Identification of Single-Nucleotide Polymorphisms in Differentially Expressed Genes Favoring Soybean Meal Tolerance in Higher-Growth Zebrafish (Danio rerio).

Genetic variability within the same fish species could confer soybean meal (SBM) tolerance in some individuals, thus favoring growth. This study investigates the single-nucleotide polymorphisms (SNPs) in differentially expressed genes (DEGs) favoring SBM tolerance in higher-growth zebrafish (Danio rerio). In a previous work, nineteen families of zebrafish were fed a fish meal diet (100FM control diet) or SBM-based diets supplemented with saponin (50SBM + 2SPN-experimental diet), from juvenile to adult stages. Individuals were selected from families with a genotype-by-environment interaction higher (170 ± 18 mg) or lower (76 ± 10 mg) weight gain on 50SBM + 2SPN in relation to 100FM. Intestinal transcriptomic analysis using RNA-seq revealed six hundred and sixty-five differentially expressed genes in higher-growth fish fed 50SBM + 2SPN diet. In this work, using these results, 47 SNPs in DEGs were selected. These SNPs were genotyped by Sequenom in 340 zebrafish that were fed with a 50SBM + 2SPN diet or with 100FM diet. Marker-trait analysis revealed 4 SNPs associated with growth in 3 immunity-related genes (aif1l, arid3c, and cst14b.2) in response to the 50SBM + 2SPN diet (p-value < 0.05). Two SNPs belonging to aif1l y arid3c produce a positive (+19 mg) and negative (-26 mg) effect on fish growth, respectively. These SNPs can be used as markers to improve the early selection of tolerant fish to SBM diet or other plant-based diets. These genes can be used as biomarkers to identify SNPs in commercial fish, thus contributing to the aquaculture sustainability.

SNP genotyping revealed a hybrid zone between Mytilus chilensis and M. platensis in southern South America (the Strait of Magellan, Isla Grande de Tierra del Fuego and the Falkland Islands).

Hybrid zones among mussel species have been extensively studied in the northern hemisphere. In South America, it has only recently become possible to study the natural hybrid zones, due to the clarification of the taxonomy of native mussels of the Mytilus genus. Analysing 54 SNP markers, we show the genetic species composition and admixture in the hybrid zone between M. chilensis and M. platensis in the southern end of South America. Bayesian, non-Bayesian clustering and re-assignment algorithms showed that the natural hybrid zone between M. chilensis and M. platensis in the Strait of Magellan, Isla Grande de Tierra del Fuego and the Falkland Islands shows clinal architecture. The hybrid zone can be divided into three different areas: the first one is on the Atlantic coast where only pure M. platensis and hybrid were found. In the second one, inside the Strait of Magellan, pure individuals of both species and mussels with variable degrees of hybridisation coexist. In the last area at the Strait in front of Punta Arenas City, fjords on the Isla Grande de Tierra del Fuego, and at the Beagle Channel, only M. chilensis and a low number of hybrids were found. According to the proportion of hybrids, bays with protected conditions away from strong currents would give better conditions for hybridisation. We do not find evidence of any other mussel species such as M. edulis, M. galloprovincialis, M. planulatus or M. trossulus in the zone.

Comparison of two commercial methods for smooth-shelled mussels (Mytilus spp.) species identification.

Seafood international trade has increased the labeling requirements in standards and regulations to include product information that enable traders and consumers to make informed choices. The European Union (EU) Regulation No. 1379/2013 imposes the declaration of an official commercial designation and scientific names for all the fishery and aquaculture products to be offered for sale to the final consumers. DNA analyses are used to enforce this regulation and to test authenticity in processed foods. We compared the performance of two mono-locus approaches for species identification (SI) in 61 Mytilus mussels: the high-resolution melting analysis of the polyphenolic adhesive protein gene and the partial sequencing of the histone H1C gene. The H1C sequences were analyzed with five different methods. Both approaches show discrepancies in the identification of putative hybrids (0.0 < κ < 0.687 and 0.0 < MCC < 0.724). Excluding putative hybrids, methods show substantial to perfect agreement (0.772 < κ < 1.0 and 0.783 < MCC < 1.0). This study highlights the need to use standardized molecular tools, as well as to use multi-locus methods for SI of Mytilus mussels in testing laboratories.

