MassArray Genotyping as a Selection Tool for Extending the Shelf-Life of Fresh Gilthead Sea Bream and European Seabass.

In modern aquaculture, genomics-driven breeding programs have emerged as powerful tools for optimizing fish quality. This study focused on two emblematic Mediterranean fish species, the European seabass (Dicentrarchus labrax) and the gilthead sea bream (Sparus aurata), with a primary aim of exploring the genetic basis of white muscle/fillet degradation in fresh fish following harvest. We identified 57 and 44 missense SNPs in gilthead sea bream and European seabass, respectively, located within genes encoding for endogenous proteases responsible for fillet quality. These SNPs were cherry-picked based on their strategic location within the catalytic/regulatory domains of endogenous proteases that are expressed in the white muscle. Using MassArray technology, we successfully associated differentiated enzymatic activity of those endogenous proteases post-harvest as a phenotypic trait with genetic polymorphism of six SNPs in gilthead sea bream and nine in European seabass. These findings can be valuable attributes in selective breeding programs toward the extension of freshness and shelf life of these species. The integration of MassArray technology into breeding programs offers a cost-effective strategy for harnessing the potential of these genetic variants to enhance the overall quality of the final product. Recognizing that fresh fish perishability is a challenge, extending shelf-life is pivotal in reducing losses and production costs.

Core microbiome profiles and their modification by environmental, biological, and rearing factors in aquaculture hatcheries.

16S rRNA gene sequencing and bacteria- and genus-specific quantitative PCR was used to profile microbial communities and their associated functions in water, live feed (microalgae, Artemia, and rotifer), and European sea bass and gilthead sea bream larvae from hatcheries in Greece and Italy. The transfer to larvae of genus containing potential pathogens of fish was more likely with Artemia and rotifer than with microalgae or water, irrespective of geographic location. The presence of potentially pathogenic bacteria (Vibrio and Pseudoalteromonas) in the core microbiota of water, live feed, and fish larvae, the enrichment of different bacterial resistance pathways and biofilm formation, and the overall low beneficial bacteria load during larval ontogeny emphasizes the risk for disease outbreaks. The present data characterizing microbiota in commercial aquaculture hatcheries provides a baseline for the design of strategies to manage disease and to model or remediate potential adverse environmental impacts.

Investigating the Impact of a Curse: Diseases, Population Isolation, Evolution and the Mother's Curse.

The mitochondrion was characterized for years as the energy factory of the cell, but now its role in many more cellular processes is recognized. The mitochondrion and mitochondrial DNA (mtDNA) also possess a set of distinct properties, including maternal inheritance, that creates the Mother's Curse phenomenon. As mtDNA is inherited from females to all offspring, mutations that are harmful to males tend to accumulate more easily. The Mother's Curse is associated with various diseases, and has a significant effect on males, in many cases even affecting their reproductive ability. Sometimes, it even leads to reproductive isolation, as in crosses between different populations, the mitochondrial genome cannot cooperate effectively with the nuclear one resulting in a mito-nuclear incompatibility and reduce the fitness of the hybrids. This phenomenon is observed both in the laboratory and in natural populations, and have the potential to influence their evolution and speciation. Therefore, it turns out that the study of mitochondria is an exciting field that finds many applications, including pest control, and it can shed light on the molecular mechanism of several diseases, improving successful diagnosis and therapeutics. Finally, mito-nuclear co-adaptation, paternal leakage, and kin selection are some mechanisms that can mitigate the impact of the Mother's Curse.

Variability of key-performance-indicators in commercial gilthead seabream hatcheries.

