The Northern Adriatic Sea (FAO Geographical Sub-Area 17) is one of the most productive fishing areas of the Mediterranean Sea and it includes a broad diversity of habitats. In the Northern Adriatic ...basin, the Pomo Pit (200-273 m of depth) is one of the most important areas of aggregation for some demersal stocks shared in the Adriatic Sea and it is an important spawning/nursery area of the European hake (Merluccius merluccius). Through a metabarcoding approach we investigated the feeding habits of European hake, both inside and outside the Pomo Pit, and their temporal variability comparing samples collected in 2016 and 2014. Our analyses proved the presence of an ontogenetic shift from a diet based mainly on crustaceans in juveniles to a more piscivorous feeding behaviour in adult hakes and suggested the presence of a specific niche partitioning and food preferences between hakes living inside and outside the Pomo Pit. The main differences among adult hakes refer to the presence of molluscs in the stomachs of hakes collected within the Pomo Pit and the presence of high depth prey species (i.e., Micromesistius poutassou). Metabarcoding revealed the relevant ecological role played by the Pomo Pit in M. merluccius feeding behaviour and ontogenetic development, promoting a careful ecosystem-based management of fisheries in this area through focused conservation measures.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Nematodes inhabiting benthic deep-sea ecosystems account for >90% of the total metazoan abundances and they have been hypothesised to be hyper-diverse, but their biodiversity is still largely ...unknown. Metabarcoding could facilitate the census of biodiversity, especially for those tiny metazoans for which morphological identification is difficult. We compared, for the first time, different DNA extraction procedures based on the use of two commercial kits and a previously published laboratory protocol and tested their suitability for sequencing analyses of 18S rDNA of marine nematodes. We also investigated the reliability of Roche 454 sequencing analyses for assessing the biodiversity of deep-sea nematode assemblages previously morphologically identified. Finally, intra-genomic variation in 18S rRNA gene repeats was investigated by Illumina MiSeq in different deep-sea nematode morphospecies to assess the influence of polymorphisms on nematode biodiversity estimates. Our results indicate that the two commercial kits should be preferred for the molecular analysis of biodiversity of deep-sea nematodes since they consistently provide amplifiable DNA suitable for sequencing. We report that the morphological identification of deep-sea nematodes matches the results obtained by metabarcoding analysis only at the order-family level and that a large portion of Operational Clustered Taxonomic Units (OCTUs) was not assigned. We also show that independently from the cut-off criteria and bioinformatic pipelines used, the number of OCTUs largely exceeds the number of individuals and that 18S rRNA gene of different morpho-species of nematodes displayed intra-genomic polymorphisms. Our results indicate that metabarcoding is an important tool to explore the diversity of deep-sea nematodes, but still fails in identifying most of the species due to limited number of sequences deposited in the public databases, and in providing quantitative data on the species encountered. These aspects should be carefully taken into account before using metabarcoding in quantitative ecological research and monitoring programmes of marine biodiversity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Fishery genetics have greatly changed our understanding of population dynamics and structuring in marine fish. In this study, we show that the Atlantic Bluefin tuna (ABFT, Thunnus thynnus), an ...oceanic predatory species exhibiting highly migratory behavior, large population size, and high potential for dispersal during early life stages, displays significant genetic differences over space and time, both at the fine and large scales of variation. We compared microsatellite variation of contemporary (n = 256) and historical (n = 99) biological samples of ABFTs of the central-western Mediterranean Sea, the latter dating back to the early 20th century. Measures of genetic differentiation and a general hétérozygote deficit suggest that differences exist among population samples, both now and 96-80 years ago. Thus, ABFTs do not represent a single panmictic population in the Mediterranean Sea. Statistics designed to infer changes in population size, both from current and past genetic variation, suggest that some Mediterranean ABFT populations, although still not severely reduced in their genetic potential, might have suffered from demographic declines. The short-term estimates of effective population size are straddled on the minimum threshold (effective population size = 500) indicated to maintain genetic diversity and evolutionary potential across several generations in natural populations.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Atlantic Bluefin Tuna (ABFT) shows complex demography and ecological variation in the Mediterranean Sea. Genetic surveys have detected significant, although weak, signals of population structuring; ...catch series analyses and tagging programs identified complex ABFT spatial dynamics and migration patterns. Here, we tested the hypothesis that the genetic structure of the ABFT in the Mediterranean is correlated with mean surface temperature and salinity.
We used six samples collected from Western and Central Mediterranean integrated with a new sample collected from the recently identified easternmost reproductive area of Levantine Sea. To assess population structure in the Mediterranean we used a multidisciplinary framework combining classical population genetics, spatial and Bayesian clustering methods and a multivariate approach based on factor analysis.
