Novel tools for conservation genetics in marine fish: population structure and evolution of the European hake (Merluccius merluccius) inferred by SNP variation and applications to traceability

The research presented in my PhD thesis is part of a wider European project, FishPopTrace, focused on traceability of fish populations and products. My work was aimed at developing and analyzing novel genetic tools for a widely distributed marine fish species, the European hake (Merluccius merluccius), in order to investigate population genetic structure and explore potential applications to traceability scenarios. A total of 395 SNPs (Single Nucleotide Polymorphisms) were discovered from a massive collection of Expressed Sequence Tags, obtained by high-throughput sequencing, and validated on 19 geographic samples from Atlantic and Mediterranean. Genome-scan approaches were applied to identify polymorphisms on genes potentially under divergent selection (outlier SNPs), showing higher genetic differentiation among populations respect to the average observed across loci. Comparative analysis on population structure were carried out on putative neutral and outlier loci at wide (Atlantic and Mediterranean samples) and regional (samples within each basin) spatial scales, to disentangle the effects of demographic and adaptive evolutionary forces on European hake populations genetic structure. Results demonstrated the potential of outlier loci to unveil fine scale genetic structure, possibly identifying locally adapted populations, despite the weak signal showed from putative neutral SNPs. The application of outlier SNPs within the framework of fishery resources management was also explored. A minimum panel of SNP markers showing maximum discriminatory power was selected and applied to a traceability scenario aiming at identifying the basin (and hence the stock) of origin, Atlantic or Mediterranean, of individual fish. This case study illustrates how molecular analytical technologies have operational potential in real-world contexts, and more specifically, potential to support fisheries control and enforcement and fish and fish product traceability.

Fish quality assessment through the application of chemico-physical, sensory and microbiological analyses

The quality of fish products is indispensably linked to the freshness of the raw material modulated by appropriate manipulation and storage conditions, specially the storage temperature after catch. The purpose of the research presented in this thesis, which was largely conducted in the context of a research project funded by Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF), concerned the evaluation of the freshness of farmed and wild fish species, in relation to different storage conditions, under ice (0°C) or at refrigeration temperature (4°C). Several specimens of different species, bogue (Boops boops), red mullet (Mullus barbatus), sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), during storage, under the different temperature conditions adopted, have been examined. The assessed control parameters were physical (texture, through the use of a dynamometer; visual quality using a computer vision system (CVS)), chemical (through footprint metabolomics 1H-NMR) and sensory (Quality Index Method (QIM). Microbiological determinations were also carried out on the species of hake (Merluccius merluccius). In general obtained results confirmed that the temperature of manipulation/conservation is a key factor in maintaining fish freshness. NMR spectroscopy showed to be able to quantify and evaluate the kinetics for unselected compounds during fish degradation, even a posteriori. This can be suitable for the development of new parameters related to quality and freshness. The development of physical methods, particularly the image analysis performed by computer vision system (CVS), for the evaluation of fish degradation, is very promising. Among CVS parameters, skin colour, presence and distribution of gill mucus, and eye shape modification evidenced a high sensibility for the estimation of fish quality loss, as a function of the adopted storage conditions. Particularly the eye concavity index detected on fish eye showed a high positive correlation with total QIM score.

Demersal communities in the Mediterranean Sea: a case study of Triglidae (Osteichthyes, Scorpaeniformes) on the conservation and sustainable use of marine resources

An appropriate management of fisheries resources can only be achieved with the continuous supply of information on the structure and biology of populations, in order to predict the temporal fluctuations. This study supports the importance of investigating the bio-ecology of increasingly exploited and poorly known species, such as gurnards (Osteichthyes, Triglidae) from Adriatic Sea (Mediterranean), to quantify their ecological role into marine community. It also focuses on investigate inter and intra-specific structuring factor of Adriatic population. These objectives were achieved by: 1) investigating aspects of the population dynamics; 2) studying the feeding biology through the examination of stomach contents; 3) using sagittal otoliths as potential marker of species life cycle; 4) getting preliminary data on mDNA phylogeny. Gurnards showed a specie-specific “critical size” coinciding with the start of sexual maturity, the tendency to migrate to greater depths, a change of diet from crustaceans to fish and an increase of variety of food items eaten. Distribution of prey items, predator size range and depth distribution were the main dimensions that influence the breadth of trophic niche and the relative difference amongst Adriatic gurnards. Several feeding preferences were individuated and a possible impact among bigger-size gurnards and other commercial fishes (anchovy, gadoids) and Crustacea (such as mantis prawn and shrimps) were to be necessary considered. Otolith studies showed that gurnard species have a very fast growth despite other results in other areas; intra-specific differences and the increase in the variability of otolith shape, sulcus acusticus shape, S:O ratios, sulcus acusticus external crystals arrangement were shown between juveniles and adults and were linked to growth (individual genetic factors) and to environmental conditions (e.g. depth and trophic niche distribution). In order to facilitate correct biological interpretation of data, molecular data were obtained for comparing morphological distance to genetic ones.

The environmental DNA in the risk assessment and decision making processes for the invasive species management in agri-food sector, hydraulic security and biodiversity conservation

The use of environmental DNA (eDNA) analysis as a monitoring tool is becoming more and more widespread. The eDNA metabarcoding methods allow rapid community assessments of different target taxa. This work is focused on the validation of the environmental DNA metabarcoding protocol for biodiversity assessment of freshwater habitats. Scolo Dosolo was chosen as study area and three sampling points were defined for traditional and eDNA analyses. The gutter is a 205 m long anthropic canal located in Sala Bolognese (Bologna, Italy). Fish community and freshwater invertebrate metazoans were the target groups for the analysis. After a preliminary study in summer 2019, 2020 was devoted to the sampling campaign with winter (January), spring (May), summer (July) and autumn (October) surveys. Alongside with the water samplings for the eDNA study, also traditional fish surveys using the electrofishing technique were performed to assess fish community composition; census on invertebrates was performed using an entomological net and a surber sampler. After in silico analysis, the MiFish primer set amplifying a fragment of the 12s rRNA gene was selected for bony fishes. For invertebrates the FWHF2 + FWHR2N primer combination, that amplifies a region of the mitochondrial coi gene, was chosen. Raw reads were analyzed through a bioinformatic pipeline based on OBITools metabarcoding programs package and QIIME2. The OBITools pipeline retrieved seven fish taxa and 54 invertebrate taxa belonging to six different phyla, while QIIME2 recovered eight fish taxa and 45 invertebrate taxa belonging to the same six phyla as the OBITools pipeline. The metabarcoding results were then compared with the traditional surveys data and bibliographic records. Overall, the validated protocol provides a reliable picture of the biodiversity of the study area and an efficient support to the traditional methods.