MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in a wide variety of physiological processes. They can control both temporal and spatial gene expression and are believed to regulate 30 to 70% of the genes. Data are however limited for fish species, with only 9 out of the 30,000 fish species present in miRBase. The aim of the current study was to discover and characterize rainbow trout (Oncorhynchus mykiss) miRNAs in a large number of tissues using next-generation sequencing in order to provide an extensive repertoire of rainbow trout miRNAs.

The family of forkhead box (Fox) transcription factors regulates gonadogenesis and embryogenesis, but the role of foxr1 in reproduction is unknown. Evolutionary history of foxr1 in vertebrates was examined and the gene was found to exist in most vertebrates, including mammals, ray-finned fish, amphibians, and sauropsids. By quantitative PCR and RNA-seq, we found that foxr1 had an ovarian-specific expression in zebrafish, a common feature of maternal-effect genes. In addition, it was demonstrated using in situ hybridization that foxr1 was a maternally-inherited transcript that was highly expressed even in early-stage oocytes and accumulated in the developing eggs during oogenesis. We also analyzed the function of foxr1 in female reproduction using a zebrafish CRISPR/cas9 knockout model. It was observed that embryos from the foxr1-deficient females had a significantly lower survival rate whereby they either failed to undergo cell division or underwent abnormal division that culminated in growth arrest at around the mid-blastula transition and early death. These mutant-derived eggs contained dramatically increased levels of p21, a cell cycle inhibitor, and reduced rictor, a component of mTOR and regulator of cell survival, which were in line with the observed growth arrest phenotype. Our study shows for the first time that foxr1 is an essential maternal-effect gene and may be required for proper cell division and survival via the p21 and mTOR pathways. These novel findings will broaden our knowledge on the functions of specific maternal factors stored in the developing egg and the underlying mechanisms that contribute to reproductive success.

Aberrant signaling between germ cells and somatic cells can lead to reproductive disease and depends on diffusible signals, including transforming growth factor-beta (TGFB) -family proteins. The TGFB-family protein Gsdf (gonadal soma derived factor) controls sex determination in some fish and is a candidate for mediating germ cell/soma signaling.

The Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) constitute the only vertebrate taxon that fails to produce red blood cells. These fishes can be paired with closely related, but erythrocyte-producing, notothenioids to discover erythropoietic genes via representational difference analysis. Using a B30.2-domain-encoding DNA probe so derived from the hematopoietic kidney (pronephros) of a red-blooded Antarctic rockcod, Notothenia coriiceps, we discovered a related, novel gene, bloodthirsty (bty), that encoded a 547-residue protein that contains sequential RING finger, B Box, coiled-coil, and B30.2 domains. bty mRNA was expressed by the pronephric kidney of N. coriiceps at a steady-state level 10-fold greater than that found in the kidney of the icefish Chaenocephalus aceratus. To test the function of bty, we cloned the orthologous zebrafish gene from a kidney cDNA library. Whole-mount in situ hybridization of zebrafish embryos showed that bty mRNA was present throughout development and, after the mid-blastula transition, was expressed in the head and in or near the site of primitive erythropoiesis in the tail just prior to red cell production. One- to four-cell embryos injected with two distinct antisense morpholino oligonucleotides (MOs) targeted to the 5'-end of the bty mRNA failed to develop red cells, whereas embryos injected with 4- and 5-bp mismatch control MOs produced wild-type quantities of erythrocytes. The morphant phenotype was rescued by co-injection of synthetic bty mRNA containing an artificial 5'-untranslated region (UTR) with the antisense MO that bound the 5'-UTR of the wild-type bty transcript. Furthermore, the expression of genes that mark terminal erythroid differentiation was greatly reduced in the antisense-MO-treated embryos. We conclude that bty is likely to play a role in differentiation of the committed red cell progenitor.

Pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. Despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. Notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing Antarctic climate. Notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy.

