Effect of precocious high temperature treatments on sex differentiation in a highly and weakly thermo-sensitive selection line of Nile tilapia, Oreochromis niloticus
The present study investigates the influence of rearing temperatures during the precocious larval development on the phenotypic sex of mixed sexed Nile tilapia progenies. The brood stock which produced the progenies for this experiment had been selected for the sex ratio in temperature treated groups, showing male percentages of >90% in the high-line and <60% males in the low-line. The progenies obtained from either line were subjected to an early temperature treatment at 34Â°C, 35Â°C and 36Â°C starting at 12 hours post fertilisation. The experimental temperatures were reached within 5 hours and were applied till hatching (52 Â± 3 hours). The corresponding full sib control groups were kept at 28Â°C throughout the experiment. Temperatures above 34Â°C led to total mortalities in all progenies tested (n=10), whereas temperatures of 34Â°C allowed survival rates that were comparable to those of the controls after nine days, except for the high-line. Irrespective of the line the progenies belonged to, the sex ratios in all progenies were not affected by temperature treatment, which is in contrast to all-female progenies (XX) for which a masculinisation with a rising masculinising potential with increasing temperature (34-36Â°C) was reported earlier.
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Tilapias constitute an excellent model to study the evolution of sex determination. They show large variation in the mechanisms of sex determination among and within species. In Nile tilapia (Oreochromis niloticus) the phenotypic sex can be determined through (interaction of) minor autosomal factors (Müller-Belecke & Hörstgen-Schwark1995), a major male determining factor on linkage group 1 (Cnaani et al., 2008), and environmental factors, such as temperature (Baroiller et al., 1995a, b; Tessema et al., 2006; Wessels and Hörstgen-Schwark, 2007; Baroiller et al., 2009). The temperature sensitivity of Nile tilapia during sex differentiation varies among and within populations and it can be selected for, as a quantitative trait (Tessema et al., 2006; Wessels and Hörstgen-Schwark, 2007; Baroiller et al., 2009).
However, there is not much information on the action of rearing temperature during very precocious stages of larval development (12 hpf till hatching). Whether temperature acts on sex determination or -differentiation and on which tissue (brain, gonads, or both) is still unclear (Baroiller et al., 2009).
The only study focussing on high larval rearing temperatures in Nile tilapia, Rougeot et al. (2008), showed that temperatures of 35-36Â°C applied from around 12 hpf till hatching (52 hpf) had a significant effect on the proportion of males (8-27%, n=4) of genetically all-female (XX) progenies. During this stage, primordial germ cells are present (31 hpf) and the brain still is rudimentary (46 hpf) (Morrison et al., 2001). Some evidence for an early influence on the phenotypic sex in Nile tilapia seems to exist (Rougeot et al., 2008).
Therefore the goal of the present study was to determine whether thermal responsiveness during the course of sex differentiation (from 10 till 20 dpf) correlates with a high precocious temperature sensitivity (from 12 hpf till hatching) using Nile tilapia breeders from a highly thermo-sensitive and a weakly thermo-sensitive line (Wessels and Hörstgen-Schwark, 2007).
Material and Methods
The experiments were carried out in the warm water recirculation system of the Department of Animal Sciences, University of Göttingen. Nile tilapia (O. niloticus) breeders used in the present study originated from the Manzala population, and had been selected for their sex ratios in temperature treated groups (from 10-20 dpf/ 36Â°C) (Wessels & Hörstgen-Schwark, 2007). Breeders from the high-line were chosen from families giving male proportions of ~90%, whereas breeders from the low-line were taken from families giving sex ratios ~50% in temperature treated groups. Both lines originated from the same base population, giving mean progeny sex ratio of 65.6% when reared at 36Â°C for ten days. The brood stock fish were fed standard carp pellets twice per day (Skretting C2 pro Aqua K18, Norway; crude protein: 36%). Water parameters were within the following range throughout the experiment: temperature 27-28Â°C; oxygen > 7 mg/l; pH 6.5 - 7.5; NH+4 < 0.4 mg/l; NO-2 < 0.2 mg/l. All brood stock fish were Pit-tagged and could be individually recognized. The spawners were kept in 300-l glass aquaria at a stocking density of 15 kg/m3. A light program was set to 12 hours light and 12 hours darkness. Eggs could be collected through manual stripping and were then artificially fertilised. Three half sib-families were generated for the high- and low-line using one male and three females (n=6 batches). Inseminated eggs were counted, equal proportions were divided into a control (28Â°C) and treatment (34Â°C; 35Â°C; 36Â°C) groups, and then incubated at 28Â°C water temperature. The temperature treatment was initiated 12 hpf and the experimental temperatures were reached at 17 hpf after a gradual temperature increase. Water temperatures of 35 and 36Â°C led to total mortalities of fertilised eggs before hatching (n=10 batches). Fry from the 34Â°C treatment groups were readapted to 28Â°C water temperature after they hatched (51Â±3hpf). The control groups were kept at 28Â°C throughout the experiment. At swim up (9 dpf) fry were counted again and 110 fish per group were raised in 80-liter aquaria till sexing (120 dpf). Fish were sexed by microscopic inspection of gonad squashes (Guerrero and Shelton, 1974). Survival rates were assessed from fertilisation till swim up and from swim up till sexing.
