DNA metabarcoding suggests dietary niche partitioning in the Adriatic European hake.

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.

Unite research with what citizens do for fun: "recreational monitoring" of marine biodiversity.

Institutes often lack funds and manpower to perform large-scale biodiversity monitoring. Citizens can be involved, contributing to the collection of data, thus decreasing costs. Underwater research requires specialist skills and SCUBA certification, and it can be difficult to involve volunteers. The aim of this study was to involve large numbers of recreational divers in marine biodiversity monitoring for increasing the environmental education of the public and collecting data on the status of marine biodiversity. Here we show that thousands of recreational divers can be enrolled in a short time. Using specially formulated questionnaires, nonspecialist volunteers reported the presence of 61 marine taxa encountered during recreational dives, performed as regular sport dives. Validation trials were carried out to assess the accuracy and consistency of volunteer-recorded data, and these were compared to reference data collected by an experienced researcher. In the majority of trials (76%) volunteers performed with an accuracy and consistency of 50-80%, comparable to the performance of conservation volunteer divers on precise transects in other projects. The recruitment of recreational divers involved the main diving and tour operators in Italy, a popular scientific magazine, and mass media. During the four-year study, 3825 divers completed 18757 questionnaires, corresponding to 13539 diving hours. The volunteer-sightings-based index showed that in the monitored area the biodiversity status did not change significantly within the project time scale, but there was a significant negative correlation with latitude, suggesting improved quality in the southernmost areas. This trend could be related to the presence of stressors in the northern areas and has been supported by investigations performed by the Italian Ministry of the Environment. The greatest limitation with using volunteers to collect data was the uneven spatial distribution of samples. The benefits were the considerable amounts of data collected over short time periods and at low costs. The successful development of citizen-based monitoring programs requires open-mindedness in the academic community; advantages of citizen involvement in research are not only adding large data sets to the ecological knowledge base but also aiding in the environmental education of the public.

Environmental influence on calcification of the bivalve Chamelea gallina along a latitudinal gradient in the Adriatic Sea.

Environmental factors are encoded in shells of marine bivalves in the form of geochemical properties, shell microstructure and shell growth rate. Few studies have investigated how shell growth is affected by habitat conditions in natural populations of the commercial clam Chamelea gallina. Here, skeletal parameters (micro-density and apparent porosity) and growth parameters (bulk density, linear extension and net calcification rates) were investigated in relation to shell sizes and environmental parameters along a latitudinal gradient in the Adriatic Sea (400 km). Net calcification rates increased with increasing solar radiation, sea surface temperature and salinity and decreasing Chlorophyll concentration in immature and mature shells. In immature shells, which are generally more porous than mature shells, enhanced calcification was due to an increase in bulk density, while in mature shells was due to an increase in linear extension rates. The presence of the Po river in the Northern Adriatic Sea was likely the main driver of the fluctuations observed in environmental parameters, especially salinity and Chlorophyll concentration, and seemed to negatively affect the growth of C. gallina.

A metabarcoding approach for the feeding habits of European hake in the Adriatic Sea.

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.

Morphology and Species Composition of Southern Adriatic Sea Leptocephali Evaluated Using DNA Barcoding.

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.

Shell properties of commercial clam Chamelea gallina are influenced by temperature and solar radiation along a wide latitudinal gradient.

Phenotype can express different morphologies in response to biotic or abiotic environmental influences. Mollusks are particularly sensitive to different environmental parameters, showing macroscale shell morphology variations in response to environmental parameters. Few studies concern shell variations at the different scale levels along environmental gradients. Here, we investigate shell features at the macro, micro and nanoscale, in populations of the commercially important clam Chamelea gallina along a latitudinal gradient (~400 km) of temperature and solar radiation in the Adriatic Sea (Italian cost). Six populations of clams with shells of the same length were analyzed. Shells from the warmest and the most irradiated population were thinner, with more oval shape, more porous and lighter, showing lower load fracture. However, no variation was observed in shell CaCO3 polymorphism (100% aragonite) or in compositional and textural shell parameters, indicating no effect of the environmental parameters on the basic processes of biomineralization. Because of the importance of this species as commercial resource in the Adriatic Sea, the experimentally quantified and significant variations of mass and fracture load in C. gallina shells along the latitudinal gradient may have economic implications for fisheries producing different economical yield for fishermen and consumers along the Adriatic coastline.

