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Dietary lipid levels have a remarkable impact on the expression of growth-related genes in Senegalese sole (Solea senegalensis Kaup)

SUMMARY In Senegalese sole (Solea senegalensis Kaup), growth is negatively correlated to dietary lipid levels. To understand the molecular basis of this effect a molecular toolbox of 12 genes, including fgf6, fst, mstn1, myf5, mrf4, myod1, myod2, myog, myHC, mylc2, igf1r and insr, was developed. The expression profiles of these genes were investigated in white muscle and liver of fish fed with three dietary lipid levels (4%, 12% and 20%). The expression of igf-I and igf-II was also examined. MRFs and myosins were only expressed in the muscle and, except for myf5, the general trend was a decrease in expression with an increase in dietary lipids. Fgf6 was identified for the first time in liver and its expression augmented in hepatic tissues with increasing dietary lipid levels. A similar tendency was observed for mstn1 and igf-I. The opposite was observed for igf1r expression in muscle and liver. Myog, mrf4, mylc2 and igf1r were highly correlated with growth and nutrient utilisation indices. In addition to its practical implications, this work provides a valuable contribution towards our understanding of the genetic networks controlling growth in teleosts.

microRNA in teleost fish

MicroRNAs (miRNAs) are transcriptional and posttranscriptional regulators involved in nearly all known biological processes in distant eukaryotic clades. Their discovery and functional characterization have broadened our understanding of biological regulatory mechanisms in animals and plants. They show both evolutionary conserved and unique features across Metazoa. Here, we present the current status of the knowledge about the role of miRNA in development, growth, and physiology of teleost fishes, in comparison to other vertebrates. Infraclass Teleostei is the most abundant group among vertebrate lineage. Fish are an important component of aquatic ecosystems and human life, being the prolific source of animal proteins worldwide and a vertebrate model for biomedical research. We review miRNA biogenesis, regulation, modifications, and mechanisms of action. Specific sections are devoted to the role of miRNA in teleost development, organogenesis, tissue differentiation, growth, regeneration, reproduction, endocrine system, and responses to environmental stimuli. Each section discusses gaps in the current knowledge and pinpoints the future directions of research on miRNA in teleosts.

Sex-biased miRNA expression in Atlantic halibut (Hippoglossus hippoglossus) brain and gonads

The role of miRNA in fish sexual development is not elucidated yet. We profiled miRNAs in gonads and brains of Atlantic halibut using SOLiD sequencing technology. We found tissue- and sexually dimorphic expression of several miRNAs, including miR-29a, miR-34, miR-143, miR-145, miR-202-3p, miR-451, and miR-2188. miR-9 and miR-202 were abundant in brain and gonads, respectively. In the next step, we selected some miRNAs showing differential expression patterns between sexes and performed RT-qPCR on 3 age groups: juveniles, 3-year-, and 5-year-olds. In brains, miR-451 was significantly down-regulated in juveniles compared to adults. let-7a, miR-143, and miR-202-3p were up-regulated in gonads of mature males compared to immature females at the same age. We investigated the effect of suppressing aromatase cytochrome P450 enzyme on miRNA expression at the onset of sex differentiation through masculinization with Fadrozole or 17-α-methyltestosterone. We found significant differences in miRNA expression between masculinized individuals and untreated controls. miR-202-3p was significantly down-regulated in female juveniles compared to male juveniles. The expression levels of let-7a and miR-451 were restored after termination of the masculinization treatment. Our data give a first insight into miRNA involvement in sexual development in teleosts.

RNA deep sequencing of the Atlantic cod transcriptome

The Atlantic cod (Gadus morhua) is an emerging aquaculture species. Efforts to develop and characterize its genomic recourses, including draft-grade genome sequencing, have been initiated by the research community. The transcriptome represents the whole complement of RNA transcripts in cells and tissues and reflects the expressed genes at various life stages, tissue types, physiological states, and environmental conditions. We are investigating the Atlantic cod transcriptome by Roche 454, Illumina GA, and ABI SOLiD deep sequencing platforms and corresponding bioinformatics. Both embryonic developmental stages and adult tissues are studied. Here we summarize our recent progress in the analyses of nuclear and mitochondrial polyA mRNAs, non-protein-coding intermediate RNAs, and regulatory microRNAs.