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Sex ratio was analysed by fitting a generalized linear mixed model using the GLIMMIX macro (binomial error distribution, logit link function) in SAS 9.1 with sex coded as a binary trait (0=female, 1=male) (Mclean et al., 1991). As fixed factors the line (high, low), treatment (28Â°C, 34Â°C), and the interaction of line and treatment were included in the model. Effects on survival rates were analysed using a general linear model (Proc GLM) in SAS. The sex ratios in each control and treatment group were tested for deviations from the expected 1:1 distribution using the chi-square test. Differences between control and treatment groups were tested using a 2 x 2 contingency chi-square test
Three batches per line (high/low) were subjected to the experimental procedure and sexed after 120 days. The overall number of sexed fish was 1090, giving an average number of 91 sexed individuals per group. The results summarizing survival rates, number of (sexed) individuals and male percentages are illustrated in table 1. Survival rates from fertilisation till 9 dpf tended to be higher in the control (58.2 %) than in the temperature treated groups (40.5 %), without being statistically significant (P > 0.1758). In controls of progenies from the high-line the survival rates from fertilisation till 9 dpf also tended to be higher (68.3 % vs. 47.8 %), when compared to progenies from low-line families. Generally, the standard deviations for survival after nine days were large. The control groups in the low-line showed the largest variation followed by the treatment groups of both lines. In the high-line the control groups were found to exhibit the least variation in this regard. When differences in survival rate at 9 dpf in the high-line were analysed separately, the effect of the temperature treatment over the control was significant (P < 0.05). Otherwise, the differences observed were not found to be statistically significant.
The survival rates from 9 dpf till sexing did not differ significantly, neither between control and treatment groups (85.2 % vs. 84.2%, P > 0.85) nor between progenies from the high- and low-line (80.2 % vs. 89.1 %, P > 0.1565).
The male proportions in the six families tested were not affected by the precocious temperature treatment (P > 0.9085). Sex ratios in the control groups were in the range of 46.1 - 61.7% males (mean sex ratio = 55.5 Â± 5.5), whereas in the treatment groups the range was more narrow (51.7 - 58.9% males; mean sex ratio = 55.2 Â± 2.9). In one control group, sired by father 2 and mother 4 belonging to the low-line, sex ratios differed from the expected 1:1 distribution (61.7 %; n=107; chi-square > 3.84). All the other sex ratios showed a 1:1 distribution. Two thirds of the treatment groups showed a slightly lower male proportion than their corresponding controls, no matter if they were from the high- or low-line. The differences between control and treatment groups were in the magnitude of -3,9 - +0.4% males, i.e. sex ratios were comparable. Only one family showed a deviation between control and treatment group sex ratios of more than that (difference = 12.1%). However, this difference was not statistically significant, due to the low numbers of fish in both the control (n=76) and the treatment group (n=55). The difference might therefore be attributed to a larger binomial sampling error and not to the influence of the thermal treatment. Thus the mean male proportions of control and treatment groups in the high- and low-line were highly comparable (high-line 54.0 Â± 7.1 vs. 55.7 Â± 2.8; low-line 56.1 Â± 4.8 vs. 54.8 Â± 3.7).
When the data were analysed using the generalized linear mixed model, neither the fixed factors "treatment" and "line" nor the interaction term (treatment x line) had a significant influence on the male proportions (treatment P > 0.9085, line P > 0.7545, treatment x line P > 0.7456).
The goal of the present study was to study whether Nile tilapia breeders (XX/XY), which give high or low male proportions after a 10 day temperature treatment at 36Â°C (10 - 20 dpf), also give progenies that respond to a very precocious temperature treatment (from 12+5 hpf till hatching, 34Â°C) regarding their male proportions.
In an earlier study, Rougeot et al. (2008) showed that high water temperatures (34Â°C, 35Â°C and 36Â°C) applied from 12+3 hpf till hatching (52 hpf) had a significant effect on the proportion of males (8-27%, n=4) of genetically all-female (XX) Nile tilapia progenies. The authors demonstrated that survival rates of fry subjected to temperatures above 35Â°C decreased drastically but did not lead to total mortalities. In the present study, already temperatures above 34Â°C from 12+5 hpf onwards led to total mortalities before hatching (n=10 progenies). The fact that Rougeot et al. (2008) collected the fry from the mouth of the females after natural reproduction of breeders might be the reason for the differences observed, as the exact age of the fry in this experiment could not be determined. Hence, the fry might have been older than 15 h (collection at 12 hpf, onset of temperatures treatment at 15 hpf) when the temperature treatment began, which could have led to an improved resistance towards high temperatures due to the ontogenetic more advanced stage. In the present study, the age of the fry at the onset of the temperature treatment could be exactly determined and never exceeded 15 hpf, due to the fact that breeders were reproduced artificially. Although the experimental temperatures (34, 35 or 36Â°C) in Rougeot et al. (2008) were reached within three hours, the slower temperature increase (5h) in the present study did not allow for surviving fry at experimental temperatures above 34Â°C, indicating either differences between genotypes (i.e. XX Rougeot et al. 2008 vs. XX/XY in the present study; different temperature susceptibilities) or differences in timing of the experiment and the ontogenetic stage. Although in both studies the Lake Manzala strain had been used the differences in survival rates at high temperature (34-36Â°C) between the two studies might be related to the strain due to different selection history of the strain samples or genetic drift.
