An integrated genomic approach for the study of mandibular prognathism in the European seabass (Dicentrarchus labrax)

Skeletal anomalies in farmed fish are a relevant issue affecting animal welfare and health and causing significant economic losses. Here, a high-density genetic map of European seabass for QTL mapping of jaw deformity was constructed and a genome-wide association study (GWAS) was carried out on a total of 298 juveniles, 148 of which belonged to four full-sib families. Out of 298 fish, 107 were affected by mandibular prognathism (MP). Three significant QTLs and two candidate SNPs associated with MP were identified. The two GWAS candidate markers were located on ChrX and Chr17, both in close proximity with the peaks of the two most significant QTLs. Notably, the SNP marker on Chr17 was positioned within the Sobp gene coding region, which plays a pivotal role in craniofacial development. The analysis of differentially expressed genes in jaw-deformed animals highlighted the "nervous system development" as a crucial pathway in MP. In particular, Zic2, a key gene for craniofacial morphogenesis in model species, was significantly down-regulated in MP-affected animals. Gene expression data revealed also a significant down-regulation of Sobp in deformed larvae. Our analyses, integrating transcriptomic and GWA methods, provide evidence for putative mechanisms underlying seabass jaw deformity

Reproduction and immunology: transcriptomic approaches to improve bivalves farming

Over the last years, there has been an increasing demand of shellfish for consumption. The ability of traditional capture fisheries to supply bivalves is unlikely to increase significantly because of the drops in natural recruitments of seed, which are mainly due to overexploitation of natural stocks. Production of bivalve seed in hatcheries is a relatively new industry for which empirical approaches were developed, adapting methods across species and measuring the resulting effect in terms of growth and survival. To date, only a few bivalve species of major aquacultural importance in Europe have benefited from newly developed genomic resources (e.g. microarrays and RNA sequencing) and gene silencing approaches, which are both expected to significantly improve the biological knowledge on these commercially important species in the coming years. The present PhD thesis aimed firstly at increasing, through Next Generation Sequencing, genomic information available for two emerging species, Venerupis decussata and Pecten maximus. The second aim was to investigate two main bottlenecks hampering the bivalve production in hatchery: efficiency of reproduction and susceptibility to pathogens. By means of microarray analysis, a gene expression study on V .decussata oocytes at different maturation stages was performed and the major biological processes involved in gamete maturation were identified. A RNA sequencing experiment was conducted on pathogen-challenged hemocytes and unchallenged controls to study the P. maximus immune transcriptome. mRNAs encoding proteins with a known immune function were detected and a global analysis of differential expression comparing gene-expression levels in stimulated hemocytes against controls provided evidence on a large set of transcripts involved in P. maximus immune response. Finally, reverse genetic and real-time PCR were implemented to investigate the role of IkB2 in the Crassostrea gigas immune response against Ostreid herpesvirus type 1. Following the injection of a dsRNA targeting IkB2, juveniles were infected with OsHV-1 and mRNA levels of four immune genes (IkB1, IkB2, Rel, SOCS) were evaluated in gonads and gills demonstrating their implication in the Pacific oyster antiviral response.Nel corso degli ultimi anni, si assistito ad una crescita della domanda di molluschi destinati al consumo alimentare, alla quale però la pesca tradizionale non ha saputo far fronte, principalmente a causa dell’impoverimento degli stock naturali e alla conseguente scarsità di seme. La produzione di seme di bivalvi in schiuditoio è un’attività relativamente recente e le attuali metodiche di allevamento non sono altro che protocolli già usati in altre specie, adattati e verificati in termini di crescita e sopravvivenza. Ad oggi, ad aver beneficiato delle recenti risorse genomiche, come microarry ed RNA sequencing, e delle sofisticate strategie di silenziamento genico sono state solo alcune specie di bivalvi di notevole interesse alimentare a livello europeo. Tuttavia, si pensa che, nel corso dei prossimi anni, questi approcci molecolari possano costituire una risorsa importante per lo studio della biologia di numerose specie di bivalvi non ancora allevate su larga scala ma di crescente interesse commerciale. Il primo obiettivo della presente tesi di dottorato è stato quello di incrementare, mediante tecniche di Next Generation Sequencing, le risorse genomiche attualmente disponibili per due specie emergenti: la vongola verace Venerupis decussata e la cappasanta atlantica Pecten maximus. In secondo luogo, si sono considerati due importanti fattori che ostacolano l’allevamento dei bivalvi in schiuditoio: l’efficienza riproduttiva e la suscettibilità ai patogeni. Utilizzando una piattaforma microarray, sono stati valutati i livelli di espressione genica di oociti di V. decussata a due diversi stadi di maturazione. L’analisi dei dati ha quindi permesso di identificare i principali processi biologici coinvolti nella maturazione dei gameti femminili. Inoltre, al fine di studiare il trascrittoma immunitario di P. maximus, è stato effettuato un esperimento di RNA sequencing su emociti immuno-stimolati e di controllo. Questo studio ha permesso di identificare trascritti di mRNA che codificano per importanti proteine del sistema immunitario. In aggiunta l’individuazione di geni differenzialmente espressi in emociti stimolati e controlli ha messo in evidenza un set di trascritti potenzialmente implicati nella risposta immunitaria di P. maximus. Infine, allo scopo di valutare il ruolo di IkB2 nella risposta immunitaria di Crassostrea gigas all’infezione da herpesvirus di tipo 1, è stato effettuato un esperimento di RNA interference. In seguito all’iniezione di un RNA a doppio filamento codificante una porzione del trascritto IkB2, individui giovani di C. gigas sono stati infettati con un omogenato contenente il virus HV-1. Infine, per valutare l’importanza di quattro geni della risposta immunitaria (IkB1, IkB2, Rel, SOCS), i loro livelli di espressione sono stati valutati in due diversi tessuti: le gonadi e le branchie

