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- Factors driving bacterial microbiota of eggs from commercial hatcheries of European Seabass and Gilthead SeabreamPublication . Najafpour, Babak; Pinto, Patricia IS; Moutou, Katerina A.; Canario, A.V.M.; Power, Deborah MaryA 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.
- Specific evolution and gene family expansion of complement 3 and regulatory factor H in fishPublication . Najafpour, Babak; Cardoso, João CR; Canario, Adelino; Power, DeborahThe complement system comprises a large family of plasma proteins that play a central role in innate and adaptive immunity. To better understand the evolution of the complement system in vertebrates and the contribution of complement to fish immunity comprehensive in silico and expression analysis of the gene repertoire was made. Particular attention was given to C3 and the evolutionary related proteins C4 and C5 and to one of the main regulatory factors of C3b, factor H (Cfh). Phylogenetic and gene linkage analysis confirmed the standing hypothesis that the ancestral c3/c4/c5 gene duplicated early. The duplication of C3 (C3.1 and C3.2) and C4 (C4.1 and C4.2) was likely a consequence of the (1R and 2R) genome tetraploidization events at the origin of the vertebrates. In fish, gene number was not conserved and multiple c3 and cfh sequence related genes were encountered, and phylogenetic analysis of each gene generated two main clusters. Duplication of c3 and cfh genes occurred across the teleosts in a species-specific manner. In common, with other immune gene families the c3 gene expansion in fish emerged through a process of tandem gene duplication. Gilthead sea bream (Sparus aurata), had nine c3 gene transcripts highly expressed in liver although as reported in other fish, extra-hepatic expression also occurs. Differences in the sequence and protein domains of the nine deduced C3 proteins in the gilthead sea bream and the presence of specific cysteine and N-glycosylation residues within each isoform was indicative of functional diversity associated with structure. The diversity of C3 and other complement proteins as well as Cfh in teleosts suggests they may have an enhanced capacity to activate complement through direct interaction of C3 isoforms with pathogenic agents.
- Core microbiome profiles and their modification by environmental, biological, and rearing factors in aquaculture hatcheriesPublication . Najafpour, Babak; Pinto, Patricia IS; Sanz, Eric Climent; Martinez-Blanch, Juan F.; Canario, Adelino; Moutou, Katerina A.; Power, Deborah16S 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 Pseudoalter-omonas) 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.
- Transcriptome datasets and histological profiles of critical larval stages in gilthead seabreamPublication . NAJAFPOUR, BABAK; Canario, Adelino; Power, Deborah MaryThe transcriptome of the seabream larvae farmed in different European commercial hatcheries was analysed during critical larval stages. The complementary data herein presented support the findings reported in the associated research article "Insights into core molecular changes associated with metamorphosis in gilthead seabream larvae across diverse hatcheries". Samples were collected from gilthead seabream ( Sparus aurata ) hatcheries in Greece (site Gr), Italy (site It), and France (site Fr). RNA was extracted from larvae with different weights, mainly at the flexion (23 and 25 dph) and mid-metamorphosis stages (43, 50, 52, 56, and 60 dph). RNAseq libraries were sequenced using Illumina HiSeq xten. The paired-end sequenced raw reads were deposited in the NCBISRA database with the accession number PRJNA956882. Differential expression and function of genes were obtained by comparing transcriptome profiles of larvae at different developmental stages.
- Insights into core molecular changes associated with metamorphosis in gilthead seabream larvae across diverse hatcheriesPublication . NAJAFPOUR, BABAK; Santos, Soraia; Manchado, Manuel; Vidal, Aurora; Tsipourlianos, Andreas; Canario, Adelino; Moutou, Katerina A.; Power, Deborah MaryEarly development is a critical period in fish aquaculture and is influenced by biotic and abiotic factors (e.g., temperature, feed) that can vary significantly between hatcheries, making it difficult to identify core factors determining quality. Many of the existing larval transcriptome studies are small-scale and occur under specific rearing conditions that do not mirror the diversity of larviculture practices at an industrial level. In the present transcriptome study, gilthead seabream at the larval to juvenile transition (metamorphosis) from several hatcheries in Europe (Greece, Italy, and France) were analysed in a large-scale RNA-seq study. The aim was to uncover the most significant molecular modifications occurring during metamorphosis, irrespective of differences in biotic or abiotic factors, to address knowledge gaps associated with critical early developmental stages under industrial hatchery conditions. Commonly modified gene transcripts between larval stages were identified based on the clustering of gene expression profiles of 25 gilthead seabream libraries from different hatcheries in a PCA analysis. When larvae at flexion were compared to larvae at mid-metamorphosis, 2243 differentially expressed genes (DEGs) were identified, and when larvae at early to mid-metamorphosis were compared to mid to late-metamorphosis, 2299 DEGs were identified. Comparative analysis across the developmental stages of gilthead seabream revealed genes of importance for the metamorphic transition and adaptation to rearing conditions, including genes related to the nervous system at flexion (24 days post hatch), enteroendocrine cell differentiation, and lipid homeostasis at early to mid-metamorphosis (46 dph), and enrichment of genes indicative of immune competence at mid to late-metamorphosis (51-54 dph). The differential expression of some endocrine-associated genes, dio1, dio2, cldn1, ing4, Pou3f4, and fgf22, highlights their importance in metamorphosis. Meta-analysis of the transcriptomes from two species, the gilthead seabream and Senegalese sole, that have differing symmetry and ecology uncovered common molecular expression patterns that underlie larvae maturation during metamorphosis, and we propose that these represent core gene markers of metamorphosis in these two fish species.
