Effect of high temperatures on sex ratio and differential expression analysis (RNA-seq) of sex-determining genes in <i>Oreochromis niloticus</i> from different river basins in Benin

peer reviewedAbstract The high temperature sex reversal process leading to functional phenotypic masculinization during development has been widely described in Nile tilapia (Oreochromis niloticus) under laboratory or aquaculture conditions and in the wild. In this study, we selected five wild populations of O. niloticus from different river basins in Benin and produced twenty full-sib families of mixed-sex (XY and XX) by natural reproduction. Progenies were exposed to room temperature or high (36.5°C) temperatures between 10 and 30 days post-fertilization (dpf). In control groups, we observed sex ratios from 40 to 60% males as expected, except for 3 families from the Gobé region which showed a bias towards males. High temperature treatment significantly increased male rates in each family up to 88%. Transcriptome analysis was performed by RNA-sequencing (RNA-seq) on brains and gonads from control and treated batches of six families at 15 dpf and 40 dpf. Analysis of differentially expressed genes, differentially spliced genes, and correlations with sex reversal was performed. In 40 dpf gonads, genes involved in sex determination such as dmrt1, cyp11c1, amh, cyp19a1b, ara, and dax1 were upregulated. In 15 dpf brains, a negative correlation was found between the expression of cyp19a1b and the reversal rate, while at 40 dpf a negative correlation was found between the expression of foxl2, cyp11c1, and sf1 and positive correlation was found between dmrt1 expression and reversal rate. Ontology analysis of the genes affected by high temperatures revealed that male sex differentiation processes, primary male sexual characteristics, autophagy, and cilium organization were affected. Based on these results, we conclude that sex reversal by high temperature treatment leads to similar modifications of the transcriptomes in the gonads and brains in offspring of different natural populations of Nile tilapia, which thus may activate a common cascade of reactions inducing sex reversal in progenies

Genetic and environmental interactions involved in early sex determination in a teleost model: epigenetics, heritability and adaptation

