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Sexual dimorphism of aquaculture traits is common for farmed fish. The Nile tilapia is the second most important farmed species with a production 6 million tons in 2020. Intensive farming relies on the production of all-males due to males' higher growth rate, and as a way to avoid uncontrolled reproductions. Currently the large majority of the all-male productions are obtained through androgen treatments. We aim to use more sustainable procedures to produce all-males such as the use of YY males. Until now, the use of YY males has not been reliable. This is because sex determination in Nile tilapia is complex and controlled by several factors. Although sex determinism follows an XX/XY system, the linkage group (LG) carrying the major sex determinant gene has been assigned to either LG1 or LG23, depending on the domesticated strain. Minor parental factors can also be implicated and in addition, high temperatures can override the genetic determinism. It is not clear to what extent these differences in sex determination are due to natural diversity in the mechanisms of sex determination or due to processes of domestication. It is therefore necessary to better understand the genetic basis of sex determinism in order to use this approach to generate all-males. For this, we decided to work on wild populations in Africa that have not suffered domestic manipulations. We underwent a study of sex determination in several wild populations from West (Lake Volta, Lake Kou) and East Africa (Lake Koka and Lake Hora). We used complementary genomic approaches of ddRAD, whole genome sequencing and long Nanopore reads. We were able to determine that the amh region present on LG23 is the major sex-determining region in most of these populations. Nevertheless, our results also show that there is high polymorphism in this SD region. Furthermore, there are populations that lack the male-specific amh duplication on LG23. Hence, there are no universal Y markers for Nile tilapia. It is necessary to work at the population level to identify and validate sex markers, in order to allow the local production of YY males.

We have developed important genomic tools and methods of comparative genomics in the Nile tilapia, Oreochromis niloticus. The physical comparative map generated with BACs allowed us to calculate that there were about one rearrangement of intra-chromosomal every 3 Mb. For most chromosomes (about 40-48 Mb) there were about 16 breakpoints with ie eight inversions per chromosome. When extrapolated to the large bivalent chromosome Chr 1-LG3 (about 100-120 Mb), we estimate probably 40 breakpoints and 20 inversions. Our work suggests that intrachromosomal rearrangements with 160 inversions have occurred since the divergence of tilapia with stickleback, the species found to be closely related to tilapia. We have also developed a high resolution RH map containing 1296 markers distributed over the 22 chromosomes, that are now identifiable by cytogenetic analysis. The linkage groups of the genetic map were anchored with 149 microsatellites to chromosomes. A inverted zone was found by BAC-FISH close to the centromere in one of the Chr1-LG3 chromosomes of XY males and in 2 Chr1-LG3 chromosomes of YY males, but absent in XX females. This inversion is not in the region where recombination is suppressed. These results suggest that the Chr1-LG3 chromosome is still acting as a sex chromosome. The Nile tilapia appears to be in the midst of a transition between ZW to XY systems. In the Chr1-LG3 chromosome, 515 protein-encoding genes were found with 152 being tilapia-specific, while in the inversed region 19 genes were found including the gene dmrtA1. Highly conserved and ordered syntenies have been identified with the genes of the inversion zone, not only with the stickleback (Chr 7 and Chr9) but also with the chicken Z chromosome, the platypus X5 Chr and also with human Chr9, in which genes involved in sex determination are located. (Texte integral)

