Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.).

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.

Genetic monitoring on the world's first MSC eco-labeled common octopus (O. vulgaris) fishery in western Asturias, Spain.

Octopus vulgaris (Cuvier, 1797) is a cephalopod species with great economic value. In western Asturias (northwest of Spain), O. vulgaris artisanal fisheries are relatively well monitored and conditionally eco-labeled by the Marine Stewardship Council (MSC). Despite this, the Asturian octopus stocks have not been genetically assessed so far. In order to improve the current fishery plan and contrast the octopus eco-label validity in Asturias, 539 individuals from five regions of the O. vulgaris geographic distribution, including temporal samplings in Asturias, were collected and genotyped at thirteen microsatellite loci. All the samples under analysis were in agreement with Hardy-Weinberg expectations. Spatial levels of genetic differentiation were estimated using F-statistics, multidimensional scaling, and Bayesian analyses. Results suggested that the O. vulgaris consists of at least four genetically different stocks coming from two ancestral lineages. In addition, temporal analyses showed stability in terms of genetic variation and high NE (> 50) for several generations in different localities within Asturias, pointing out to indeed sustainable fishery exploitation levels. Even though, the current Asturias fishery plan shows no significant genetic damages to the stocks, the regional-specific management plans need systematic genetic monitoring schemes as part of an efficient and preventive regional fishery regulation strategy.

Heterozygosity-fitness correlations in the gilthead sea bream Sparus aurata using microsatellite loci from unknown and gene-rich genomic locations.

Heterozygosity-fitness correlations (HFC) were assessed for a sample of a gilthead sea bream Sparus aurata population. Two hundred and seventy-one fish were genotyped at 22 known and novel microsatellite loci, from which correlations between the multilocus heterozygosity index (I(MLH) ) and various fitness traits (fork length, mass and specific growth rates) were calculated. Significant global HFCs were found in this sample (0·02 ≤r(2) ≤ 0·08). In addition, all the significant correlations found in this work were negative, indicating that heterozygotes had lower fitness than their homozygote counterparts. Marker location could not explain the observed HFCs. Evidence of inbreeding, outbreeding or population and family structuring was not found in this work. The presence of undetected general effects that may lead to the appearance of HFCs, however, cannot be ruled out. These results seem to be best explained by the occurrence of local effects (due to linkage) or even by possible direct locus advantages.

Building on gAMBI in ports for a challenging biological invasions scenario: Blue-gNIS as a proof of concept.

The status of aquatic ecosystems has historically been monitored by the use of biotic indices. However, few biotic measures consider the presence of non-indigenous species as a sign of anthropogenic pollution and habitat disturbance even when this may seriously affect the metric scores and ecological status classifications of an environment. Today, biological invasions are currently one of the greatest threats to biodiversity and sustainable blue economies around the world. In this work, environmental assessments were conducted in the Port of Gijon, Northern Spain, using eDNA metabarcoding, and the gAMBI (genetics based AZTI Marine Biotic Index) was estimated. Results indicate a high/good ecological status within the port. However, nine non-indigenous species and five invasive species were found, and a modification of the gAMBI that includes species invasiveness was proposed: Blue-gNIS. The index was preliminary tested against existing validated indices such as gAMBI, BENTIX (based on the ecology of macroinvertebrates) and ALEX (based on the invasiveness of the species). Blue-gNIS classified the port in a good ecological status and showed its potential usefulness to achieve more complete water quality assessments of ports.

Perspectives on the marine environment and biodiversity in recreational ports: The marina of Gijon as a case study.

Recreational ports are known to be sources of pollution to the coastal marine environment due to the pouring of pollutants or the transfer of invasive species to neighboring areas. Nonetheless, the responsibility of protecting the marine environment does not lie solely on the users of the ports, but also affects the rest of citizens. Thus, an effective communication is necessary between scientists and citizens to avoid the lack of knowledge and boost cooperation against these environmental problems. In this study, (focused on the marina of Gijon, Northwestern Spain) citizens set education and social media as the main sources of information, rarely considering science outreach. Also, their environmental knowledge showed to be based on a visual perception, rather than on a cognitive one, as marine litter was considered a great environmental problem, while invasive species and biofouling went unnoticed, remarking the lack of an effective communication from scientific sources.

Food control and a citizen science approach for improving teaching of Genetics in universities.

A Citizen Science approach was implemented in the laboratory practices of Genetics at the University of Oviedo, related with the engaging topic of Food Control. Real samples of food products consumed by students at home (students as samplers) were employed as teaching material in three different courses of Genetics during the academic year 2014-2015: Experimental Methods in Food Production (MBTA) (Master level), and Applied Molecular Biology (BMA) and Conservation Genetics and Breeding (COMGE) (Bachelor/Degree level). Molecular genetics based on PCR amplification of DNA markers was employed for species identification of 22 seafood products in COMGE and MBTA, and for detection of genetically modified (GM) maize from nine products in BMA. In total six seafood products incorrectly labeled (27%), and two undeclared GM maize (22%) were found. A post-Laboratory survey was applied for assessing the efficacy of the approach for improving motivation in the Laboratory Practices of Genetics. Results confirmed that students that worked on their own samples from local markets were significantly more motivated and better evaluated their Genetic laboratory practices than control students (χ(2) = 12.11 p = 0.033). Our results suggest that citizen science approaches could not be only useful for improving teaching of Genetics in universities but also to incorporate students and citizens as active agents in food control. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(5):450-462, 2016.

