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

The high temperature sex reversal process leading to functional phenotypic masculinization during development has been widely described in Nile tilapia (Oreochromis n iloticus) 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.

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

[This corrects the article DOI: 10.1371/journal.pone.0212504.].

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.

Temperature Preference and Sex Differentiation in African Catfish, Clarias gariepinus.

The African catfish Clarias gariepinus has a genetic sex determination system in which high temperature induces masculinization. The thermosensitive period for sex differentiation is short and occurs very early (from 6 to 8 days posthatching [dph]). As young juveniles can encounter high masculinizing temperature (36.5°C) in African water points, we aimed to determine the thermal preference of sexually undifferentiated juveniles and investigate if they spontaneously move toward high masculinizing temperature. Experiments were carried out in an environmental continuum (28-28-28°C and 28-32-36.5°C) made up of three 50-L aquariums connected together. Four hundred larvae from 10 different full-sib progenies were reared successively from 2 to 14 dph in these facilities. Before and after thermal treatments, fish were reared at 28°C until sex ratio determination at 70 dph. In the control continuum, fish were nearly equally distributed in the three compartments. Conversely, in the thermal continuum, compartment occupation significantly differed with progeny and period. During the highly thermosensitive period, two of five progenies significantly preferred (54.7% and 39.8% occupation) the 36.5°C compartment. All tested progenies reared in thermal continuum and separated 36.5°C aquarium showed a skewed sex ratio toward the male phenotype (78-100%). Nevertheless, no correlation was found between 36.5°C compartment occupation and sex ratio in thermal continuum groups. As masculinization temperature could be encountered in African water points during the spawning season, we discussed the adaptive advantage for the African catfish to display a sex differentiation process controlled by a temperature effect.

Do sex reversal procedures differentially affect agonistic behaviors and sex steroid levels depending on the sexual genotype in Nile tilapia?

In Nile tilapia Oreochromis niloticus, phenotypic males and females with different sexual genotypes (XX, XY, YY) have particular behavioral and physiological traits. Compared to natural XX females and XY males, XY and YY females and XX males expressed higher level of aggressiveness that could be related to higher levels of 17ß-estradiol and 11-ketotestosterone, respectively. Our results suggest that the presence of a Y chromosome increases aggressiveness in females. However, since the same relationship between aggressiveness and the Y chromosome is not observed in males, we can hypothesize that the differences in aggressiveness are not directly dependent on the genotype but on the sex reversal procedures applied on young fry during their sexual differentiation to produce these breeders. These hormonal treatments could have permanently modified the development of the brain and consequently influenced the behavior of adults independently of their genotype. In both hypotheses (genotype or sex reversal influence), the causes of behavioral modifications have to be searched in an early modification of the brain sexual differentiation.

Brief exposure of embryos to steroids or aromatase inhibitor induces sex reversal in Nile tilapia (Oreochromis niloticus).

This study aimed to develop sex reversal procedures targeting the embryonic period as tools to study the early steps of sex differentiation in Nile tilapia with XX, XY, and YY sexual genotypes. XX eggs were exposed to masculinizing treatments with androgens (17α-methyltestosterone, 11-ketotestosterone) or aromatase inhibitor (Fadrozole), whereas XY and YY eggs were subjected to feminizing treatments with estrogen analog (17α-ethynylestradiol). All treatments consisted of a single or double 4-hr immersion applied between 1 and 36 hour post-fertilization (hpf). Concentrations of active substances were 1000 or 2000 µg l(-1) in XX and XY, and 2000 or 6500 µg l(-1) in YY. Masculinizing treatments of XX embryos achieved a maximal sex reversal rate of 10% with an exposure at 24 hpf to 1000 µg l(-1) of 11-ketotestosterone or to 2000 µg l(-1) of Fadrozole. Feminization of XY embryos was more efficient and induced up to 91% sex reversal with an exposure to 2000 µg l(-1) of 17α-ethynylestradiol. Interestingly, similar treatments failed to reverse YY fish to females, suggesting either that a sex determinant linked to the Y chromosome prevents the female pathway when present in two copies, or that a gene present on the X chromosome is needed for the development of a female phenotype.

The sensitive period for male-to-female sex reversal begins at the embryonic stage in the Nile tilapia and is associated with the sexual genotype.