Characterization of Novel Precursor miRNAs Using Next Generation Sequencing and Prediction of miRNA Targets in Atlantic Halibut

Background microRNAs (miRNAs) are implicated in regulation of many cellular processes. miRNAs are processed to their mature functional form in a step-wise manner by multiple proteins and cofactors in the nucleus and cytoplasm. Many miRNAs are conserved across vertebrates. Mature miRNAs have recently been characterized in Atlantic halibut (Hippoglossus hippoglossus L.). The aim of this study was to identify and characterize precursor miRNA (pre-miRNAs) and miRNA targets in this non-model flatfish. Discovery of miRNA precursor forms and targets in non-model organisms is difficult because of limited source information available. Therefore, we have developed a methodology to overcome this limitation. Methods Genomic DNA and small transcriptome of Atlantic halibut were sequenced using Roche 454 pyrosequencing and SOLiD next generation sequencing (NGS), respectively. Identified pre- miRNAs were further validated with reverse–transcription PCR. miRNA targets were identified using miRanda and RNAhybrid target prediction tools using sequences from public databases. Some of miRNA targets were also identified using RACE-PCR. miRNA binding sites were validated with luciferase assay using the RTS34st cell line. Results We obtained more than 1.3 M and 92 M sequence reads from 454 genomic DNA sequencing and SOLiD small RNA sequencing, respectively. We identified 34 known and 9 novel pre-miRNAs. We predicted a number of miRNA target genes involved in various biological pathways. miR-24 binding to kisspeptin 1 receptor-2 (kiss1-r2) was confirmed using luciferase assay. Conclusion This study demonstrates that identification of conserved and novel pre-miRNAs in a non-model vertebrate lacking substantial genomic resources can be performed by combining different next generation sequencing technologies. Our results indicate a wide conservation of miRNA precursors and involvement of miRNA in multiple regulatory pathways, and provide resources for further research on miRNA in non-model animals.

Maternal gene expression in Atlantic halibut (Hippoglossus hippoglossus L.) and its relation to egg quality

BackgroundThe commercial production of Atlantic halibut (Hippoglossus hippoglossus L.) suffers from a major bottleneck due to the low success of producing juveniles for on-growing. Atlantic halibut females are routinely hand-stripped and incorrect timing of stripping can result in low quality eggs due to post-ovulatory aging. Post-ovulatory aging leads to compositional changes in eggs that include maternally provided proteins and RNAs. There have been few studies of the maternally provided mRNA transcripts that control early development in commercially important fish species. The present study aimed to study maternal gene expression in Atlantic halibut and its relation to egg quality parameters including blastomere symmetry and hatching success.ResultsA maternal EST library containing 2341 sequences was constructed by suppressive subtractive hybridisation. Thirty genes were selected for expression studies; 23 novel genes and 7 genes with documented roles in early development. The expressions of twenty-one selected genes were measured by qPCR from fertilization to the 10-somite stage. Three genes were identified as strictly maternal genes that were expressed until the start of gastrulation; askopos (kop), si:dkey-30j22.9 (Tudor family member), and Tudor 5 protein (Tdrd5). The expressions of 18 genes at the 8-cell stage were correlated with egg quality parameters. The majority of genes showed either no or very minor correlations with egg quality parameter. However, two genes correlated positively with hatching success (r> 0.50, HHC00353: r = 0.58, p < 0.01; HHC01517: r = 0.56, p < 0.01) and one gene (HHC00255) was negatively correlated with the percentage of normal blastomeres (r = -0.62, p < 0.05).ConclusionsDuring this study we have related maternal levels of gene expression to hatching success in fish. Poor hatching success was not correlated with a general decrease in transcript abundance but with low transcript levels of some specific genes. Thus, the molecular mechanisms leading to low Atlantic halibut egg quality cannot be entirely explained by post-ovulatory aging.

Transcriptome of Atlantic Cod (Gadus morhua L.) Early Embryos from Farmed and Wild Broodstocks

Significant efforts have been made to elucidate factors affecting egg quality in fish. Recently, we have shown that eggs originating from wild broodstock (WB) of Atlantic cod (Gadus morhua L.) are of superior quality to those derived from farmed broodstock (FB), and this is associated with differences in the chemical composition of egg yolk. However, maternal transcripts, accumulated during oogenesis, have not been studied extensively in fish. The aim of the present study was to characterize putative maternal mRNA transcriptome in fertilized eggs of Atlantic cod and to compare transcript pools between WB and FB in order to investigate the relation between egg developmental potential and putative maternal mRNA deposits. We performed high-throughput 454 pyrosequencing. For each WB and FB group, five cDNA libraries were individually tagged and sequenced, resulting in 98,687 (WB) and 119,333 (FB) average reads per library. Sequencing reads were de novo assembled, annotated, and mapped. Out of 13,726 identified isotigs, 238 were differentially expressed between WB and FB, with 155 isotigs significantly upregulated in WB. The sequence reads were mapped to 11,340 different Atlantic cod transcripts and 158 sequences were differentially expressed between the 2 groups. Important transcripts involved in fructose metabolism, fatty acid metabolism, glycerophospholipid metabolism, and oxidative phosphorylation were differentially represented between the two broodstock groups, showing potential as biomarkers of egg quality in teleosts. Our findings contribute to the hypothesis that maternal mRNAs affect egg quality and, consequently, the early development of fish.