Skeletal abnormalities are one of the most important key-performance-indicators (KPIs) in finfish hatcheries. Coping with the problem of skeletal abnormalities relies on the understanding of the link between the variability in the rearing conditions, and the variability in abnormalities incidence. Here, 74 seabream larval populations, from four commercial hatcheries, were examined for the presence of abnormalities and monitored with respect to the applied conditions. The inward folding of gill-cover and pugheadedness were the most frequent abnormalities present, with a mean (± SD) frequency of 11.3 ± 17.9 and 6.0 ± 7.2%, respectively. Other abnormalities were observed at very low mean rates (≤ 1%). A new abnormality type, ray-resorption syndrome, was also found. The recorded rate of normally inflated swimbladder was 92.3 ± 7.4% and mean survival rate was 25.9 ± 21.0%. Classification tree analysis indicated six rearing variables as potentially important predictors for pugheadedness, six variables for caudal-fin abnormalities and 10 variables for survival rate. Complementary genetic analysis, revealed differentiating genetic diversity and significant genetic distances among participating hatcheries, suggestive of the role of company-specific management of genetic resources in KPIs' variability. The results are discussed with respect to their potential use in the control of skeletal abnormalities by commercial hatcheries, as well as for benchmarking among different hatcheries.

Oviductal epithelial cells transcriptome and extracellular vesicles characterization during thermoneutral and heat stress conditions in dairy cows.

In this study, the transcriptome of oviductal epithelial cells and certain characteristics of their extracellular vesicles of dairy cows were described under thermoneutral and heat stress conditions. Twenty cows were compared in springtime at THI = 65.6 ± 0.90 and in summertime at THI = 78.36 ± 2.73. During each season, the estrous cycles of the cows were synchronized, and on day 3 of the ensuing cycle, a blood sample was collected for progesterone determination, while their oviducts were collected after slaughter. Epithelial cells and oviductal fluid were collected from the oviduct ipsilateral and contralateral to the corpus, respectively. For the gene expression study, a comparative transcriptomic approach, using RNASeq, was performed on cells collected from the ipsilateral and the contralateral oviducts. The size and the concentration of extracellular vesicles (EVs) at both seasons were analyzed using Transmission Electron Microscopy and Nanoparticle tracking analysis and specific proteins were detected by Western blotting. Progesterone concentration was higher during the thermoneutral period. Between seasons, divergent expression of genes related to immune system, contractility, gamete protection and lncRNAs was found. The size and the concentration of the EVs did not differ between seasons, however, the concentration in the ipsilateral oviduct tended to be lower (p = 0.09) from the contralateral one in the summer, but not in the spring. Our results show for the first time that HS could be involved with alterations in the oviductal cells' gene expression and in the changes in concentration of EVs in the oviductal lumen. Our results imply that the altered oviductal environment during HS could be associated with the suppressed summer fertility in dairy cows.

Factors Driving Bacterial Microbiota of Eggs from Commercial Hatcheries of European Seabass and Gilthead Seabream.

A comprehensive understanding of how bacterial community abundance changes in fishes during their lifecycle and the role of the microbiota on health and production is still lacking. From this perspective, the egg bacterial communities of two commercially farmed species, the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata), from different aquaculture sites were compared, and the potential effect of broodstock water microbiota and disinfectants on the egg microbiota was evaluated. Moreover, 16S ribosomal RNA gene sequencing was used to profile the bacterial communities of the eggs and broodstock water from three commercial hatcheries. Proteobacteria were the most common and dominant phyla across the samples (49.7% on average). Vibrio sp. was the most highly represented genus (7.1%), followed by Glaciecola (4.8%), Pseudoalteromonas (4.4%), and Colwellia (4.2%), in eggs and water across the sites. Routinely used iodine-based disinfectants slightly reduced the eggs' bacterial load but did not significantly change their composition. Site, species, and type of sample (eggs or water) drove the microbial community structure and influenced microbiome functional profiles. The egg and seawater microbiome composition differed in abundance but shared similar functional profiles. The strong impact of site and species on egg bacterial communities indicates that disease management needs to be site-specific and highlights the need for species- and site-specific optimization of disinfection protocols.

HLA class II peptide-binding-region analysis reveals funneling of polymorphism in action.