Development and Validation of a Multi-Locus PCR-HRM Method for Species Identification in Mytilus Genus with Food Authenticity Purposes.

DNA-based methods using informative markers such as single nucleotide polymorphism (SNPs) are suitable for reliable species identification (SI) needed to enforce compliance with seafood labelling regulations (EU No.1379/2013). We developed a panel of 10 highly informative SNPs to be genotyped by PCR-High resolution melting (HRM) for SI in the Mytilus genus through in silico and in vitro stages. Its fitness for purpose and concordance were assessed by an internal validation process and by the transference to a second laboratory. The method was applicable to identify M. chilensis, M. edulis, M. galloprovincialis and M. trossulus mussels, fresh, frozen and canned with brine, oil and scallop sauce, but not in preserves containing acetic acid (wine vinegar) and tomato sauce. False-positive and negative rates were zero. Sensitivity, expressed as limit of detection (LOD), ranged between 5 and 8 ng/µL. The method was robust against small variations in DNA quality, annealing time and temperature, primer concentration, reaction volume and HRM kit. Reference materials and 220 samples were tested in an inter-laboratory assay obtaining an "almost perfect agreement" (κ = 0.925, p < 0.001). In conclusion, the method was suitable for the intended use and to be applied in the seafood industry.

Intestinal Transcriptome Analysis Reveals Enrichment of Genes Associated with Immune and Lipid Mechanisms, Favoring Soybean Meal Tolerance in High-Growth Zebrafish (Danio Rerio).

The molecular mechanisms underlying fish tolerance to soybean meal (SBM) remain unclear. Identifying these mechanisms would be beneficial, as this trait favors growth. Two fish replicates from 19 experimental families were fed fishmeal-(100FM) or SBM-based diets supplemented with saponin (50SBM + 2SPN) from juvenile to adult stages. Individuals were selected from families with a genotype-by-environment interaction higher (HG-50SBM + 2SPN, 170 ± 18 mg) or lower (LG-50SBM + 2SPN, 76 ± 10 mg) weight gain on 50SBM + 2SPN for intestinal transcriptomic analysis. A histological evaluation confirmed middle intestinal inflammation in the LG- vs. HG-50SBM + 2SPN group. Enrichment analysis of 665 differentially expressed genes (DEGs) identified pathways associated with immunity and lipid metabolism. Genes linked to intestinal immunity were downregulated in HG fish (mpx, cxcr3.2, cftr, irg1l, itln2, sgk1, nup61l, il22), likely dampening inflammatory responses. Conversely, genes involved in retinol signaling were upregulated (rbp4, stra6, nr2f5), potentially favoring growth by suppressing insulin responses. Genes associated with lipid metabolism were upregulated, including key components of the SREBP (mbtps1, elov5l, elov6l) and cholesterol catabolism (cyp46a1), as well as the downregulation of cyp7a1. These results strongly suggest that transcriptomic changes in lipid metabolism mediate SBM tolerance. Genotypic variations in DEGs may become biomarkers for improving early selection of fish tolerant to SMB or others plant-based diets.

High resolution melting analysis for identification of commercially-important Mytilus species.

Mytilus are edible mussels, including commercially-significant species such as M. chilensis, M. galloprovincialis and M. edulis. The scientific name of the species must be indicated on commercial products to satisfy labelling and traceability requirements. Species identification using morphological criteria is difficult due the plasticity of these characteristics and the absence of shells in processed products, and conventional PCR-based methods are laborious and time-intensive. As alternative, we propose high resolution melting (HRM) analysis as a simple tool to detect and identify SNP (single nucleotide polymorphisms) and length polymorphisms in Mytilus spp. We designed HRM-specific primers for the Mytilus genus to identify M. chilensis, M. galloprovincialis, M. edulis and their hybrids through clearly-distinguishable melting curves. HRM analysis showed high sensitivity (0.9639), specificity (1.0000) and precision (1.0000) compared to a conventional PCR-RFLP test. HRM is a fast and low cost method, being a reliable tool for species identification within the Mytilus genus.