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In zebrafish, Danio rerio, a polygenic pattern of sex determination or a female heterogamety with possible influences of environmental factors is assumed. The present study focuses on the effects of an elevated water temperature (35°C) during the embryonic development on sex determination in zebrafish. Eggs derived from 3 golden females were fertilized by the same mitotic gynogenetic male and exposed to a water temperature of 35°C, applied from 5 to 10 h post fertilization (hpf), from 5 to 24 hpf, and from 5 to 48 hpf, which correspond to the following developmental stages: gastrula, gastrula to segmentation, and gastrula to pharyngula stage, respectively. Hatching and survival rates decreased with increasing exposure to high water temperatures. Reductions in the hatching and survival rates were not responsible for differences in sex ratios. Accordingly, exposition of the fertilized eggs to a high temperature (35°C) leads to an increase of the male proportion from 22.0% in the controls to a balanced sex ratio (48.3, 47.5, and 52.6%) in the gastrula, segmentation, and pharyngula groups, respectively. These results prove the possibility to change the pathway of sexual determination during early embryonic stages in zebrafish by exposure to a high water temperature.

Temperature effects on sex determination or differentiation exist in many fish species, with high temperatures predominantly producing more males. The present study aimed at elucidating the genetic background of temperature effects on sex differentiation in zebrafish. Experimental fish were generated by matings between 4 or 6 golden females and a normal or a mitotic gynogenetic male, respectively. All the larvae were reared at 28.5°C until they were divided into 3 groups per full-sib family, a control group raised at 28.5°C and 2 treatment groups reared at 35°C from 20 to 30 dpf or 25 to 35 dpf, respectively. Backcross progenies, reared at 28.5°C, were derived from F1 temperature-treated sons (35°C, 25–35 dpf) that were sired by a mitotic gynogenetic male and their corresponding mothers. No significant differences were observed regarding the survival rate between the control and treatment groups. Significant differences in the phenotypic male proportions from the controls were observed in groups treated at 35°C. The sex ratio in zebrafish was influenced by the male spawner, the female spawner, and a significant interaction of genotype by temperature. Backcross experiments point to a continuum of major genetic, minor genetic, and environmental factors in the expression of the phenotypic sex in zebrafish.

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Owing to the demand for sustainable sex-control protocols in aquaculture, research in tilapia sex determination is gaining momentum. The mutual influence of environmental and genetic factors hampers disentangling the complex sex determination mechanism in Nile tilapia (Oreochromis niloticus). Previous linkage analyses have demonstrated quantitative trait loci for the phenotypic sex on linkage groups 1, 3, and 23. Quantitative trait loci for temperature-dependent sex reversal similarly reside on linkage group 23. The anti-Müllerian hormone gene (amh), located in this genomic region, is important for sexual fate in higher vertebrates, and shows sexually dimorphic expression in Nile tilapia. Therefore this study aimed at detecting allelic variants and marker-sex associations in the amh gene. Sequencing identified six allelic variants. A significant effect on the phenotypic sex for SNP ss831884014 (p<0.0017) was found by stepwise logistic regression. The remaining variants were not significantly associated. Functional annotation of SNP ss831884014 revealed a non-synonymous amino acid substitution in the amh protein. Consequently, a fluorescence resonance energy transfer (FRET) based genotyping assay was developed and validated with a representative sample of fish. A logistic linear model confirmed a highly significant effect of the treatment and genotype on the phenotypic sex, but not for the interaction term (treatment: p<0.0001; genotype: p<0.0025). An additive genetic model proved a linear allele substitution effect of 12% in individuals from controls and groups treated at high temperature, respectively. Moreover, the effect of the genotype on the male proportion was significantly higher in groups treated at high temperature, giving 31% more males on average of the three genotypes. In addition, the groups treated at high temperature showed a positive dominance deviation (+11.4% males). In summary, marker-assisted selection for amh variant ss831884014 seems to be highly beneficial to increase the male proportion in Nile tilapia, especially when applying temperature-induced sex reversal.