Anti-müllerian hormone (AMH) is expressed in male embryos and represses development of müllerian ducts during testis differentiation in mammals, birds and reptiles. Amh orthologues have been identified in teleosts despite them lacking müllerian ducts. Previously we found sexually dimorphic aromatase activity in tilapia brains before ovarian differentiation. This prompted us to search for further dimorphisms in tilapia brains during sex differentiation and see whether amh is expressed. We cloned the tilapia amh gene and found that it contains 7 exons but no spliced forms. The putative protein presents highest homologies with Amh proteins of pejerrey and medaka as compared to other Perciformes. We analysed amh expression in adult tissues and found elevated levels in testes, ovary and brain. Amh expression was dimorphic with higher levels in XY male brains at 10–15 dpf, when the gonads were still undifferentiated and gonadal amh was not dimorphic. Male brains had 2.7-fold higher amh expression than gonads. Thereafter, amh levels decreased in the brain while they were up-regulated in differentiating testes. Our study indicates that amh is transcribed in male brains already at 10 dpf, suggesting that sexual differentiation may be occurring earlier in tilapia brain than in gonads. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Within the tilapia group, the sex determining locus has been located on linkage group 1 (LG1) in Oreochromis niloticus, whereas in O. aureus both LG1 and LG3 are sex-linked. Using specific BAC clones as probes for FISH, LG3 and LG1 have been located on the largest and on a smaller chromosome pair, respectively. The largest pair presents various traits of a relatively old sex chromosome, whereas LG1 seems to be at an early stage of sex chromosome evolution. Evidence for interactions between the two sex linked loci has been demonstrated at least in O. aureus , with minor factors (genetic and environmental factors) also modulating sex ratios. We have taken advantage of the 13-fold larger size of one of the chromosome chromosome pairs to microdissect it in order to search for genes specific to this large pair. This chromosome was microdissected from metaphase preparations of homogametic genotypes (XX and YY for O. niloticus, and ZZ for O. aureus). Microdissected chromosomes were amplified by DOP-PCR and the DOP-products were then used to screen a gonadal cDNA library. Positive clones were hybridized by FISH on metaphase spreads of different genotypes from both species. We evidenced positive signals located on the large chromosomes of both strands indicating specificity of the probes, despite the presence of large amounts of repetitive sequences on these large sex chromosomes. The conservation of the structure of the large pair between the two species is discussed. (Texte intégral)

Sex in the Nile tilapia, Oreochromis niloticus, is governed by the interactions between a complex genetic sex determination system and the influence of temperature. High temperatures applied during a critical period of sex differentiation can induce masculinisation in some progenies originating from both domestic and wild stocks. Differences in thermo-sensitivity were observed between the wild populations and both paternal and maternal effects have been demonstrated, suggesting that it is a heritable trait. This has been confirmed by the development of divergent lines for thermo-sensitivity. Two thermo-sensitive divergent lines were selected with regard to their sex ratios showing high (=90% males) and low responses (only 54%) when treated at 36°C temperatures. At least one of the mechanisms by which temperature exerts its effects on sex differentiation is through a downregulation of aromatase Cyp19. Expression profiles were analysed in monosex XX populations treated at 27°C and 36°C temperatures showing that temperature up-regulates Sox9s, Amh, IGFs, Dax1 and down-regulates other genes such as aromatase Cyp19-1a. Variation in these gene expression levels was associated with the percentage of males obtained by temperature treatments. The divergent thermo-sensitive lines have been used to complement our study done on the monosex populations. Expression of Cyp19-1a was also analysed in these two divergent lines. (Texte intégral)

Cichlid fish of the Tilapia group are an ideal model for studying sex determination. Sequencing of the Tilapia (Oreochromis niloticus) genome has started in USA and France and genetic and physical linkage maps based on high resolution fingerprints of BAC clones are produced. We have in charge the chromosomal anchoring of some sex linked BACs from the genetic mapping. Some BAC libraries constructed from a male O. niloticus have been assembled into a physical map. Generally, species with a major sex determination locus on linkage group 1 (LG1) are male heterogametic and species with a major sex determination locus on linkage group 3 (LG3) are female heterogametic. In order to confirm that LG1 and LG3 are on two different, unlinked chromosomes, we physically mapped the sex-linked markers by chromosomal FISH on the chromosomes of two closely related Tilapia species, O. niloticus and O. aureus. These experiments demonstrated that LG3 corresponds to the largest chromosome in the Tilapia karyotype, and that LG1 corresponds to a different, smaller chromosome. The chromosome pair corresponding to LG3 in O. niloticus has been manually microdissected and a series of cDNAs isolated from this microdissected product have been cloned, sequenced and selected for chromosomal FISH. Their hybridization helps to validate the results. (Texte intégral)