Despite the popularity of zebrafish as a research model, its sex determination (SD) mechanism is still unknown. Most cytogenetic studies failed to find dimorphic sex chromosomes and no primary sex determining switch has been identified even though the assembly of zebrafish genome sequence is near to completion and a high resolution genetic map is available. Recent publications suggest that environmental factors within the natural range have minimal impact on sex ratios of zebrafish populations. The primary aim of this study is to find out more about how sex is determined in zebrafish. Using classical breeding experiments, we found that sex ratios across families were wide ranging (4.8% to 97.3% males). On the other hand, repeated single pair crossings produced broods of very similar sex ratios, indicating that parental genotypes have a role in the sex ratio of the offspring. Variation among family sex ratios was reduced after selection for breeding pairs with predominantly male or female offspring, another indication that zebrafish sex is regulated genetically. Further examinations by a PCR-based “blind assay" and array comparative genomic hybridization both failed to find universal sex-linked differences between the male and female genomes. Together with the ability to increase the sex bias of lines by selective breeding, these data suggest that zebrafish is unlikely to utilize a chromosomal sex determination (CSD) system. Taken together, our study suggests that zebrafish sex is genetically determined with limited, secondary influences from the environment. As we have not found any sign for CSD in the species, we propose that the zebrafish has a polygenic sex determination system.

In this review, we provide a detailed overview of studies on the elusive sex determination (SD) and gonad differentiation mechanisms of zebrafish (Danio rerio). We show that the data obtained from most studies are compatible with polygenic sex determination (PSD), where the decision is made by the allelic combinations of several loci. These loci are typically dispersed throughout the genome, but in some teleost species a few of them might be located on a preferential pair of (sex) chromosomes. The PSD system has a much higher level of variation of SD genotypes both at the level of gametes and the sexual genotype of individuals, than that of the chromosomal sex determination systems. The early sexual development of zebrafish males is a complicated process, as they first develop a ‘juvenile ovary’, that later undergoes a transformation to give way to a testis. To date, three major developmental pathways were shown to be involved with gonad differentiation through the modulation of programmed cell death. In our opinion, there are more pathways participating in the regulation of zebrafish gonad differentiation/transformation. Introduction of additional powerful large-scale genomic approaches into the analysis of zebrafish reproduction will result in further deepening of our knowledge as well as identification of additional pathways and genes associated with these processes in the near future.

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species’ native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics. We describe the genome assembly of Asian seabass (Lates calcarifer), a marine teleost with aquaculture relevance. Though >500 eukaryotic genome sequences are available in public repositories, the majority are highly fragmented with incomplete assemblies, which explains why considerable effort and resources are often spent to improve their quality after publication. In our study, we employed long read sequencing combined with genetic and optical mapping, and syntenic information to produce a chromosomal level assembly. The largely continuous genome assembly will be useful for comparative genomics and offers an opportunity to look into regions less explored such as tandem repeats (the core component of centromeres and telomeres). In addition, population structure of the species was analysed based on low-coverage genome sequence information from 61 individuals representing diverse geographic locations stretching from North-Western India across South-East Asia and Australia to Papua New Guinea.

Significance: Fish exhibit remarkable sexual plasticity. However, the underlying mechanism of heat-induced sex reversal is still unclear. Here we first established the conditions for heat-induced reprogramming of sexual phenotypes in zebrafish through sex ratio analysis and gonad transcriptomics. Sex ratio response to heat was family-specific and resulted in masculinization. We observed two heat-induced gonadal transcriptomic profiles per sex in adults, among them neomales and, strikingly, females with an ovary but a “male-like” transcriptome. The latter indicates major transcriptomic reprogramming with preserved organ structure, an interesting observation in vertebrates. In all heat-treated juveniles, we also observed a male-like transcriptome. Overall, this study reveals novel lasting thermal effects on fish gonads, with practical implications for studying the effects of global warming in natural populations.

