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Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

Nine common sole Solea vulgaris samples from rive fishery Management Units (MUs) of the central Mediterranean exhibited differences in a control region sequence marker. Parsimony network showed two low-divergent haplotype phylogroups. The former predominated in samples from the Ligurian, Tyrrhenian, Adriatic and north-western Ionian Seas (MUs 9, 10, 17, 18 and northern part of 19, respectively), whereas the latter was abundant in the south-western Ionian (southern part of MU 19). The geographical clustering of maternal lineages accounts for population structuring and indicates geographical isolation of common sole stocks in the Mediterranean. Several life-history traits of common sole and hydro-geographical features of the basin might support this pattern of differentiation. Haplotype frequency differences were detected among samples either from different MUs or within the same MU. This situation indicates the presence of partially subdivided or nearly panmictic population units, whose exploitation should be based on appropriate bio-ecological features. The usefulness of control region sequence marker enhances the routinely use of the genetic stock structure analysis in low-dispersal demersal marine resources. © 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.

The performance of different molecular markers in the assessment of population structure was tested using samples of Solea vulgaris collected in the Mediterranean within and outside the hypothetical dispersal ability of the species. A total of 172 individuals belonging to four population samples were analysed using 15 microsatellites [simple sequence repeats (SSRs)] and 153 amplified fragment length polymorphisms (AFLPs). Considering the global qualitative patterns, we found a correlation between SSRs and AFLPs in detecting genetic differentiation among samples. However, on a small geographical scale, AFLPs were able to discriminate individuals from neighbouring populations whereas SSRs were not, and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with SSRs. The high number of loci analysed with the AFLP technique could increase the probability to include outlier loci in the analysis; however, the neutrality test performed on our data set did not show evidence of selection acting on the S. vulgaris samples. Even if the choice of the molecular marker depends mainly on the biological question to be addressed, the higher power of discrimination and the comparative technical ease of obtaining data from AFLPs with respect to SSRs suggest the use of AFLPs for many population genetics studies.

Because of its complex life cycle and due to multiple stress factors, the European eel is suffering a dramatic decline and has been declared Critically Endangered by the IUCN. A possible way to address this problem could be relieve the pressure on natural stocks by implementing its mass production by artificial breeding protocols. A previous study based on direct observation and parentage assignment underlined the presence of an allegedly hierarchic structure among European eel males in semi-natural mating conditions, with a consequent bias in F1 proportion assigned to each one. The aim of the present study is to attest if a different fertilization protocol based on the artificial mixing of female eggs with different males’ milt in equal volume (1 F:4 M) could represent a solution to the disproportion observed in semi-natural conditions. For this purpose, six families of European eels were generated, and 10 species-specific microsatellite loci were used to infer offspring composition by paternity assignment on 280 samples. Due to the asynchronous ovarian development of female European eels, the per- centage of fertilized eggs for each female showed a great variability, ranging from 4.70% to 94.50%. A pro- portion of 94.02% of genotyped offspring were assigned with high confidence to their true parents. As regard males’ fertilization pattern, no substantial differences from natural mating were observed: a single male accounted for most offspring, which was just mostly composed of full sibs. Concluding, the obtained results suggest that the admixture of an equal volume of different males’ milt seems to contribute to the single-locus genetic variability (observed heterozygosity higher than expected in 7 out 9 loci), but it is not sufficient to ensure all the males the same chance to transmit their gene pool, and new fertilization strategies must be developed.

The high nutritional quality of common sole Solea solea increases its value for the fishery industry and for the aquaculture sector. To ensure the expansion of its production, it is necessary to implement farming and broodstock management technique to produce high quality eggs and larvae. This work summarizes eight years of study on reproductive performances, growth and parental contribution of a common sole broodstock from the Mediterranean-North Adriatic Sea. The broodstock (11 females and 13 males) reached the peak of fecundity after 5/6 years of captivity, with a production of 296.48 and 376.54 × 103 of total eggs kg−1 female with a fertilization rate of 31.6 ± 18.3 and 41.9 ± 23.8 %, respectively. Results shows that variations in temperature cycles are pivotal for a successful breeding season, and body condition during the first 3 years of captivity was the most important parameter positively correlated to parental contribution for both males and females. Parental contribution was assessed by eight species-specific microsatellite loci during the first 3 years of reproduction. Although the two sexes displayed different temporal trends, parentage assessment demonstrated an overall increase in the number of active breeders. However, only 13 couples out of 50 produced the 70.4 % of the larvae in year 5, confirming the same reproduction behavioural pattern in Mediterranean common sole than that observed both in Senegalese sole and in common sole of Atlantic origin. This work provides valuable data needed to understand the mechanisms behind the maturation characteristics of this species and support future strategy for the establishment and management of Mediterranean common sole aquaculture.