BACKGROUND: HLA-class II proteins hold important roles in key physiological processes. The purpose of this study was to compile all class II alleles reported in human population and investigate patterns in pocket variants and their combinations, focusing on the peptide-binding region (PBR). METHODS: For this purpose, all protein sequences of DPA1, DQA1, DPB1, DQB1 and DRB1 were selected and filtered, in order to have full PBR sequences. Proportional representation was used for pocket variants while population data were also used. RESULTS: All pocket variants and PBR sequences were retrieved and analyzed based on the preference of amino acids and their properties in all pocket positions. The observed number of pocket variants combinations was much lower than the possible inferred, suggesting that PBR formation is under strict funneling. Also, although class II proteins are very polymorphic, in the majority of the reported alleles in all populations, a significantly less polymorphic pocket core was found. CONCLUSIONS: Pocket variability of five HLA class II proteins was studied revealing favorable properties of each protein. The actual PBR sequences of HLA class II proteins appear to be governed by restrictions that lead to the establishment of only a fraction of the possible combinations and the polymorphism recorded is the result of intense funneling based on function.

SNP Identification through Transcriptome Analysis of the European Brown Hare (Lepus europaeus): Cellular Energetics and Mother's Curse.

The European brown hare (Lepus europaeus, Pallas 1778) is an important small game species in Europe. Due to its size and position in the food chain, as well as its life history, phenotypic variation and the relatively recent speciation events, brown hare plays an important role in the structure of various ecosystems and has emerged as an important species for population management and evolutionary studies. In order to identify informative SNPs for such studies, heart and liver tissues of three samples from the European lineage and a three-sample pool from the Anatolian lineage were subjected to RNA-Sequencing analysis. This effort resulted in 9496 well-assembled protein-coding sequences with close homology to human. After applying very stringent filtering criteria, 66185 polymorphic sites were identified in 7665 genes/cds and 2050 of those polymorphic sites are potentially capable of distinguishing the European from the Anatolian lineage. From these distinguishing mutations we focused on those in genes that are involved in cellular energy production, namely the glycolysis, Krebs cycle and the OXPHOS machinery. A selected set of SNPs was also validated by Sanger sequencing. By simulating the three European individuals as one pool, no substantial informative-SNP identification was lost, making it a cost-efficient approach. To our knowledge this is the first attempt to correlate the differentiation in both nuclear and mitochondrial genome between the two different lineages of L. europaeus with the observed spatial partitioning of the lineages of the species, proposing a possible mechanism that is maintaining the reproductive isolation of the lineages.

Transient up- and down-regulation of expression of myosin light chain 2 and myostatin mRNA mark the changes from stratified hyperplasia to muscle fiber hypertrophy in larvae of gilthead sea bream (Sparus aurata L.).

Hyperplasia and hypertrophy are the two mechanisms by which muscle develops and grows. We study these two mechanisms, during the early development of white muscle in Sparus aurata, by means of histology and the expression of structural and regulatory genes. A clear stage of stratified hyperplasia was identified early in the development of gilthead sea bream but ceased by 35 dph when hypertrophy took over. Mosaic recruitment of new white fibers began as soon as 60 dph. The genes mlc2a and mlc2b were expressed at various levels during the main phases of hyperplasia and hypertrophy. The genes myog and mlc2a were significantly up-regulated during the intensive stratified formation of new fibers and their expression was significantly correlated. Expression of mstn1 and igf1 increased at 35 dph, appeared to regulate the hyperplasia-to-hypertrophy transition, and may have stimulated the expression of mlc2a, mlc2b and col1a1 at the onset of mosaic hyperplasia. The up-regulation of mstn1 at transitional phases in muscle development indicates a dual regulatory role of myostatin in fish larval muscle growth.

Genetic variation in the major histocompatibility complex of the European brown hare (Lepus europaeus) across distinct phylogeographic areas.