In this study, we sought to determine the mechanism of early sex reversal in a teleost by applying 4 hr feminization treatments to XY (17α-ethynylestradiol 2000 µg L(-1) ) and YY (6500 µg L(-1) ) Nile tilapia embryos on the first day post-fertilization (dpf). We then searched for changes in the expression profiles of some sex-differentiating genes in the brain (cyp19a1b, foxl2, and amh) and in sex steroids (testosterone, 17ß-estradiol, and 11-ketotestosterone) concentrations during embryogenesis and gonad differentiation. No sex reversal was observed in YY individuals, whereas sex-reversal rates in XY progeny ranged from 0-60%. These results, together with the clearance profile of 17α-ethynylestradiol, confirmed the existence of an early sensitive period for sex determination that encompasses embryonic and larval development and is active prior to any sign of gonad differentiation. Estrogen treatment induced elevated expression of cyp19a1b and higher testosterone and 17ß-estradiol concentrations at 4 dpf in both XY and YY individuals. foxl2 and amh were repressed at 4 dpf and their expression levels were not different between treated and control groups at 14 dpf, suggesting that foxl2 did not control cyp19a1b in the brains of tilapia embryos. Increased cyp19a1b expression in treated embryos could reflect early brain sexualization, although this difference alone cannot account for the observed sex reversal as the treatment was ineffective in YY individuals. The differential sensitivity of XY and YY genotypes to embryonic induced-feminization suggests that a sex determinant on the sex chromosomes, such as a Y repressor or an X activator, may influence sex reversal during the first steps of tilapia embryogenesis.

Physiological and proteomic responses to single and repeated hypoxia in juvenile Eurasian perch under domestication--clues to physiological acclimation and humoral immune modulations.

We evaluated the physiological and humoral immune responses of Eurasian perch submitted to 4-h hypoxia in either single or repeated way. Two generations (F1 and F5) were tested to study the potential changes in these responses with domestication. In both generations, single and repeated hypoxia resulted in hyperglycemia and spleen somatic index reduction. Glucose elevation and lysozyme activity decreased following repeated hypoxia. Complement hemolytic activity was unchanged regardless of hypoxic stress or domestication level. A 2D-DIGE proteomic analysis showed that some C3 components were positively modulated by single hypoxia while C3 up- and down-regulations and over-expression of transferrin were observed following repeated hypoxia. Domestication was associated with a low divergence in stress and immune responses to hypoxia but was accompanied by various changes in the abundance of serum proteins related to innate/specific immunity and acute phase response. Thus, it appeared that the humoral immune system was modulated following single and repeated hypoxia (independently of generational level) or during domestication and that Eurasian perch may display physiological acclimation to frequent hypoxic disturbances.

Cortisol is responsible for positive and negative effects in the ovarian maturation induced by the exposure to acute stressors in Nile tilapia, Oreochromis niloticus.

The aim of the present study was to evaluate the effect of acute stress and cortisol injection on oocyte final maturation process in female Nile tilapia (Oreochromis niloticus). Handling followed by a prophylactic treatment (0.3 mL L(-1) H(2)O(2), 5 g L(-1) NaCl solution during 30 min) and an environmental change (transfer from a 2 m(3) fibreglass square tank to 50 L aquaria) were used as acute stressors and compared to a single cortisol injection (0.5 or 5 mg kg(-1) body weight). For both acute stress and cortisol injection (0.5 mg kg(-1) body weight), serum cortisol level was significantly increased from 2.3 to 134.1 ng mL(-1) 1 h post-stress/injection and returned to a resting basal value 24 h after the stress/injection. In fish injected with 5 mg kg(-1) body weight cortisol, mean serum cortisol level reached a peak up to 2500 ng mL(-1) 1 h after injection. 63 % of the females (mean body weight: 242 ± 4 g) submitted to the acute stress ovulated within 72 h after the stress. In the same way, cortisol injection (5 mg kg(-1) body weight) at the 10th day of the maturation cycle led to a twofold reduction of the time before ovulation compared to vehicle injected control fish. Relative and total fecundity were significantly decreased in females submitted to an acute stress or cortisol injected at 5 mg kg(-1) body weight, but not fertilization or hatching rates. In conclusion, acute stress and cortisol induction exert both positive and negative effects on the final reproductive process in O. niloticus, and cortisol is the endocrine mediator causing these changes.

Comparative study of the reproductive characteristics of XY male and hormonally sex-reversed XX male Eurasian perch, Perca fluviatilis.

In order to compare the reproductive capacity of XY male versus XX male (neomales) Eurasian perch (Perca fluviatilis), we determined the sperm quality (sperm concentration and motility) and reproductive characteristics such as gonadosomatic index (GSI), fertilization rate and sex steroid levels (testosterone, T; 17beta-estradiol, E2 and 11-ketotestosterone, 11KT) during the reproductive season. Median GSI was not significantly different between XY males (7.9%) and XX males (7.5%). Fertilization rates ranged between 30.0 and 98.0%. Sperm concentration ranged between 27.9 x 10(9) and 42.0 x 10(9) spermatozoa ml(-1). Median level of T, 11KT and E2 levels increased in the middle of the reproductive season (2136.0, 2409.0 and 3252.0 pg ml(-1), respectively) and decreased at the end (1657.0, 2006.6 and 431.0 pg ml(-1), respectively). Sperm motility was assessed by CASA and expressed by the curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), percentage of motile sperm (% MOT) and motile concentration (MOC). Overall, there were not any significant differences between XY and XX males. In conclusion, no differences of reproductive capacities were observed between XY males and XX males suggesting that the last can be crossed with females to improve the productivity of Eurasian perch by producing all-female stock.