Le Tilapia du Nil, Oreochromis niloticus, est une espèce de poisson tropicale gonochorique d’origine africaine d’un grand intérêt économique. Cette espèce présente un déterminisme du sexe complexe, dépendant de facteurs génétiques majeurs (système XX|XY), de facteurs génétiques mineurs et de facteurs environnementaux, en particulier de la température. Il a en effet été montré que des hautes températures (supérieures à 32°C) appliquées durant le développement induisent une masculinisation des individus dans des proportions variables. De grandes inconnues existent cependant quant à l’intégration de ce signal et à son interaction avec les autres facteurs. Les objectifs de ce travail ont été articulés autour de 4 grands axes de recherche : (1) La remise en contexte de l’inversion sexuelle par les hautes températures, (2) la recherche de nouveaux gènes (3) l’étude de potentiels mécanismes de régulation épigénétique et (4) d’un potentiel rôle du cerveau dans l’inversion sexuelle par les hautes températures. Une expérience de preferendum thermique a permis de montrer que des juvéniles à génotype sexuel XX choisissaient volontairement des températures masculinisantes, ce qui entraine une déviation du sexe ratio de la population. Cela donne du poids quant à l’existence du phénomène de masculinisation par les hautes températures dans le milieu naturel. Des études d’inversion sexuelle thermique tardive sur des individus à génotype sexuel YY ont permis de remettre en évidence un phénomène de féminisation par les hautes températures, ce qui pousse à développer un modèle plus complexe pour expliquer l’intégration du signal température lors du déterminisme du sexe chez O. niloticus et a amené à intégrer la féminisation par les hautes températures aux différents axes de recherche de ce travail. Lors des différentes expériences d’inversion sexuelle thermique, il a été observé que les répétitions d’un croisement identique donnaient des progénitures avec une réponse variable à la température. Il a également été observé que l’origine du mâle utilisé (inversion spontanée, thermique ou hormonale) a une influence sur la thermosensibilité de la progéniture. Ces deux observations ont mené à l’hypothèse de l’implication de mécanismes épigénétiques dans l’inversion sexuelle à la haute température. Le méthylome des gonades d’individus XX et XY à 15 jpf et 40 jpf a donc été étudié par séquençage Oxford Nanopore Technology®. Aucune différence de méthylation des gènes connus pour être impliqués dans le déterminisme du sexe n’a été observée dans le cadre de notre étude. Des différences de méthylation dans des gènes impliqués dans le métabolisme ont cependant été mises en évidence. Le transcriptome des gonades et des têtes à 15 jpf et à 40 jpf d’individus XX et XY élevés en condition contrôle (28°C) et en traitement par la haute température (36,5°C) a été analysé par RNA-seq dans le but de trouver des gènes cibles impliqués dans la masculinisation et dans la féminisation par les hautes températures. Beaucoup de gènes cibles ont été identifiés et des critères de sélection ont été appliqués pour identifier les gènes d’intérêt les plus pertinents. Dans les gonades, foxl2, un gène codant pour un facteur de transcription activateur de la voie de la stéroïdogenèse femelle a pu a été identifié comme étant sous-exprimé lors de la masculinisation et surexprimé lors de la féminisation. Dans les têtes à 15 jpf, il a été observé que le gène dbi était surexprimé lors de la masculinisation et sous-exprimé lors de la féminisation. Ce gène code pour plusieurs neuropeptides potentiellement circulants qui sont connus pour être impliqués dans l’import de cholestérol dans les mitochondries, qui est la première étape de la stéroïdogenèse. La mise en évidence de dbi[2] dans les têtes couplées aux résultats d’inversion thermique précoce, aux déplacement précoces des juvéniles vers les hautes températures dans l’expérience de preferendum thermique et à la remise en évidence de la féminisation nous ont amenés à émettre l’hypothèse de l’implication du cerveau dans l’intégration du signal température. Pour illustrer cette hypothèse, nous avons développé un modèle général en 3 parties : (1) l’intégration du signal par le cerveau, (2) le moyen de communication entre le cerveau et les gonades et (3) la réponse spécifique de la gonade entrainant la différenciation sexuelle. En conclusion, ce travail a permis de mieux caractériser certains phénomènes liés à l’inversion sexuelle chez le tilapia du Nil, mais aussi de trouver, par des études de séquençage à haut-débit, de nouveaux gènes cibles dont l’étude approfondie permettraient de mieux comprendre l’intégration du signal température lors du déterminisme sexuel de O. niloticus.SRL Fish Sex Ratio Lability: Mechanisms and their Morphological, Behavioural and Ecological Consequence

On the necessity and biological significance of threshold-free regulon prediction outputs

The in silico prediction of cis-acting elements in a genome is an efficient way to quickly obtain an overview of the biological processes controlled by a trans-acting factor, and connections between regulatory networks. Several regulon prediction web tools are available, designed to identify DNA motifs predicted to be bound by transcription factors using position weight matrix-based algorithms. In this paper we expose and discuss the conflicting objectives of software creators (bioinformaticians) and software users (biologists), who aim for reliable and exhaustive prediction outputs, respectively. Software makers, concerned with providing tools that minimise the number of false positive hits, often impose a stringent threshold score for a sequence to be included in the list of the putative cis-acting sites. This rigidity eventually results in the identification of strongly reliable but largely straightforward sites, i.e. those associated with genes already anticipated to be targeted by the studied transcription factor. Importantly, this biased identification of strongly bound sequences contrasts with the biological reality where, in many circumstances, a weak DNA-protein interaction is required for the appropriate gene's expression. We show here a series of transcriptionally controlled systems involving weakly bound cis-acting elements that could never have been discovered because of the policy of preventing software users from modifying the screening parameters. Proposing only trustworthy prediction outputs thus prevents biologists from fully utilising their knowledge background and deciding to analyse statistically irrelevant hits that could nonetheless be potentially involved in subtle, unexpected, though essential cis-trans relationships