Reproduction and immunology: transcriptomic approaches to improve bivalves farming

Over the last years, there has been an increasing demand of shellfish for consumption. The ability of traditional capture fisheries to supply bivalves is unlikely to increase significantly because of the drops in natural recruitments of seed, which are mainly due to overexploitation of natural stocks. Production of bivalve seed in hatcheries is a relatively new industry for which empirical approaches were developed, adapting methods across species and measuring the resulting effect in terms of growth and survival. To date, only a few bivalve species of major aquacultural importance in Europe have benefited from newly developed genomic resources (e.g. microarrays and RNA sequencing) and gene silencing approaches, which are both expected to significantly improve the biological knowledge on these commercially important species in the coming years. The present PhD thesis aimed firstly at increasing, through Next Generation Sequencing, genomic information available for two emerging species, Venerupis decussata and Pecten maximus. The second aim was to investigate two main bottlenecks hampering the bivalve production in hatchery: efficiency of reproduction and susceptibility to pathogens. By means of microarray analysis, a gene expression study on V .decussata oocytes at different maturation stages was performed and the major biological processes involved in gamete maturation were identified. A RNA sequencing experiment was conducted on pathogen-challenged hemocytes and unchallenged controls to study the P. maximus immune transcriptome. mRNAs encoding proteins with a known immune function were detected and a global analysis of differential expression comparing gene-expression levels in stimulated hemocytes against controls provided evidence on a large set of transcripts involved in P. maximus immune response. Finally, reverse genetic and real-time PCR were implemented to investigate the role of IkB2 in the Crassostrea gigas immune response against Ostreid herpesvirus type 1. Following the injection of a dsRNA targeting IkB2, juveniles were infected with OsHV-1 and mRNA levels of four immune genes (IkB1, IkB2, Rel, SOCS) were evaluated in gonads and gills demonstrating their implication in the Pacific oyster antiviral response.Nel corso degli ultimi anni, si assistito ad una crescita della domanda di molluschi destinati al consumo alimentare, alla quale però la pesca tradizionale non ha saputo far fronte, principalmente a causa dell’impoverimento degli stock naturali e alla conseguente scarsità di seme. La produzione di seme di bivalvi in schiuditoio è un’attività relativamente recente e le attuali metodiche di allevamento non sono altro che protocolli già usati in altre specie, adattati e verificati in termini di crescita e sopravvivenza. Ad oggi, ad aver beneficiato delle recenti risorse genomiche, come microarry ed RNA sequencing, e delle sofisticate strategie di silenziamento genico sono state solo alcune specie di bivalvi di notevole interesse alimentare a livello europeo. Tuttavia, si pensa che, nel corso dei prossimi anni, questi approcci molecolari possano costituire una risorsa importante per lo studio della biologia di numerose specie di bivalvi non ancora allevate su larga scala ma di crescente interesse commerciale. Il primo obiettivo della presente tesi di dottorato è stato quello di incrementare, mediante tecniche di Next Generation Sequencing, le risorse genomiche attualmente disponibili per due specie emergenti: la vongola verace Venerupis decussata e la cappasanta atlantica Pecten maximus. In secondo luogo, si sono considerati due importanti fattori che ostacolano l’allevamento dei bivalvi in schiuditoio: l’efficienza riproduttiva e la suscettibilità ai patogeni. Utilizzando una piattaforma microarray, sono stati valutati i livelli di espressione genica di oociti di V. decussata a due diversi stadi di maturazione. L’analisi dei dati ha quindi permesso di identificare i principali processi biologici coinvolti nella maturazione dei gameti femminili. Inoltre, al fine di studiare il trascrittoma immunitario di P. maximus, è stato effettuato un esperimento di RNA sequencing su emociti immuno-stimolati e di controllo. Questo studio ha permesso di identificare trascritti di mRNA che codificano per importanti proteine del sistema immunitario. In aggiunta l’individuazione di geni differenzialmente espressi in emociti stimolati e controlli ha messo in evidenza un set di trascritti potenzialmente implicati nella risposta immunitaria di P. maximus. Infine, allo scopo di valutare il ruolo di IkB2 nella risposta immunitaria di Crassostrea gigas all’infezione da herpesvirus di tipo 1, è stato effettuato un esperimento di RNA interference. In seguito all’iniezione di un RNA a doppio filamento codificante una porzione del trascritto IkB2, individui giovani di C. gigas sono stati infettati con un omogenato contenente il virus HV-1. Infine, per valutare l’importanza di quattro geni della risposta immunitaria (IkB1, IkB2, Rel, SOCS), i loro livelli di espressione sono stati valutati in due diversi tessuti: le gonadi e le branchie