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The fact that in the present study breeders were chosen from the high- or low-line did not influence the survival rates significantly, but after 9 dpf the average survival rates tended to be higher in progenies belonging to control groups of the high-line. In the treatment groups, no such effect could be observed; here survival rates at 9 dpf were almost equal.
A higher survival rate after the precocious temperature treatment in either of the lines could indicate a selective/adaptational dis-/advantage to high temperatures due to higher or lower degree of thermo-sensitivity. If temperature dependent sex determination is prevalent (i.e. in the high-line) the ability to tolerate high temperatures might be enhanced. However, regarding the outcome of the present study this theory could not be confirmed. The fact that the survival rates at 9 dpf tended to be higher in the high-line, when compared to the low-line, might at least be looked at as interesting, as it could stand for an advantage in fitness in the high-line. Nevertheless, low batch numbers and non-significant differences do not allow drawing this conclusion, but it will be tested in further experiments.
The most striking difference between the present study and Rougeot et al. (2008) is the absence of masculinising potential of the precocious temperature treatment. The authors found significant differences between the control and the 34Â°C treatment groups in three out of four batches. When a water temperature of 35Â°C was applied, the male proportions were significantly different from the control in all families. In contrast, in our experiment the male proportions in the precociously temperature treated groups did neither differ from a 1:1 distribution nor from their corresponding controls. As Rougeot et al. (2008) used all-female (XX) populations, a temperature dependent masculinisation of the fry is easier to detect as if mixed sexed progenies are used. Rougeot et al. (2008) reported differences in the male percentage between controls (which always showed 0% males) and treatment groups, in the magnitude of 3.6 to 12.7 %. In the present study the male proportions in the control and treatment groups ranged from 46.1 to 61.7%, respectively 51.7 to 58.9%, thus showing even a more narrow range in the treatment groups. Furthermore, two thirds of the treatment groups showed a slightly lower male proportion than their corresponding controls. Consequently, all sex ratios in the treatment groups were not significantly skewed in the male direction, the influence of the breeders' thermal responsiveness (high- vs. low-line) did obviously not influence the masculinising potential of the precocious temperature treatment.
One possible effect of precocious, high rearing temperatures might be the influence on sex differentiation through survival of primordial germ cells, which are present at 32 hpf, as observed in various fish species. In the Medaka which has an XX/XY sex determination system with DMY as the sex determining gene, a temperature increase from 27Â°C to 32Â°C from the beginning of sex differentiation (stage 25) till hatching (stage 36) clearly led to sex reversal from XX-females to functional males (Selim et al., 2009). The authors state that high temperatures inhibited the proliferation and development of germ cells and conclude that in the Medaka the degenerative character of high temperatures is comparably low. In the puffer fish (Takifugu rubripes) precocious high temperature treatments (32Â°C) induced gonadal degeneration leading to gonads without germ cells (Lee et al., 2009). Morphologically the high-temperature treatments did not alter sexual differentiation as ovarian characteristics were maintained. But gene expression studies showed that the male-specific gene Dmrt1 was expressed in ovarian tissues with degenerated germ cells, thus showing a masculinising effect of precocious high temperatures. Siegfried and Nüsslein-Vollhardt (2008) demonstrated that the germ line is required for the ovary versus testis fate in zebra fish. When the germ line was absent, the gonad underwent testis fate and normal somatic structures were developed. Primordial germ cells are sensitive to high water temperature, and apoptosis might lead to the development of male somatic tissues.
In the present study gonads from temperature treated fish showed a normal appearance but were not analysed for the number of germ cells. High temperature treatments might also act on germ cell survival or other key triggers like aromatase in Nile tilapia, which then lead to functional male development, like observed in the study of Rougeot et al. (2008).
Hence, the role of high temperature on germ cell survival might also play an important role in Nile tilapia and should be analysed in future gene expression studies using germ cell markers, like Vasa. Further light should also be shed on differences between genetic male and female progenies with regard to the precocious temperature responsiveness.
The authors thank Mrs. Birgit Reinelt and Mr. Uwe Schipke for their technical assistance. This work was supported by the German Research Foundation (Ho 838/5-2)