Study of gene expression during high temperature sex reversal in Oreochromis niloticus

peer reviewe

Towards a sustainable aquaponic farm model in Belgium : productivity and economical analysis of two constrating fish production models (tilapia vs pikeperch)

editorial reviewe

Temperature preference of Nile tilapia (Oreochromis niloticus) juveniles induces spontaneous sex reversal

Nile tilapia (Oreochromis niloticus) is an African freshwater fish that displays a genetic sex determination system (XX|XY) where high temperatures (above 32°C to 36.5°C) induce masculinization. In Nile tilapia, the thermosensitive period was reported from 10 to 30 days post fertilization. In their natural environment, juveniles may encounter high temperatures that are above the optimal temperature for growth (27–30°C). The relevance of the thermal sex reversal mechanism in a natural context remains unclear. The main objective of our study is to determine whether sexually undifferentiated juveniles spontaneously prefer higher, unfavorable temperatures and whether this choice skews the sex ratio toward males. Five full-sib progenies (from 100% XX crosses) were subjected to (1) a horizontal three-compartment thermal step gradient (thermal continuum 28°C– 32°C– 36.5°C) during the thermosensitive period, (2) a control continuum (28°C– 28°C– 28°C) and (3) a thermal control tank (36.5°C). During the first days of the treatment, up to an average of 20% of the population preferred the masculinizing compartment of the thermal continuum (36.5°C) compared to the control continuum. During the second part of the treatment, juveniles preferred the lower, nonmasculinizing 32°C temperature. This short exposure to higher temperatures was sufficient to significantly skew the sex ratio toward males, compared to congeners raised at 28°C (from 5.0 ± 6.7% to 15.6 ± 16.5% of males). The proportion of males was significantly different in the thermal continuum, thermal control tank and control continuum, and it was positively correlated among populations. Our study shows for the first time that Nile tilapia juveniles can choose a masculinizing temperature during a short period of time. This preference is sufficient to induce sex reversal to males within a population. For the first time, behavior is reported as a potential player in the sex determination mechanism of this species

A Realistic Mixture of Persistent Organic Pollutants Affects Zebrafish Development, Behavior, and Specifically Eye Formation by Inhibiting the Condensin I Complex

peer reviewedPersistent organic pollutants (POPs) are posing major environmental and health threats due to their stability, ubiquity, and bioaccumulation. Most of the numerous studies of these compounds deal with single chemicals, although real exposures always consist of mixtures. Thus, using different tests, we screened the effects on zebrafish larvae caused by exposure to an environmentally relevant POP mixture. Our mixture consisted of 29 chemicals as found in the blood of a Scandinavian human population. Larvae exposed to this POP mix at realistic concentrations, or sub-mixtures thereof, presented growth retardation, edemas, retarded swim bladder inflation, hyperactive swimming behavior, and other striking malformations such as microphthalmia. The most deleterious compounds in the mixture belong to the per- and polyfluorinated acids class, although chlorinated and brominated compounds modulated the effects. Analyzing the changes in transcriptome caused by POP exposure, we observed an increase of insulin signaling and identified genes involved in brain and eye development, leading us to propose that the impaired function of the condensin I complex caused the observed eye defect. Our findings contribute to the understanding of POP mixtures, their consequences, and potential threats to human and animal populations, indicating that more mechanistic, monitoring, and long-term studies are imperative.PROTECTion against Endocrine Disruptor

Osteoblast populations and bone extracellular matrix proteins during skeletal development in zebrafish.