Missense single nucleotide variants affecting CYP3A catalytic activity are present in Limousine cattle

Cytochrome P450 (CYP) 3A is one of the most important subfamily of drug metabolising enzymes. Genetic factors such as breed and allelic variants might modify CYP3A expression and enzyme activity and lead to inter- and intra-individual variability in clinical response or toxicity. CattleCYP3Agene cluster has been recently re-sequenced in 300 Piedmontese cattle by deep targeted sequencing. Thirteen missense single nucleotide variants (SNVs) were identified, and for five of them an impact on CYP3A activity was demonstratedin vitro. In the present work, we assessed the genetic frequency of these five SNVs in Limousine, a cattle breed widely raised for meat production in Veneto Region. A total of 215 cows were genotyped using specific melting curve assays. Only two missense SNVs out of five, both located onCYP3A28coding sequence, were detected:rs384467435 andrs454167819. The former mutant allele was present in homozygosis in the 4% of tested cows, while in heterozygosis in the 24% of animals. The second SNV was detected only in heterozygosis in 11 cows out of 215 (i.e. 4.9%). These findings suggest the presence of missense SNVs, proved to halve CYP3A28 catalytic activity, in both Limousine and Piedmontese meat cattle breeds. As a consequence, these SNVs might impact on the kinetics of xenobiotics metabolised by CYP3A, including drugs and natural toxins like ivermectin and aflatoxins, thereby resulting in toxicity or accumulation of harmful residues in foodstuffs. This result paves the way for new considerations on the fate of xenobiotics in cattle farming

Gene transcription and biomarker responses in the clam Ruditapes philippinarum after exposure to ibuprofen

Pharmaceuticals are a class of emerging environmental contaminants that continuously enter aquatic environments. Presently, little information is available about the effects of these substances on non-target organisms, such as bivalves. We investigated the effects of ibuprofen (IBU) on the clam Ruditapes philippinarum. Clams were exposed for 1, 3, 5 and 7 days to 0, 100 and 1000 \ub5g IBU/L, and biomarker responses (haemolymph lysozyme, gill acetylcholinesterase and digestive gland superoxide dismutase activities) as well as digestive gland transcriptome were evaluated. A two-way ANOVA revealed significant effects of both \u201cIBU concentration\u201d and \u201cexposure duration\u201d on biomarker responses. Overall, the enzyme activities were generally lower in IBU-exposed clams than in controls. Although limited knowledge of the mollusc transcriptome makes it difficult to interpret the effects of IBU on clams, the gene transcription analysis using DNA microarrays enabled the identification of the putative molecular mode of action of the IBU. The functional analysis of differentially expressed genes revealed that IBU can interfere with various signalling pathways in clams, such as arachidonic acid metabolism, apoptosis, peroxisomal proliferator-activated receptors, and nuclear factor-kappa B. In addition, several genes involved in the metabolism of xenobiotics (e.g., glutathione S-transferase, sulfotransferase, cytochrome P450) were also found to be significantly affected by IBU exposure. In summary, the integrated approach of gene transcription analysis and biomarker responses facilitated the elucidation of the mechanisms of action of IBU in non-target species