The major histocompatibility complex is one of the best studied systems in vertebrates providing evidence for the long-term action of selection. Here, we examined the intra- and inter-population genetic diversity of the MHC class II DRB locus in European brown hare (Lepus europaeus) and correlated the results with genetic variability already estimated from the MHC DQA locus and from maternally (mitochondrial DNA (mtDNA)) and biparentally (allozymes, microsatellites) inherited loci. L. europaeus showed remarkable genetic polymorphism in both DQA and DRB1 loci. The Anatolian populations exhibited the highest genetic polymorphism for both loci. Balancing selection has established increased variability in the European populations despite the founder effects after the last glaciation. Different evolutionary rates were traced for DRB1 and DQA loci, as evidenced by the higher number of common DRB1 than DQA alleles and the greater differences between DRB1 alleles with common origin in comparison with DQA alleles. The high number of rare alleles with low frequencies detected implies that frequency-dependent selection drives MHC evolution in the brown hare through the advantage of rare alleles. Both loci were under the influence of positive selection within the peptide-binding region. The functional polymorphism, recorded as amino acid substitutions within the binding pockets, fell also within distinct geographic patterns, yet it was much narrower than the genetic polymorphism. We hypothesize that certain structural and functional characteristics of the binding pockets set limitations to the actual shape of genetic polymorphism in MHC.

Polarisation of major histocompatibility complex II host genotype with pathogenesis of European Brown Hare syndrome virus.

A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV) in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC) host genotype. Liver samples were examined from 170 brown hares (hunted, found sick or dead), collected between 2004 and 2009. Macroscopical and histopathological findings consistent with EBHS were detected in 24 (14.1%) hares; 35 (20.6%) had liver lesions not typical of the syndrome, 50 (29.4%) had lesions in other tissues and 61 (35.9%) had no lesions. Sixty five (38.2%) of 170 samples were found to be EBHSV-positive (RT-PCR, VP60 gene). In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180) was lower than expected (H e = 0.5835). The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively) were lower in Denmark than those assessed in other European countries (8.3% and 16.9%, respectively). Within the peptide binding region codons the number of nonsynonymous substitutions (dN) was much higher than synonymous substitutions (dS), which would be expected for MHC alleles under balancing selection. Allele frequencies did not significantly differ between EBHSV-positive and -negative hares. However, allele Leeu-DQA*30 was detected in significantly higher (P = 0.000006) frequency among the positive hares found dead with severe histopathological lesions than among those found sick or apparently healthy. In contrast, the latter group was characterized by a higher frequency of the allele Leeu-DQA*14 as well as the proportion of heterozygous individuals (P = 0.000006 and P = 0.027). These data reveal a polarisation between EBHSV pathogenesis and MHC class II genotype within the European brown hare in Denmark.

Domestication does not narrow MHC diversity in Sus scrofa.

The Major Histocompatibility Complex (MHC) is a multigene family of outstanding polymorphism. MHC molecules bind antigenic peptides in the peptide-binding region (PBR) that consists of five binding pockets (P). In this study, we compared the genetic diversity of domestic pigs to that of the modern representatives of their wild ancestors, the wild boar, in two MHC loci, the oligomorphic DQA and the polymorphic DRB1. MHC nucleotide polymorphism was compared with the actual functional polymorphism in the PBR and the binding pockets P1, P4, P6, P7, and P9. The analysis of approximately 200 wild boars collected throughout Europe and 120 domestic pigs from four breeds (three pureblood, Pietrain, Leicoma, and Landrace, and one mixed Danbred) revealed that wild boars and domestic pigs share the same levels of nucleotide and amino acid polymorphism, allelic richness, and heterozygosity. Domestication did not appear to act as a bottleneck that would narrow MHC diversity. Although the pattern of polymorphism was uniform between the two loci, the magnitude of polymorphism was different. For both loci, most of the polymorphism was located in the PBR region and the presence of positive selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous substitutions in the PBR. P4 and P6 were the most polymorphic binding pockets. Functional polymorphism, i.e., the number and the distribution of pocket variants within and among populations, was significantly narrower than genetic polymorphism, indicative of a hierarchical action of selection pressures on MHC loci.

Oxidative stress in Cyprinus carpio to analyze microcystin impact in eutrophic shallow lakes: a preliminary study.