peer reviewedUnderstanding osteoblast differentiation and their function in bone matrix deposition and mineralization is central to the comprehension of bone development and of various bone pathologies. It is crucial to not only follow the expression of some landmark transcription factors or ECM proteins, but to also investigate the status of signaling pathways and factors regulating these processes. This can only be achieved by assessing the entire transcriptome of these cells. We employed developing Tg(sp7:gfp-cr) zebrafish larvae to study the osteoblast transcriptome by isolating the entire osteoblast population at 4dpf, focusing on the possible existence of specific subpopulations. RNA-seq. Analysis revealed two populations at this stage and the differentially expressed gene list that showed genes related to bone and cartilage development. Further, GO term analysis gave us hits very specific to bone extracellular matrix, skeletal development, cartilage and bone specific genes, transcription factors and Calcium regulating hormones etc. to name a few. The expression and function of specific genes of bone extracellular matrix, revealed by these analyses, were investigated using mutant lines and state of the art technologies. Thereafter, characterized for bone and cartilage staining’s at various stages of development. Taken together, these different approaches should allow us to better understand the function of the different genes and the regulatory circuits that are involved in skeletal differentiation, morphogenesis and mineralization, specifically in a non-mammalian vertebrate, but potentially also in humans

A multi-trait evaluation framework to assess the consequences of polyculture in fish production: An application for pikeperch in recirculated aquaculture systems

Polyculture offers an alternative to monoculture in recirculated aquaculture systems (RAS). Nevertheless, RAS polyculture can potentially result in beneficial as well as detrimental consequences. This places a premium on assessing consequences of a particular polyculture prior to its implementation in farming production. In fish aquaculture, most of previous assessments of polyculture consequences have been carried out through multi-trait approach by analysing independently species traits related to a single biological function. Yet, polyculture can impact many biological functions and the overall consequences are the result of multiple interactions from different functions. Therefore, an integrative analysis is required to synthesise the polyculture consequences at the multi-trait levels. Here we propose a multi-trait evaluation framework (i.e. considering traits related to multiple functions), for the polyculture consequence assessment. We analyse this framework through multivariate analysis prior integrating results for each trait. As test case, we used pikeperch (Sander lucioperca), reared in monoculture and in polyculture with European perch (Perca fluviatilis), common carp (Cyprinus carpio) and black-bass (Micropterus salmoides). Our results revealed the importance of applying multi-trait framework to obtain a reliable assessment of fish polycultures. The divergence that might occur in the expression of some traits in the same polyculture has been highlighted to support this argument. Through the integration method, it was possible to find the suitable combination(s) of fish and to exclude the unsuitable ones. For future researches, we suggest to support the multi-trait assessment by weighting the traits involved in the multivariate analysis

Temperature preference of Nile tilapia (Oreochromis niloticus) juveniles induces spontaneous sex reversal.

Nile tilapia (Oreochromis niloticus) is an African freshwater fish that displays a genetic sex determination system (XX|XY) where high temperatures (above 32°C to 36.5°C) induce masculinization. In Nile tilapia, the thermosensitive period was reported from 10 to 30 days post fertilization. In their natural environment, juveniles may encounter high temperatures that are above the optimal temperature for growth (27-30°C). The relevance of the thermal sex reversal mechanism in a natural context remains unclear. The main objective of our study is to determine whether sexually undifferentiated juveniles spontaneously prefer higher, unfavorable temperatures and whether this choice skews the sex ratio toward males. Five full-sib progenies (from 100% XX crosses) were subjected to (1) a horizontal three-compartment thermal step gradient (thermal continuum 28°C- 32°C- 36.5°C) during the thermosensitive period, (2) a control continuum (28°C- 28°C- 28°C) and (3) a thermal control tank (36.5°C). During the first days of the treatment, up to an average of 20% of the population preferred the masculinizing compartment of the thermal continuum (36.5°C) compared to the control continuum. During the second part of the treatment, juveniles preferred the lower, nonmasculinizing 32°C temperature. This short exposure to higher temperatures was sufficient to significantly skew the sex ratio toward males, compared to congeners raised at 28°C (from 5.0 ± 6.7% to 15.6 ± 16.5% of males). The proportion of males was significantly different in the thermal continuum, thermal control tank and control continuum, and it was positively correlated among populations. Our study shows for the first time that Nile tilapia juveniles can choose a masculinizing temperature during a short period of time. This preference is sufficient to induce sex reversal to males within a population. For the first time, behavior is reported as a potential player in the sex determination mechanism of this species