Background: Mitochondrial DNA-derived sequences have become popular markers for evolutionary studies, as their comparison may yield significant insights into the evolution of both the organisms and their genomes. From the more than 24,000 teleost species, only 254 complete mtDNA sequences are available (GenBank status on 06 Sep 2006). In this paper, we report the complete mitochondrial genome sequence of Asian arowana, a basal bonytongue fish species, which belongs to the order of Osteoglossiformes. Results: The complete mitochondrial genomic sequence (mtDNA) of Asian arowana (Scleropages formosus) was determined by using shotgun sequencing method. The length of Asian arowana mtDNA is ca. 16,650 bp (its variation is due to polymorphic repeats in the control region), containing 13 protein-coding genes, 22 tRNA and 2 rRNA genes. Twelve of the thirteen protein coding genes were found to be encoded by the heavy strand in the order typically observed for vertebrate mitochondrial genomes, whereas only nad6 was located on the light strand. An interesting feature of Asian arowana mitogenome is that two different repeat arrays were identified in the control region: a 37 bp tandem repeat at the 5' end and an AT-type dinucleotide microsatellite at the 3' end. Both repeats show polymorphism among the six individuals tested; moreover the former one is present in the mitochondrial genomes of several other teleost groups. The TACAT motif described earlier only from mammals and lungfish was found in the tandem repeat of several osteoglossid and eel species. Phylogenetic analysis of fish species representing Actinopterygii and Sarcopterygii taxa has shown that the Asian arowana is located near the baseline of the teleost tree, confirming its status among the ancestral teleost lineages. Conclusion: The mitogenome of Asian arowana is very similar to the typical vertebrate mitochondrial genome in terms of gene arrangements, codon usage and base composition. However its control region contains two different types of repeat units at both ends, an interesting feature that to our knowledge has never been reported before for other vertebrate mitochondrial control regions. Phylogenetic analysis using the complete mtDNA sequence of Asian arowana confirmed that it belongs to an ancestral teleost lineage.

Background: The Asian seabass(Lates calcarifer)is a protandrous hermaphrodite that typically matures as a male at approximately 2–4 years of age and then changes sex in subsequent years. Although several sexual maturation stages have been described histologically for both testis and ovary, the underlying gene expression profiles remain lacking. The development of a gene expression platform is therefore necessary to improve our understanding of the gonad development of this cultured teleost species.Methods: Thirty Asian seabass gonads were collected from farms in Singapore, examined histologically and staged according to their sex and gonadal maturation status. Partial coding sequences of 24 sex-related genes were cloned using degenerate primers and were sequenced. Additional 13 cDNA sequences were obtained through next-generation sequencing. A real-time qPCR was then performed using the microfluidic-based Fluidigm 48.48 Dynamic arrays.Results: We obtained 17 ovaries and 13 testes at various stages of sexual maturation. Of the 37 genes that were tested, 32 (86%) showed sexually dimorphic expression. These genes included sex-related genes,sox9,wt1,amh,nr5a2,dmrt1andnr0b1, which showed testis-enhanced expression similar to other vertebrate species. Known male- and female-enhanced germ cells markers, which were established from studies in other species, similarly showed testis- and ovary-enhanced expression, respectively, in the Asian seabass. Three pro-Wnt signaling genes were also upregulated in the ovary, consistent with existing studies that suggested the role of Wnt signaling in ovarian differentiation in teleosts and mammals. The expression patterns of genes involved in steroidogenesis, retinoic acid metabolism, apoptosis and NF-κB signaling were also described. We were able to classify gonads according to sex and gonadal maturation stages, based on their small-scale transcriptomic profiles, and to uncover a wide variation in expression profiles among individuals of the same sex.Conclusions: The analysis of a selected set of genes related to reproduction and in sufficient number of individuals using a qPCR array can elucidate new insights into the molecular mechanisms involved in Asian seabass gonad development. Given the conservation of gene expression patterns found in this study, these insights may also help us draw parallels with other teleosts.