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions., peer-reviewed

Growing demands for marine fish products is leading to increased pressure on already depleted wild populations and a rise in the aquaculture production. Consequently, more captive bred fish are released into the wild through accidental escape or deliberate restocking, stock enhancement and sea ranching programs. The increased mixing of captive bred fish with wild conspecifics may affect the ecological and/or genetic integrity of wild fish populations. From a fisheries management perspective unambiguous identification tools for captive bred fish will be highly valuable to manage risks. Additionally there is great potential to use these tools in wildlife forensics (i.e. tracing back escapees to their origin and determining mislabelling of seafood products). Using SNP data from captive bred and wild populations of Atlantic cod (Gadus morhua L.) and sole (Solea solea L.), we explored the efficiency of population and parentage assignment techniques for the identification and tracing of captive bred fish. Simulated and empirical data were used to correct for stochastic genetic effects. Overall, parentage assignment performed well when a large effective population size characterizes the broodstock and escapees originate from early generations of captive breeding. Consequently, parentage assignments are particularly useful from a fisheries management perspective to monitor the effects of deliberate releases of captive bred fish on wild populations. Population assignment proved to be more efficient after several generations of captive breeding, which makes it a useful method in forensic applications for well-established aquaculture species. We suggest the implementation of a case by case strategy when choosing the best method.

The objective of this study was to analyse over time the evolution of E. coli O157:H7 faecal shedding in a dairy herd producing raw milk for direct human consumption. The study was performed between October 2012 and September 2013 in an average size Italian dairy farm where animals are housed inside the barn all over the year. The farm housed about 140 animals during the study – 70 cows and 70 calves and heifers. Twenty-six animals were randomly selected from both the cows and young animals group, and faecal sampling was performed rectally six times two months apart in each animal. Eleven animals were culled during the study and a total of 285 faecal samples were collected. At each faecal sampling, three trough water samples and two trough feed samples were also collected for a total of 36 water samples and 24 feed samples. Samples were analysed by real time polymerase chain reaction (RT-PCR) and culture. Overall, 16 (5.6%) faecal samples were positive for E. coli O157 by RT-PCR. Cultural examination found 9 (3.1%) samples positive for E. coli O157; all the isolates were positive for stx1, stx 2 and eae genes. One (4.1%) feed sample was positive for E. coli O157 by RT-PCR; none of the water samples was positive for E. coli O157. The model highlighted a general significant reduction of the number of positive samples observed during the study from the first to the sixth sampling (P=0.000) and a positive relation between the presence of positive samples and average environmental temperature (P=0.003). The results of the study showed that in an Italian dairy farm housing animals all year, faecal shedding of E. coli O157 followed the same temporal trend reported for other types of farming. The enhanced faecal shedding during warmer months may have a significant impact on environmental contamination and the safety of raw milk and its byproducts.

Lo scopo della ricerca è stato quello di valutare, tramite uno studio longitudinale, le modifiche temporali nella eliminazione fecale di E. coli O157:H7 in un allevamento di bovine da latte che commercializza latte crudo per il consumo umano diretto. Lo studio è stato effettuato tra ottobre 2012 e settembre 2013 in una tipica stalle di medie dimensioni. L’allevamento era costituito da circa 140 animali (70 capi adulti e 70 giovani). Ventisei animali di ciascuno dei due gruppi (adulti e giovani) sono stati scelti casualmente e sono stati effettuati da ciascun animale 6 campionamenti di feci a distanza di 2 mesi l’uno dall’altro (in totale 284). A ogni campionamento sono stati effettuati, per ciascun gruppo, 3 campioni di acqua (in totale 36) dagli abbeveratoi e 2 campioni di mangime dalla greppia (in totale 24). I campioni sono stati analizzati tramite real time PCT (RTPCR) ed esame colturale. In totale 16 (5,6%) campioni di feci sono risultati positivi tramite RT-PCR e 9 tramite esame colturale. In tutti gli isolati è stata dimostrata la presenza dei geni stx1, stx 2 e eae. Un campione di mangime è risultato positivo tramite RT-PCR; nessun campione di acqua è risultato positivo. L’elaborazione dei dati ha evidenziato in generale una riduzione del numero di campioni positivi nel corso dello studio e una relazione tra la prevalenza dei campioni positivi e la temperatura media ambientale. I risultati dello studio dimostrano che, in una tipica azionda di bovini da latte Italiana, l’eliminazione fecale di E. coli O157:H7 segue il medesimo andamento osservato in altre situazioni; l’aumento della eliminazione fecale nel periodo estivo ha un impatto significativo sulla contaminazione ambientale e e sulla sicurezza dei prodotti alimentari in particolare il latte venduto e consumato crudo

In the Mediterranean flatfish are valuable fishery resources, with potential for aquaculture production. The assessment and monitoring of the stocks status are crucial to achieve environment-friendly management. However, the coexistence of two cryptic Solea species, S. solea and S. aegyptiaca, might lead to misidentification of specimens during the collection of fishery data, especially in areas where they live in sympatry. Using recently developed molecular markers, which have enabled their reliable identification, we provide new insights on the evolutionary history of the Mediterranean soles by analyzing species distribution, population structure, demography and inter-specific reproductive interactions with multiple molecular datasets. The analyses reveal a wider distribution of S. aegyptiaca in the Mediterranean Sea than previously described, hence pointing to a possible range expansion. In both species all markers consistently revealed significant population structuring according to a longitudinal gradient and demographic expansion. Despite their sympatric distribution in various regions, nuclear data confirmed the lack of inter-specific hybridization or introgression. Further multidisciplinarity in research (combining morphology, reproductive biology, life history traits analysis with genetics) are needed to unravel the possible range expansion of S. aegyptiaca according to a general hypothesis of Mediterranean tropicalization and its ecological interactions with the sympatric S. solea.