VY6, a ß-lactoglobulin-derived peptide, altered metabolic lipid pathways in the zebra fish liver.

Today enormous research efforts are being focused on alleviating the massive, adverse effects of obesity. Short peptides are key targets for research as they can be generated from natural proteins, like milk. Here we conducted trypsinogen digestion of beta-lactoglobulin (ß-lg), the major mammalian milk protein, to release the hexamer VY6. It was assayed in vivo for its activities on lipid metabolism using zebra fish as a vertebrate model. Zebra fish juveniles were injected with two different doses of the peptide: 100 and 800 µg per g fish and left for 5 days before sacrificing. Lipid measurements showed significant reduction in liver triglycerides and free cholesterol, as well as increased liver HDL cholesterol. Dose-dependent increases of the mRNA levels of the genes coding for the enzymes acyl coenzyme A oxidase 1 (acox1) and lipoprotein lipase (lpl) were also found. The complete results suggest significant anti-obesity activity of the ß-lg-derived VY6 peptide. Its use as a nutraceutical has been discussed.

Barcodes of marine invertebrates from north Iberian ports: Native diversity and resistance to biological invasions.

Ports are gateways for many marine organisms transported by ships worldwide, especially non-indigenous species (NIS). In this study carried out in North Iberian ports (Cantabrian Sea, Bay of Biscay) we have observed 38% of exotic macroinvertebrates. Four species, namely the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus, the Pacific oyster Crassostrea gigas and the pygmy mussel Xenostrobus securis, exhibited clear signs of invasiveness. A total of 671 barcode (cytochrome oxidase subunit I or 18S rRNA) genes were obtained and confirmed the species status of some cryptic NIS. Negative and significant correlation between diversity estimators of native biota and proportion of NIS suggests biotic resistance in ports. This could be applied to management of port biota for contributing to prevent the settlement of biopollutants in these areas which are very sensitive to biological invasions.

Response of top shell assemblages to cyclogenesis disturbances. A case study in the Bay of Biscay.

Cyclones and other climate disturbances profoundly affect coastal ecosystems, promoting changes in the benthic communities that require time, sometimes even years, for a complete recovery. In this study we have analysed the morphological and genetic changes occurred in top shell (Gibbula umbilicalis and Phorcus lineatus) assemblages from the Bay of Biscay following explosive cyclogenesis events in 2014. Comparison with previous samples at short (three years before the cyclogenesis) and long (Upper Pleistocene) temporal scales served to better evaluate the extent of change induced by these disturbances in a more global dimension. A significant increase in mean size after the cyclogenesis was found for the two species, suggesting selective sweeping of small individuals weakly adhered to substrata. Loss of haplotype variants at the cytochrome oxidase subunit I gene suggests a population bottleneck, although it was not intense enough to produce significant changes in haplotype frequencies. The high population connectivity and metapopulation structuring of the two species in the area likely help the populations to recover from disturbances. At a wider temporal scale, cyclogenesis effects seemed to compensate the apparent decreasing trends in size for P. lineatus occurred after the Pleistocene-Holocene transition. Considering disturbance regimes for population baselines is recommended when the long-term effects of climate and anthropogenic pressures are evaluated.

Timing of first feeding and life-history strategies in salmon: genetic data.

To investigate the relationship between genetic composition and some physiological traits of interest (age at smoltification, precocious maturation in male parr) in early and late first feeding Atlantic salmon, genetic variation at 6 isozyme and 8 microsatellite loci was examined. Early and late first feeding salmon showed different allelic and genotypic distributions at both isozyme and microsatellite loci. A positive relation between enzymatic loci heterozygosity and precocious active alimentation and earlier smoltification (S1 cf. S2) were also found.

Correlations between fitness and heterozygosity at allozyme and microsatellite loci in the Atlantic salmon, Salmo salar L.

The relationship between heterozygosity at genetic markers (six allozyme and eight microsatellite loci), and fluctuating asymmetry (FA), length and weight was investigated in two samples of Atlantic salmon (Salmo salar L.) with different timings of first active feeding (early (EA) and late (LA) salmon). This trait had previously been related to fitness. EA fish show smaller values of FA, are longer, heavier and are more heterozygous at allozyme loci than are conspecific LA fish. Also within both samples, heterozygosity at allozyme loci was inversely related to FA and was positively related to weight and length. However, no significant differences in microsatellite diversity (heterozygosity and mean d2 measurements) were observed between samples (EA vs LA). Furthermore, no association was observed between the variability at microsatellite loci and FA, weight or length within each sample. These results suggest that allozyme loci, in themselves, influence fitness components, rather than associations arising from associative overdominance.