Dynamics of miRNA transcriptome during gonadal development of zebrafish

Studies in non-teleost vertebrates have found microRNAs (miRNAs) to be essential for proper gonadal development. However, comparatively little is known about their role during gonadal development in teleost fishes. So far in zebrafish, a model teleost, transcript profiling throughout gonadal development has not been established because of a tiny size of an organ in juvenile stages and its poor distinguishability from surrounding tissues. We performed small RNA sequencing on isolated gonads of See-Thru-Gonad line, from the undifferentiated state at 3 weeks post fertilization (wpf) to fully mature adults at 24 wpf. We identified 520 gonadal mature miRNAs; 111 of them had significant changes in abundance over time, while 50 miRNAs were either testis- or ovary-enriched significantly in at least one developmental stage. We characterized patterns of miRNA abundance over time including isomiR variants. We identified putative germline versus gonadal somatic miRNAs through differential small RNA sequencing of isolated gametes versus the whole gonads. This report is the most comprehensive analysis of the miRNA repertoire in zebrafish gonads during the sexual development to date and provides an important database from which functional studies can be performed.

See-Thru-Gonad zebrafish line: developmental and functional validation.

Zebrafish are an important model species in developmental biology. However, their potential in reproductive biology research has yet to be realized. In this study, we established See-Thru-Gonad zebrafish, a transparent line with fluorescently labeled germ cells visible throughout the life cycle, validated its gonadal development features, and demonstrated its applicability by performing a targeted gene knockdown experiment using vivo-morpholinos (VMOs). To establish the line, we crossed the zf45Tg and mitfa(w2/w2); mpv17(b18/b18) zebrafish lines. We documented the in vivo visibility of the germline-specific fluorescent signal throughout development, from gametes through embryonic and juvenile stages up to sexual maturity, and validated gonadal development with histology. We performed targeted gene knockdown of the microRNA (miRNA) miR-92a-3p through injection of VMOs directly to maturing ovaries. After the treatment, zebrafish were bred naturally. Embryos from miR-92a-3p knockdown ovaries had a significant reduction in relative miR-92a-3p expression and a higher percentage of developmental arrest at the 1-cell stage as compared with 5-base mismatch-treated controls. The experiment demonstrates that See-Thru-Gonad line can be successfully used for vertical transmission of the effects of targeted gene knockdown in ovaries into their offspring.

First feed affects the expressions of microRNA and their targets in Atlantic cod.

To our knowledge, there is no report on microRNA (miRNA) expression and their target analysis in relation to the type of the first feed and its effect on the further growth of fish. Atlantic cod (Gadus morhua) larvae have better growth and development performance when fed natural zooplankton as a start-feed, as compared with those fed typical aquaculture start-feeds. In our experiment, two groups of Atlantic cod larvae were fed reference feed (zooplankton, mostly copepods, filtered from a seawater pond) v. aquaculture feeds: enriched rotifers (Brachionus sp.) and later brine shrimp (Artemia salina). We examined the miRNA expressions of six defined developmental stages as determined and standardised by body length from first feeding for both diet groups. We found eight miRNA (miR-9, miR-19a, miR-130b, miR-146, miR-181a, miR-192, miR-206 and miR-11240) differentially expressed between the two feeding groups in at least one developmental stage. We verified the next-generation sequencing data using real-time RT-PCR. We found 397 putative targets (mRNA) to the differentially expressed miRNA; eighteen of these mRNA showed differential expression in at least one stage. The patterns of differentially expressed miRNA and their putative target mRNA were mostly inverse, but sometimes also concurrent. The predicted miRNA targets were involved in different pathways, including metabolic, phototransduction and signalling pathways. The results of this study provide new nutrigenomic information on the potential role of miRNA in mediating nutritional effects on growth during the start-feeding period in fish larvae.