Participating in a Citizen Science Monitoring Program: Implications for Environmental Education.

Tourism is of growing economical importance to many nations, in particular for developing countries. Although tourism is an important economic vehicle for the host country, its continued growth has led to on-going concerns about its environmental sustainability. Coastal and marine tourism can directly affect the environment through direct and indirect tourist activities. For these reasons tourism sector needs practical actions of sustainability. Several studies have shown how education minimizes the impact on and is proactive for, preserving the natural resources. This paper evaluates the effectiveness of a citizen science program to improve the environmental education of the volunteers, by means of questionnaires provided to participants to a volunteer-based Red Sea coral reef monitoring program (STEproject). Fifteen multiple-choice questions evaluated the level of knowledge on the basic coral reef biology and ecology and the awareness on the impact of human behaviour on the environment. Volunteers filled in questionnaires twice, once at the beginning, before being involved in the project and again at the end of their stay, after several days participation in the program. We found that the participation in STEproject significantly increased both the knowledge of coral reef biology and ecology and the awareness of human behavioural impacts on the environment, but was more effective on the former. We also detected that tourists with a higher education level have a higher initial level of environmental education than less educated people and that the project was more effective on divers than snorkelers. This study has emphasized that citizen science projects have an important and effective educational value and has suggested that tourism and diving stakeholders should increase their commitment and efforts to these programs.

First microsatellite loci of red mullet (Mullus barbatus) and their application to genetic structure analysis of Adriatic shared stock.

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.

Mitochondrial DNA sequence variation suggests the lack of genetic heterogeneity in the Adriatic and Ionian stocks of Sardina pilchardus.

A genetic stock structure analysis of 11 sardine samples from the Adriatic Sea and Ionian neighboring area was carried out through sequence variation analysis of a 307-bp cytochrome b gene fragment in order to identify self-recruiting units in the Adriatic Sardina pilchardus stock. The overall lack of genetic subdivision among samples detected by analysis of molecular variance, pairwise Phi(st) values, and the exact test of population differentiation indicates this sardine stock is part of a larger self-recruiting population whose boundaries are larger than the investigated area. This conclusion is in agreement with preliminary allozymic and mitochondrial DNA restriction fragment length polymorphism data, but contradicts the previous identification of 2 subpopulations of sardines in the Adriatic Sea argued on morphologic differences, which could be rather attributed to different hydrographic or ecologic conditions occurring in different areas of the Adriatic Sea. The reduced gene flow observed between Adriatic-Ionian and Spanish sardine geographic samples (P < 0.001) suggests that reproductively isolated populations of sardines may occur in the Mediterranean Sea.

Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

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.

Tsunami survey expedition: preliminary investigation of Maldivian coral reefs two weeks after the event.

On December 26th 2004, a earthquake west of Sumatra generated a devastating tsunami. Hundreds of thousands of people fell victim. Economic losses were greatest in those countries dependant on tourism. The impact in the Maldives on persons and things was modest. Immediately following the event and notwithstanding the lack of scientific data, the mass media gave catastrophic reports on the state of coral reefs in the area. This paper reports on the first survey on coral reefs in the Maldives after the Tsunami. Ocean walls, passes, inner reefs, and shoals in the North and South Malé atolls, were surveyed two weeks after the event. Significant damage was recorded in the passes in the South Malé atoll. Our observations showed that the damage was more or less extensive depending on latitude and topography. Sri Lanka may have broken the wave's rush, reducing the extent of the impact on northern atolls. The water's acceleration inside the passes was so intense as to cause reef collapses. The observed damage represents a minimum fraction of the entire coral reef system. Tourist perception of the area seems unchanged. These data may be used to disseminate correct information about the state of Maldives coral reefs, which would be useful in relaunching local economy.