- Quantitative PCR assays as a monitoring tool for bacterial genera in fresh fish filletsPublication . Pinto, Patricia IS; NAJAFPOUR, BABAK; Lima, Pedro; P. Machado; Aires, Tania; Engelen, Aschwin; Tsironi, T.; Anjos Guerreiro, Liliana Isabel Tomé; Power, Deborah MaryFresh fish fillets are a valuable but highly perishable food, and their rapid microbial deterioration is a drawback for food safety and sustainability of aquaculture, food and retail industries. Quantitative PCR (qPCR) assays based on 16S rRNA gene (16S) sequences were developed for the most abundant bacteria genera detected by metagenomics in fresh or processed fish fillets. The efficiency and specificity of six qPCR assays (for 16S of all bacteria or genera Shewanella, Pseudomonas, Carnobacterium, Janthinobacterium and Massilia) was verified using in silico predictions, cloning, sequencing and phylogenetic analyses of amplicons obtained from refrigerated control or high-pressure processed (HPP) European sea bass (Dicentrarchus labrax) fillets. In HPP sea bass fillets, significant decreases in total bacteria 16S and of Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium 16S compared to control fillets were confirmed by qPCR, after 11 days of refrigerated storage. The qPCR assays were successfully applied to monitor microbial contamination during refrigerated storage of fresh fillets from commercial (retail) sea bass and gilthead sea bream (Sparus aurata). Significant increases in total bacterial and Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium contamination were detected after 7-14 days. 16S copy number for total bacteria and the four target genera positively correlated with total viable counts using culture enumeration. 16S of Massilia, that is abundant in fresh fish fillets, did not significantly change during storage. The six validated qPCR assays developed are proposed as specific, sensitive, culture-independent methods for monitoring quality or processing outcomes for fish fillets during cold chain storage.
- Quantifying dominant bacterial genera detected in metagenomic data from fish eggs and larvae using genus‐specific primersPublication . Najafpour, Babak; Canario, Adelino; Power, Deborah; Pinto, Patricia ISThe goal of this study was to design genus-specific primers for rapid evaluation of the most abundant bacterial genera identified using amplicon-based sequencing of the 16S rRNA gene in fish-related samples and surrounding water. Efficient genus-specific primers were designed for 11 bacterial genera including Alkalimarinus, Colwellia, Enterovibrio, Marinomonas, Massilia, Oleispira, Phaeobacter, Photobacterium, Polarbacerium, Pseudomonas, and Psychrobium. The specificity of the primers was confirmed by the phylogeny of the sequenced polymerase chain reaction (PCR) amplicons that indicated primers were genus-specific except in the case of Colwellia and Phaeobacter. Copy number of the 16S rRNA gene obtained by quantitative PCR using genus-specific primers and the relative abundance obtained by 16S rRNA gene sequencing using universal primers were well correlated for the five analyzed abundant bacterial genera. Low correlations between quantitative PCR and 16S rRNA gene sequencing for Pseudomonas were explained by the higher coverage of known Pseudomonas species by the designed genus-specific primers than the universal primers used in 16S rRNA gene sequencing. The designed genus-specific primers are proposed as rapid and cost-effective tools to evaluate the most abundant bacterial genera in fish-related or potentially other metagenomics samples.
- Bacterial 16S ribosomal gene fingerprints as a tool to diagnose and mitigate fish larvae gut dysbiosisPublication . NAJAFPOUR, BABAK; Canario, Adelino; Power, Deborah MaryDysbiosis is associated with shifts in the diversity or relative abundance of beneficial versus harmful bacteria, leading to health issues in organisms. This study investigated gut bacterial dysbiosis associated with larval quality using 16S rRNA gene sequencing. The gut microbiome of gilthead sea bream and European sea bass, key commercial species and vertebrate models, was examined in high- and low-quality larvae batches from several European hatcheries. Larval quality, hatchery site and species influenced bacterial diversity in the gut. Individuals from larval batches that performed well had higher microbial diversity in the gut and individuals from batches that performed poorly had a gut microbiota dominated by pathogenic Vibrio (e.g., V. aestuarianus and V. cortegadensis). The bacterial dysbiosis index revealed a notable predominance of Fusobacteriota and Firmicutes phyla, Thermoanaerobacteria class and Lactobacillaceae, Moritellaceae, Clostridiaceae, Thiotrichaceae and Shewanellaceae families in good-quality larvae batches, and a prevalence of the Proteobacteria phylum, Gammaproteobacteria class, Sphingomonadaceae and Vibrionaceae families in the gut of individuals from poor-quality larvae batches. A positive dysbiosis index (cutoff >0.4) was associated with a high risk of decreased larval performance and quality. Additionally, the abundance of Clostridium_sensu_ stricto_15, Shewanellaceae_unclassified, Cetobacterium, Psychrilyobacter, Moritella and Latilactobacillus genera in the gut of good production batches, and the Vibrio genus in the gut of poor production batches, identified these genus as potential markers for diagnosing and mitigating bacterial dysbiosis in fish and potentially other vertebrates.