Microcystins (MCYSTs) are toxins produced by cyanobacteria in aquatic environment and are of high potential risk to aquatic organisms. The physiological responses and pathobiological developments that they elicit in fish have been extensively studied, mainly through acute toxicity experiments. This study was designed to examine the seasonal fluctuation of biochemical markers of oxidative stress in different tissues of a natural population of Cyprinus carpio inhabiting a shallow Mediterranean lake, along with the respective MCYSTs concentrations in blood and tissues at environmentally relevant MCYSTs values. MCYSTs content was assessed in liver, kidney, intestine, brain and muscle along with the MCYSTs in lake water and scum applying ELISA technique. Catalase activity, GSH/GSSH relative concentrations and lipid peroxidation were used as biochemical markers. Our results suggest that common carp of Lake Pamvotis exposed to naturally fluctuating concentrations of MCYST in water and scum contained stably high MCYST concentrations in all tissues that might pose a threat to public health. Liver and kidney were the primary target organs. Tissue concentrations did not correlate with the response of any of the elements of the antioxidant defence system. Hepatic catalase, GSH content and TBARS in all tissues tested followed the fluctuations of major limnological parameters, i.e. water temperature and oxygen concentration, chlorophyll-a, MCYST in water and scum, suggesting that they should be cautiously used to monitor exposure to MCYSTS in natural freshwater ecosystems.

The response of digestive proteases to abrupt salinity decrease in the euryhaline sparid Sparus aurata L.

The response of the digestive proteases to abrupt salinity change was studied in juvenile gilthead sea bream (Sparus aurata) for 15 days after transfer from 33 per thousand to 21 per thousand. Salinity decrease affected significantly neither the activity of total acid proteases in stomach, nor the activities of total alkaline proteases and major serine proteases--trypsin and chymotrypsin--in the alkaline part of the intestine. The activity of the major proteases was significantly different between the alkaline segments of the intestine, with the posterior intestine presenting the highest activities followed by the pyloric caeca. This distribution pattern remained unaffected by salinity decrease. Notably, salinity change led to significant alterations in elastase and carboxypeptidase activity. The changes were more prominent in the upper part of the intestine (pyloric caeca and anterior intestine) than in the posterior intestine. In pyloric caeca significant alteration of carboxypeptidase A and B activities was observed, elastase changes were confined to anterior intestine together with alterations in carboxypeptidase B activity, while in posterior intestine the changes were restricted to carboxypeptidase A activity. The results are discussed in relation to the osmoregulatory action of the intestinal segments and dietary protein digestion.

Sequence analysis and tissue expression pattern of Sparus aurata chymotrypsinogens and trypsinogen.

Two apparently full-length cDNA clones encoding chymotrypsinogens I and II (CHTRI, 1022 bp; CHTRII, 909 bp) and one cDNA clone encoding trypsinogen II (TRPII, 848 bp) were isolated from a cDNA library prepared from gilthead sea bream (Sparus aurata) liver. The deduced amino acid sequences of the isolated cDNAs contain highly conserved residues essential for serine protease catalytic activity and conformational maintenance. The deduced amino acid sequences of CHTRI and CHTRII are 261 aa and 277 aa long, respectively, and share only 61% identity. Sea bream CHTRII appears to be the longest of all known teleostean chymotrypsinogen forms and contains a high number of methionine residues. Compared with CHTRI, CHTRII is more hydrophobic and has a lower isoelectric point. On the other hand, deduced amino acid sequence of TRPII is 241 aa long and has a signal peptide of thirteen amino acid residues and an activation peptide of seven amino acids long. In contrast to CHTRI and CHTRII, TRPII has a low isoelectric point (4.95), which makes it anionic at neutral pH. Northern blot analysis revealed that liver is the major transcription site for all zymogens. As expected, all zymogen transcripts were detected in parts of the digestive tract (stomach, pyloric caeca, anterior and posterior intestine) and pyloric caeca presented the most intense expression. In all tissues and amongst all zymogens, TRPII constitutive expression was the highest.