In the Mediterranean, the common (Solea solea) and Egyptian (S. aegyptiaca) soles are two of the most valuable flatfish fishery resources. In the past, S. aegyptiaca was erroneously synonymised with Solea solea because of the great similarity in external morphology. Recently molecular markers have enabled their reliable identification, showing that the two species seem to co-occur in several areas of the Mediterranean, forming sympatric demes. The sympatric distribution and the close phylogenetic relationship allow a scenario for potential ecological and evolutionary interactions. Here, we aimed at providing new insights on the taxonomy, phylogeography and evolutionary interactions of the Mediterranean soles by i) developing PCR tests for the rapid screening of the two cryptic species, ii) analysing the species composition at several geographical locations, iii) assessing the phylogeographic pattern and genetic structure of populations and iv) the possible occurrence of interspecific hybridization in sympatric populations. In total 435 sole individuals were sequenced for a mitochondrial fragment (cytB gene) and 217 genotyped at two nuclear marker classes (ITS rDNA gene and 8 microsatellite loci). The analysis of species composition revealed a wider distribution of S. aegyptiaca in the Mediterranean than previously described. The observed frequent sympatry with S. solea could have lead to their frequent mis-identification. All nuclear markers however consistently revealed a clear genetic separation of the two species. Both mitochondrial and nuclear data showed the structuring of populations according to a longitudinal gradient, between the Western and Eastern basin, with different degree of differentiation in the two sole species. Higher genetic diversity was observed in S. solea for all markers. Further multidisciplinary data (combining data from morphology, reproductive biology and life history with genetic) are needed to unravel distribution and ecology of S. aegyptiaca in the Mediterranean and its ecological interactions with the cryptic species S. solea. This assessment will have important implications for sustainable management and conservation of fishery resources, because different species can differently respond to environmental pressures and changes.

Quattro campioni di Nephrops norvegicus provenienti da differenti zone dell'Adriatico sono stati analizzati tramite cinque loci microsatellitari specie specifici. E' stato individuato un buon livello di polimorfismo genetico. L'eterozigosi media osservata va da 0,447 a 0,942 e il numero medio di alleli per locus va da 9 a 40. I risultati di polimorfismo e differenziamento genetico osservati sulla base dei cinque loci microsatelliti analizzati sono a supporto dell'ipotesi di panmissia dello tock adriatico di scampo.

In the semienclosed Adriatic Sea, the shared stocks of the cephalopods Loligo vulgaris and Sepia officinalis represent important marine fisheries resources exploited by all coastal countries. The improving of knowledge on the demographic features of these shared stocks is internationally relevant for adopting responsible management and conservation of these marine resources. Analyses of microsatellite variation in geographical samples collected from all parts of the Adriatic Sea were performed using arrays of species-specific di-nucleotide and tri-nucleotide loci. In L. vulgaris the level of genetic variability was consistent with that observed in other loliginid species, whereas the S. officinalis stock showed a microsatellite variation markedly lower than that estimated for the Atlantic and Mediterranean populations collected around the Iberian peninsula. The weak spatial genetic differentiation, the discordant results of the genetic divergence estimators and the lack of any geographical cline in the spatial genetic differences suggest the occurrence of single genetically homogeneous populations within the Adriatic stocks of both species, recommending a coordinated management of the squid and cuttlefish by the Adriatic fishing countries. On the contrary, significant differences detected in temporal replicates of S. officinalis might suggest that allelic frequency can change relating to reproductive behaviour.

In order to study the genetic structure of the Adriatic shared stock of red mullet (Mullus barbatus), we developed a set of dinucleotide microsatellite markers. A dinucleotide-enriched genomic library was obtained, and 6 polymorphic dinucleotide loci were successfully optimized. The markers showed high expected heterozygosity (from 0.68 to 0.92) and allele number (from 12 to 33); thus they appear to be suitable for detecting genetic differences in the population of red mullet. Four Adriatic samples were subsequently analyzed for microsatellite variation, and the results showed subtle but statistically significant genetic differentiation, indicating that the Adriatic red mullet may group into local, genetically isolated populations. No correlation between geographic distance and genetic differentiation was observed. In addition, the evidence of recent bottlenecks in the Adriatic samples indicates that the observed population subdivision might reflect random local allelic variations, generated by reproductive success, survival rates, or fishing pressure.