Extending RAD tag analysis to microbial ecology: A comparison between MultiLocus Sequence Typing and 2b-RAD to investigate Listeria monocytogenes genetic structure

The advent of next-generation sequencing (NGS) has dramatically changed bacterial typing technologies, increasing our ability to differentiate bacterial isolates. Despite it is now possible to sequence a bacterial genome in a few days and at reasonable costs, most genetic analyses do not require whole-genome sequencing, which also remains impractical for large population samples due to the cost of individual library preparation and bioinformatics. More traditional sequencing approaches, however, such as MultiLocus Sequence Typing (mlst) are quite laborious and time-consuming, especially for large-scale analyses. In this study, a genotyping approach based on restriction site-associated (RAD) tag sequencing, 2b-RAD, was applied to characterize Listeria monocytogenes strains. To verify the feasibility of the method, an in silico analysis was performed on 30 available complete genomes. For the same set of strains, in silico mlst analysis was conducted as well. Subsequently, 2b-RAD and mlst analyses were experimentally carried out on 58 isolates collected from food samples or food-processing sites. The obtained results demonstrate that 2b-RAD predicts mlst types and often provides more detailed information on population structure than mlst. Moreover, the majority of variants differentiating identical sequence type isolates mapped against accessory fragments, thus providing additional information to characterize strains. Although mlst still represents a reliable typing method, large-scale studies on molecular epidemiology and public health, as well as bacterial phylogenetics, population genetics and biosafety could benefit of a low cost and fast turnaround time approach such as the 2b-RAD analysis proposed here

Data from: Extending RAD tag analysis to microbial ecology: a comparison between multi locus sequence typing (MLST) and 2b-RAD to investigate Listeria monocytogenes genetic structure

The advent of next-generation sequencing (NGS) has dramatically changed bacterial typing technologies, increasing our ability to differentiate bacterial isolates. Despite it is now possible to sequence a bacterial genome in a few days and at reasonable costs, most genetic analyses do not require whole-genome sequencing, which also remains impractical for large population samples due to the cost of individual library preparation and bioinformatics. More traditional sequencing approaches, however, such as MultiLocus Sequence Typing (mlst) are quite laborious and time-consuming, especially for large-scale analyses. In this study, a genotyping approach based on restriction site-associated (RAD) tag sequencing, 2b-RAD, was applied to characterize Listeria monocytogenes strains. To verify the feasibility of the method, an in silico analysis was performed on 30 available complete genomes. For the same set of strains, in silico mlst analysis was conducted as well. Subsequently, 2b-RAD and mlst analyses were experimentally carried out on 58 isolates collected from food samples or food-processing sites. The obtained results demonstrate that 2b-RAD predicts mlst types and often provides more detailed information on population structure than mlst. Moreover, the majority of variants differentiating identical sequence type isolates mapped against accessory fragments, thus providing additional information to characterize strains. Although mlst still represents a reliable typing method, large-scale studies on molecular epidemiology and public health, as well as bacterial phylogenetics, population genetics and biosafety could benefit of a low cost and fast turnaround time approach such as the 2b-RAD analysis proposed here

Deepening the Whole Transcriptomics of Bovine Liver Cells Exposed to AFB1: A Spotlight on Toll-like Receptor 2

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA-seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38β MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38β MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle

Curcumin Mitigates AFB1-Induced Hepatic Toxicity by Triggering Cattle Antioxidant and Anti-inflammatory Pathways: A Whole Transcriptomic In Vitro Study

Aflatoxin B1 (AFB1) toxicity in livestock and human beings is a major economic and health concern. Natural polyphenolic substances with antioxidant properties have proven to be effective in ameliorating AFB1-induced toxicity. Here we assessed the potential anti-AFB1 activity of curcumin (pure curcumin, C, and curcumin from Curcuma longa, CL) in a bovine fetal hepatocyte-derived cell line (BFH12). First, we measured viability of cells exposed to AFB1 in presence or absence of curcumin treatment. Then, we explored all the transcriptional changes occurring in AFB1-exposed cells cotreated with curcumin. Results demonstrated that curcumin is eective in reducing AFB1-induced toxicity, decreasing cells mortality by approximately 30%. C and CL induced similar transcriptional changes in BFH12 exposed to AFB1, yet C treatment resulted in a larger number of significant genes compared to CL. The mitigating effects of curcuminoids towards AFB1 toxicity were mainly related to molecular pathways associated with antioxidant and anti-inflammatory response, cancer, and drug metabolism. Investigating mRNA changes induced by curcumin in cattle BFH12 cells exposed to AFB1 will help us to better characterize possible tools to reduce its consequences in this susceptible and economically important food-producing species