In vertebrates, late oogenesis is a key period during which the oocyte acquires its ability to resume meiosis (i.e. maturational competence) and to develop, once fertilized, into a normal embryo (i.e. developmental competence). However, the molecular mechanisms involved in these key biological processes are far from being fully understood. In order to identify key mechanisms conserved among teleosts and amphibians, we performed a comparative analysis using ovarian tissue sampled at successive steps of the maturational competence acquisition process in the rainbow trout (Oncorhynchus mykiss) and in the clawed toad (Xenopus laevis). Our study aimed at identifying common differentially expressed genes during late oogenesis in both species. Using an existing transcriptomic analysis that had previously been carried out in rainbow trout, candidate genes were selected for subsequent quantitative PCR-based comparative analysis.

The Nme family, previously known as Nm23 or NDPK, is involved in various molecular processes including tumor metastasis and some members of the family, but not all, exhibit a Nucleoside Diphosphate Kinase (NDPK) activity. Ten genes are known in humans, in which some members have been extensively studied. In non-mammalian species, the Nme protein family has received, in contrast, far less attention. The picture of the vertebrate Nme family remains thus incomplete and orthology relationships with mammalian counterparts were only partially characterized. The present study therefore aimed at characterizing the Nme gene repertoire in vertebrates with special interest for teleosts, and providing a comprehensive overview of the Nme gene family evolutionary history in vertebrates.

MicroRNAs (miRNAs) are small, highly conserved non-coding RNAs that play important roles in the regulation of many physiological processes. However, the role of miRNAs in vertebrate oocyte formation (i.e., oogenesis) remains poorly investigated. To gain new insights into the roles of miRNAs in oogenesis, we searched for ovarian-predominant miRNAs. Using a microarray displaying 3,800 distinct miRNAs originating from different vertebrate species, we identified 66 miRNAs that are expressed predominantly in the ovary. Of the miRNAs exhibiting the highest overabundance in the ovary, 20 were selected for further analysis. Using a combination of QPCR and in silico analyses, we identified 8 novel miRNAs that are predominantly expressed in the ovary, including 2 miRNAs (miR-4785 and miR-6352) that exhibit strict ovarian expression. Of these 8 miRNAs, 7 were previously uncharacterized in fish. The strict ovarian expression of miR-4785 and miR-6352 suggests an important role in oogenesis and/or early development, possibly involving a maternal effect. Together, these results indicate that, similar to protein-coding genes, a significant number of ovarian-predominant miRNA genes are found in fish.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change.

Sustainable aquaculture production requires a greater reduction in the use of marine-derived ingredients, and one of the most promising solutions today is the augmentation in the proportion of digestible carbohydrates in aquafeed. This challenge is particularly difficult for high trophic level teleost fish as they are considered to be glucose-intolerant (growth delay and persistent postprandial hyperglycemia observed in juveniles fed a diet containing more than 20% of carbohydrates). It was previously suggested that broodstock could potentially use carbohydrates more efficiently than juveniles, probably due to important metabolic changes that occur during gametogenesis. To investigate this hypothesis, 2-year old male and female rainbow trout (Oncorhynchus mykiss) were either fed a diet containing no carbohydrates (NC) or a 35%-carbohydrate diet (HC) for an entire reproductive cycle. Zootechnical parameters as well as the activities of enzymes involved in carbohydrate metabolism were measured in livers and gonads. Fish were then reproduced to investigate the effects of such a diet on reproductive performance. Broodstock consumed the HC diet, and in contrast to what is commonly observed in juveniles, they were able to grow normally and they did not display postprandial hyperglycemia. The modulation of their hepatic metabolism, with an augmentation of the glycogenesis, the pentose phosphate pathway and a possible better regulation of gluconeogenesis, may explain their improved ability to use dietary carbohydrates. Although the HC diet did induce precocious maturation, the reproductive performance of fish was not affected, confirming that broodstock are able to reproduce when fed a low-protein high-carbohydrate diet. In conclusion, this exploratory work has shown that broodstock are able to use a diet containing digestible carbohydrates as high as 35% and can then grow and reproduce normally over an entire reproductive cycle for females and at least at the beginning of the cycle for males. These results are highly promising and suggest that dietary carbohydrates can at least partially replace proteins in broodstock aquafeed.

