Carole Rougeot

Sex control and sex determinism study in Eurasian perch, Perca fluviatilis, by use of hormonally sex-reversed male breeders

The Eurasian perch, Perca fluviatilis, displays sexual growth dimorphism in which females grow significantly faster than males. In this species, the sex-determining mechanism is not yet described. In order to control sex by steroid treatment and to study the genetic sex-determining mechanism in Eurasian perch, the first step is to optimize sex reversal using steroids. A combination of three hormonal doses (40, 60 and 80 mg of 17α-methyltestosterone kg−1 diet), four initial body weights (43, 71, 150 and 205 mg) and two treatment durations (30 and 80 days) was tested in duplicate. Complete masculinisation (100% male progenies) occurred when the treatment was applied in fish initially ranging from 40 to 71 mg body weight. Treatment applied at a larger body weight (150 and 205 mg body weight) provided variable proportions of males with two testes (45–62%), females (0–29%), XX males with a single twisted testis (16–40%), fish with ovotestis (0–18%) and sterile fish (0–27%). The sperm of XX males, determined on the basis of gonad morphology (single twisted testis), was used to fertilize eggs. The resulting offspring displayed sex ratios ranging between 95% and 100% females, while the sex ratio of most XY male progenies did not differ significantly (P>0.05) from a 50:50 balanced sex ratio. These results suggest that a female homogamety (XX)–male heterogamety (XY) chromosomal system is primarily operating in Eurasian perch. Nevertheless, because some males arise from crosses with XX males, and because two populations resulting from normal crosses display a sex ratio significantly (P<0.05) deviated towards males (60.7%) or females (65%), this suggests the action of other genetic sex-determining factors (autosomal or polygenic).

Physiological and proteomic evidences that domestication process differentially modulates the immune status of juvenile Eurasian perch (Perca fluviatilis) under chronic confinement stress.

The current study aimed to evaluate the influence of domestication process on the stress response and subsequent immune modulation in Eurasian perch juveniles (Perca fluviatilis) submitted to chronic confinement. Briefly, F1 and F4 generations were confined into small-size tanks and sampled 7 and 55 days after stocking. Cortisol and glucose levels as well as lysozyme activity and immunoglobulin level were evaluated in the serum. Spleen Somatic Index and spleen ROS production were also measured. A proteomic analysis was performed on serum sampled on day 7. Finally, both generations were genetically characterized using a microsatellite approach. Globally, results revealed that chronic confinement did not elicit a typical stress response but resulted in a prolonged immune stimulation. Proteomic results suggested that domestication process influenced the immune status of perch submitted to chronic confinement as the F1 confined fish displayed lower abundance of C3 complement component, transferrin and Apolipoprotein E. Microsatellite data showed a strong genetic drift as well as reduced genetic diversity, allelic number and heterozygosity along with domestication process. The present work is the first to report that fish under domestication can develop an immune response, assessed by a combined approach, following recurrent challenges imposed by captive environment despite a reduced genetic variation.

Does domestication process affect stress response in juvenile Eurasian perch Perca fluviatilis

The objective was to evaluate the impact of domestication process on the physiological stress response of cultured Eurasian perch confronted to a chronic stress situation. Briefly, F1 and F4 juveniles were submitted to chronic confinement and investigated on days 5, 15 and 30. Capture and 15min-anesthesia were imposed on fish to assess the effect of preceding confinement on acute stress response. On day 30, the fish were finally challenged with Aeromonas hydrophila and sampled after 5 and 10 days for immune parameter measurements. Cortisol and glucose levels were not affected by confinement but increased significantly after acute stressor exposure. Moreover, cortisol rise following capture and anesthesia was higher in F1 confined-fish, suggesting that they have previously been affected by chronic confinement. A higher HSP70 level was also observed on day 30 in F1 confined-juveniles. During bacterial challenge, regardless of confinement level, F4 juveniles displayed higher lysozyme activity and agglutination response than F1 which may indicate a higher immune capacity in domesticated fish. In conclusion, chronic confinement stressor induced few physiological responses but may increase the responsiveness to other aquacultural stressors. Domestication process also seems to improve chronic stress resistance, growth as well as the immune status of the fish.

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.