BackgroundIn Nile tilapia sex determination is governed by a male heterogametic system XX/XY either on LG1 or LG23. The latter carries a Y-specific duplicate of the amh gene, which is a testis-determining factor. Allelic variants in the amh gene demonstrated to be major triggers for autosomal and temperature-dependent sex reversal. Further, QTL on LG23 and LG20 show a temperature-responsiveness with influence on the phenotypic sex relative to the sex chromosomes. Here we present a ddRADseq based approach to identify genomic regions that show unusual large differentiation in terms of fixation index (FST) between temperature-treated pseudomales and non-masculinized females using a comparative genome-scan. Genome-wide associations were identified for the temperature-dependent sex using a genetically all-female population devoid of amh-ΔY.ResultsTwenty-two thousand three hundred ninety-two SNPs were interrogated for the comparison of temperature-treated pseudomales and females, which revealed the largest differentiation on LG23. Outlier FST-values (0.35–0.44) were determined for six SNPs in the genomic interval (9,190,077–11,065,693) harbouring the amh gene (9,602,693–9,605,808), exceeding the genome-wide low FST of 0.013. Association analysis with a set of 9104 selected SNPs confirmed that the same genomic region on LG23 exerts a significant effect on the temperature-dependent sex.ConclusionsThis study highlights the role of LG23 in sex determination, harbouring major determinants for temperature-dependent sex reversal in Nile tilapia. Furthermore FST outlier detection proves a powerful tool for detection of sex-determining regions in fish genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3930-0) contains supplementary material, which is available to authorized users.

This study for the first time screens microsatellite markers for associations with the temperature-dependent sex of Oreochromis niloticus. Previous studies revealed markers on linkage groups (LG) 1, 3, and 23 to be linked to the phenotypic sex of Oreochromis spp. at normal rearing temperatures. Moreover, candidate genes for sex determination and differentiation have been mapped to these linkage groups. Here, 6 families of a temperature-treated genetically all-female (XX) F₁-population were genotyped for 21 microsatellites on the 3 LGs. No population-wide QTL (quantitative trait loci) or marker trait associations could be detected. However, family-specific QTL were found on LG 1 flanked by UNH995 and UNH104, on LG 3 at the position of GM213, and on LG 23 next to GM283. Moreover, family-specific single marker associations for UNH995 and UNH104 on LG 1, GM213 on LG 3, as well as for UNH898 and GM283 on LG 23 were detected. Yet, marker trait associations could not explain the temperature-dependent sex of all fish in the respective families. The molecular cue for the temperature-dependent sex in Nile tilapia might partially coincide with allelic variants at major and minor genetic sex determining factors. Moreover, additional QTL contributing to variable liabilities towards temperature might persist on other LGs.

High mortality from cannibalism in crowded cultures of the noble crayfish (Astacus astacus L.) was the reason why the efforts for an intensive production of this species in the 1980s were largely stopped. In the present study, 14-to 15-month-old juveniles of A. astacus were cultured in tanks of an indoor recirculation system under constant 'summer conditions' (19°C, LD 16:8) from early August to late January. The animals continued to moult and grow, indicating that the absence of these processes in the field from autumn to late spring does not involve any endogenous programming. The experimental design (suitable hiding places in excess, highly diversified diet, and special daily and lunar light regimes) ensured high survival (>90%) and growth rates which were largely independent of stocking rate (15-60 individuals per m 2 ). Moulting occurred during daytime which allowed freshly moulted animals to escape from cannibalistic attacks of their nocturnal conspecifics. There is a first indication that an artificial moonlight cycle can synchronize moulting events (maximum around 'new moon'), in this way contributing to a further decrease in mortality. The results encourage a resumption of efforts for an intensive production of A. astacus in indoor recirculation systems.