The broodstock diet, and in particular the lipid and fatty acid composition of the diet, is known to play a key role in reproductive efficiency and survival of the progeny in fish. A major problem when replacing both fish meal and fish oil by plant sources is the lack of n-3 long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To address this problem, we studied the effect of the plant-based diet supplemented with Schizochytrium sp. microalgae, source of DHA, compared to a conventional commercial diet rich in fish meal and fish oil on reproductive performance and egg quality and the consequences on progeny, in female rainbow trout broodstock.

We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts.

Physical enrichment is known to improve living conditions of fish held in farming systems and has been shown to promote behavioral plasticity in captive fish. However, the brain's regulatory-mechanism systems underlying its behavioral effects remain poorly studied. The present study investigated the impact of a three-month exposure to an enriched environment (EE vs. barren environment, BE) on the modulation of brain function in rainbow trout (Oncorhynchus mykiss) juveniles. Using high-throughput RT-qPCR, we assessed mRNA genes related to brain function in several areas of the trout brain. These included markers of cerebral activity and plasticity, neurogenesis, synaptogenesis, or selected neurotransmitters pathways (dopamine, glutamate, GABA, and serotonin). Overall, the fish from EE displayed a series of differentially expressed genes (neurotrophic, neurogenesis, and synaptogenesis markers) essentially localized in the telencephalon, which could underpin the beneficial effects of complexifying the environment on fish brain plasticity. In addition, EE significantly affected blood plasma c-miRNA signatures, as revealed by the upregulation of four c-miRNAs (miR-200b/c-3p, miR-203a-3p, miR-205-1a-5p, miR-218a-5p) in fish blood plasma after 185 days of EE exposure. Overall, we concluded that complexifying the environment through the addition of physical structures that stimulate and encourage fish to explore promotes the trout's brain function in farming conditions.

The aim of this study was to investigate the respective contribution of maternally-inherited mRNAs and proteins to egg molecular cargo and to its developmental competence in fish using pikeperch as a model. Our study provides novel insights into the understanding of type-specific roles of maternally-inherited molecules in fish. Here we show, for the first time, that transcripts and proteins have distinct, yet complementary, functions in the egg of teleost fish. Maternally-inherited mRNAs would shape embryo neurodevelopment, while maternally-inherited proteins would rather be responsible for protecting the embryo against pathogens. Additionally, we observed that processes directly preceding ovulation may considerably affect the reproductive success by modifying expression level of genes crucial for proper embryonic development, being novel fish egg quality markers (e.g., smarca4 or h3f3a). These results are of major importance for understanding the influence of external factors on reproductive fitness in both captive and wild-type fish species.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor, beta receptor 1a (tgfbr1a) gene, gonadal somatic cell derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out, for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

Ancient bony fishes had heterocercal tails, like modern sharks and sturgeons, with asymmetric caudal fins and a vertebral column extending into an elongated upper lobe. Teleost fishes, in contrast, developed a homocercal tail characterized by two separate equal-sized fin lobes and the body axis not extending into the caudal fin. A similar heterocercal-to-homocercal transition occurs during teleost ontogeny, although the underlying genetic and developmental mechanisms for either transition remain unresolved. Here, we investigated the role of hox13 genes in caudal fin formation as these genes control posterior identity in animals. Analysis of expression profiles of zebrafish hox13 paralogs and phenotypes of CRISPR/Cas9-induced mutants showed that double hoxb13a and hoxc13a mutants fail to form a caudal fin. Furthermore, single mutants display heterocercal-like morphologies not seen since Mesozoic fossil teleosteomorphs. Relaxation of functional constraints after the teleost genome duplication may have allowed hox13 duplicates to neo- or subfunctionalize, ultimately contributing to the evolution of a homocercal tail in teleost fishes.

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

Antarctic fish have adapted to the freezing waters of the Southern Ocean. Representative adaptations to this harsh environment include a constitutive heat shock response and the evolution of an antifreeze protein in the blood. Despite their adaptations to the cold, genome-wide studies have not yet been performed on these fish due to the lack of a sequenced genome. Notothenia coriiceps, the Antarctic bullhead notothen, is an endemic teleost fish with a circumpolar distribution and makes a good model to understand the genomic adaptations to constant sub-zero temperatures.

The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.