, a molecular strategy-coupling Cytochrome Oxidase I (COI) DNA barcoding with the consolidated methodology of restriction fragment length polymorphism analysis (RFLP), was applied to search for restriction enzyme polymorphisms useful to discriminate among potential flatfish substitutes of common sole. Seven flatfish species belonging to Soleidae, Bothidae, and Citharidae families were used to construct a reference barcode library of COI sequences. The flatfish species were simultaneously discriminated through specific digestion profiles obtained with the restriction enzyme MspI. We tested the efficacy of COIBar-RFLP on 13 frozen fillets labeled as common sole purchased in local fish markets. These fillets were found to contain three species, S. solea, Solea senegalensis, and Arnoglossus laterna, successfully discriminated by COIBar-RFLP, demonstrating that this method is a rapid and simple sequencing-free molecular approach for these fish species identification in processed seafood products.