FST analysis and Bayesian clustering methods detected several subpopulations in the Mediterranean, a result also supported by multivariate analyses. In addition, we identified significant correlations of genetic diversity with mean salinity and surface temperature values revealing that ABFT is genetically structured along two environmental gradients. These results suggest that a preference for some spawning habitat conditions could contribute to shape ABFT genetic structuring in the Mediterranean. However, further studies should be performed to assess to what extent ABFT spawning behaviour in the Mediterranean Sea can be affected by environmental variation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Leptocephali are the characteristic larvae of the superorder Elopomorpha that are difficult to identify at the species level. In this study, we used DNA barcoding (i.e. short genetic sequences of DNA ...used as unique species tags) coupled with classical taxonomic methods to identify leptocephali in the southern Adriatic Sea. This information will provide an assessment of the biodiversity of the eel larvae in this region. A total of 2,785 leptocephali were collected, and using external morphology were assigned to seven morphotypes: Ariosoma balearicum, Conger conger, Gnathophis mystax, Facciolella sp., Nettastoma melanurum, Dalophis imberbis and Chlopsis bicolor. Collectively, these seven morphotypes are considered to be a good proxy for the Anguilliformes community (the main order of the Elopomorpha) in the southern Adriatic Sea (to date, seven families and sixteen species have been recorded in this region). Interestingly, the higher number of G. mystax larvae collected suggests an increased abundance of this genus. To validate the morphological identifications, we sequenced 61 leptocephali (at a 655 bp fragment from the cytochrome oxidase subunit 1 mitochondrial region) and developed barcode vouchers for the seven morphotypes. Using genetic information from reference databases, we validated three of these morphotypes. Where reference sequences were unavailable, we generated barcodes for both adult and juvenile forms to provide additional genetic information. Using this integrated approach allowed us to characterize a new species of Facciolella in the Adriatic Sea for the first time. Moreover, we also revealed a lack of differentiation, at the species level, between G. mistax and G. bathytopos, a western Atlantic Ocean species. Our morphological and barcode data have been published in the Barcoding of the Adriatic Leptocephali database. This work represents the first contribution to a wider project that aims to create a barcode database to support the assessment of leptocephali diversity in the Mediterranean Sea.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
European hake (Merluccius merluccius) is one of the most economically important fish for the Mediterranean Sea. It is an important predator of deep upper shelf slope communities currently ...characterized by growth overexploitation: the understanding of hake's diet might support next generation management tools. However, all current European hake diet studies depend on the morphological identification of prey remains in stomach content, with consequent limitations. In this study, we set up a metabarcoding approach based on cytochrome oxidase I PCR amplification and Miseq Illumina paired‐end sequencing of M. merluccius stomach content remains and compared the results to classic morphological analyses. A total of 95 stomach contents of M. merluccius sampled in the North‐Central Adriatic Sea were analyzed with both the metabarcoding and morphological approaches. Metabarcoding clearly outperformed the morphological method in the taxonomic identification of prey describing more complex trophic relationships even when considering the morphological identification of 200 stomach contents. Statistical analysis of diet composition revealed a weak differentiation among the hake's size classes, confirming an opportunistic feeding behavior. All the analyses performed showed the presence of a core of shared prey among the size classes and a cloud of size‐specific prey. Our study highlights the exceptional potential of metabarcoding as an approach to provide unprecedented taxonomic resolution in the diet of M. merluccius and potentially of other marine predators, due to the broad‐spectrum of detection of the primers used. A thorough description of these complex trophic relationships is fundamental for the implementation of an ecosystem approach to fisheries.
This study describes the experimental setup and assessment of a metabarcoding method for diet analysis, which was used to improve the knowledge of the trophic relationships of one of the most economically important fish for the Mediterranean Sea: Merluccius merluccius.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of ...environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99–100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.
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•Metabarcoding needs discriminating information in sequences and database coverage.•Requirements were tested by analyzing 16S-18S rRNA genes in 59 Alpine water bodies.•A high % of sequences and species is not included in the molecular databases.•The extent of sequence and taxonomic gaps differed in the major taxonomic groups.•Incomplete coverage of taxonomic databases hinders the application of metabarcoding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•16 EST-SSR loci in 177 Thunnus thynnnus specimens from the Mediterranean were genotyped.•The genetic structure results indicated a lack of population structuring.•The analysis of FST outliers did ...not reveal any pattern of selective pressure.•The results suggest the presence of conserved regions flanking these microsatellites.
The Atlantic Bluefin tuna (BFT, Thunnus thynnus), one of the largest top-predator fish inhabiting the pelagic ecosystems of the North Atlantic Ocean and Mediterranean Sea, has been extensively overexploited in recent decades. However, in the Mediterranean Sea, the mixing rates between the eastern, central and western basins have not yet been fully and finally resolved. To date electronic tagging, otolith and genetic markers cannot still disentangle the pattern of tuna movements and population structuring in the basin, essential background for a proper management of BFT fisheries. Here, we used Expressed Sequence Tag-linked (EST-linked) microsatellites to explore population dynamics and adaptive evolution of Mediterranean T. thynnus. For this purpose, 16 EST-linked microsatellites were genotyped in 177 tuna individuals from the Mediterranean Sea and several methods were used to explore population genetic structuring and estimate/detect signals of local adaptation. Bayesian clustering results indicated the presence of a single cluster, corroborated also by the Correspondence Analysis and pairwise FSTs. Similarly, the two methods used for the detection of outlier loci (LOSITAN and BayeScan), did not reveal any pattern suggesting the presence of selective pressure on the EST Simple Sequence Repeat (SSR) used. Our results suggest that the low level of polymorphism detected in this study could be ascribed to the presence of relatively conserved regions flanking these microsatellites. These genomic regions are probably not involved in physiological responses to local adaptation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Highlights ► Reverse vaccinology has been used to select vaccine candidates for the treatment of fish pasteurellosis. ► A new gene of PDP for a surface-exposed lipoprotein has been identified as a ...vaccine candidate. ► The lipoprotein PDP_0080 is involved in the adherence of the bacterium. ► Immunization with PDP_0080 recombinant antigen conferred protection against PDP challenge. ► PDP_0080 can be a promising vaccine candidate against infection by PDP in sea bass.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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