Ovarian steroidogenesis inhibition by constant photothermal conditions is caused by a lack of gonadotropin stimulation in Eurasian perch

In fish, the reasons for the inhibition of reproduction by constant photothermal conditions of rearing are far from clear. In an in vivo experiment, two groups of females reared under natural (4-28 degrees C) or constant photothermal conditions (20-22 degrees C, photoperiod 12/12) were investigated for gonad development, sex-steroids (testosterone-T, 17-beta-estradiol-E2 and 11 Keto-Testosterone-11KT) dynamics and brain aromatase activity in January, February and March. Two days before each sampling date, a group of females reared under constant conditions was injected with HCG (Human Chorionic Gonadotropin: 100 UI/kg) and evaluated for the same parameters. In addition, in vitro ovarian steroidogenesis capacity for each female was determined with or without stimulation by HCG and/or IGF-1 (Insulin-like Growth Factor-1). The results indicate that vitellogenesis stage is the limit ovarian stage never reached in females submitted to constant photothermal conditions. This was associated with gonadogenesis delay and low levels of circulating sex-steroids (T, E2 and 11KT). Nevertheless, HCG injections partly counteracted the plasma steroid deprivation, indicating that ovaries from fish reared under constant photothermal conditions suffer from a lack of gonadotropin stimulation, maybe caused by plasma LH suppression. Such finding was confirmed by the in vitro ovary incubation test. HCG and IGF-1 treatments induced broad testosterone and 17-beta-estradiol elevations and the exposure to constant photothermal conditions, in some cases, decreased that response to HCG. In conclusion, we show that the inhibition of reproductive cycle in Eurasian perch females by constant photothermal conditions of rearing may be related to lower sex-steroid levels and to an inhibition of ovarian regulation by gonadotropins (at least LH), probably stopping gonadogenesis before vitellogenesis stage.

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, 17β‐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 17β‐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. Mol. Reprod. Dev. 81: 1146–1158, 2014.

Induce triploidy by heat shock in Eurasian perch, Perca fluviatilis

In Eurasian perch (Perca fluviatilis), females grow significantly faster than males. Moreover, gonadal development has a significant negative impact on somatic growth and fillet yield. In order to induce sterility, triploidy induction was attempted by subjecting fertilised eggs to heat shocks. Different combinations of temperature (28, 30, 34, 35 and 36 °C), duration (2, 5, 10 and 25 min) and time of shock initiation (TI = 3, 5 and 7 min post-fertilisation) were tested. Flow cytometry analysis was used to assess ploidy level of control and heat-shocked larvae. Low intensity (28–30 °C) and long duration (10 and 25 min) shocks lead to significantly higher survival (44 ± 26%) and triploidisation (71 ± 26%) rates than high intensity (34–36 °C) and short duration (2 and 5 min) shocks (17 ± 19% and 21 ± 26%, respectively). The most effective conditions for efficient triploidy induction were low intensity shock of 30 °C, applied 5 min post-fertilisation for 25 min. This treatment led to the production of all-triploid populations (100%) with up to 43% survival rate.

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.

Influence of domestication process on immune response to repeated emersion stressors in Eurasian perch (Perca fluviatilis L.)

Domestication might be a possible way to reduce the physiological response to long-term stressors and deleterious effects on immunity. The present study aimed to evaluate the chronic immune response induced by repeated emersions and the possible impact of domestication by comparing farmed Eurasian perch with short (F1) and long (F4) captive-life history. In the first experiment, fish were exposed to a single emersion and physiological stress response was measured in the short term to characterize fish sensitivity to the tested stressor. Serum cortisol and glucose elevated within 6h post-stress and splenosomatic index (SSI) decreased within 48h, indicating that the species was affected by emersion stressor. In the second experiment, F1 and F4 generations were submitted to repeated water emersions (3 times/week during 44days). On day 9, 18 and 44, samplings were performed 48h post-stressor to highlight any sustained disruption of immune system. Serum cortisol, glucose, SSI and lysozyme activity were evaluated and serum proteome was analyzed using 2D-DIGE. Any of the tested variables were affected by repeated emersions and proteomic analysis only revealed that alpha-2 macroglobulins (a2Ms) were up-regulated in the serum of stressed individuals. Domestication also resulted in the up-regulation of five a2M isoforms and down-regulation of complement C3 and Ig light chain proteins, independently of any stressor exposure. In conclusion, the results suggested that repeated emersions are not severe stressors for Eurasian perch, probably explaining why domestication had no influence on fish responses. Changes associated with domestication are highly complex and certainly need further investigations.

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.