A model for environmental sex reversal in fish

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1 Journal of Theoretical Biology 227 (2004) A model for environmental sex reversal in fish M.A. Hurley*, P. Matthiessen, A.D. Pickering Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster, Lancashire LA1 4AP, UK Received 30 June 2003; accepted 15 October 2003 Abstract A mathematical model is presented which combines genetic XX-female/XY-male sex determination with environmental pressure for phenotypic sex reversal. This may occur when fishes are exposed to endocrine disrupters, specifically masculinization by exposure to androgens and feminization by exposure to estrogens. A generic model is derived for the sex ratio in successive generations and three special cases, with chronic and constant pressure to sex reverse, are discussed in detail. These show that, with extreme environmental pressure to masculinize, the male genotype is at risk of dying out but with less extreme pressure, masculinization will not be detectable since the proportion of phenotypic males becomes one-half. With feminization at any pressure to sex reverse, the male and female genotypes will be maintained in a stable sex ratio in which the proportion of genotypic males exceeds one-half and is close to one-half if YY offspring (eggs) are not viable. In converse, the model is also applicable to the genetic ZZ-male/ZW-female system of sex determination in fish. At present suitable data are not available with which to validate the model, but proposals are made for relevant experimental studies. r 2003 Elsevier Ltd. All rights reserved. Keywords: Endocrine disrupters; Masculinization; Feminization; Phenotypic sex ratio; Genotypic sex ratio 1. Introduction Sex determination in fish is primarily under genetic control but may also be influenced by environmental conditions. Most species of fish are gonochoristic, where each individual is either male or female throughout its life. However some, such as the cuckoo wrasse Labrus bimaculatus, are sequential, protogynous hermaphrodites spawning initially as females but then reversing to functional males (Dipper and Pullin, 1979) whereas others, such as the black porgy Acanthopagrus schlegeli are sequential, protandrous hermaphrodites, spawning initially as males but then reversing to functional sexual females (Lee et al., 2001). For gonochoristic species, the genetic control of sex may span from simple systems with genetically defined chromosomes to polyfactorial sex determination, sometimes within a single taxonomic group (Volff and Schartl, 2001). For example, the gonochoristic zebrafish Danio rerio has an all-autosomal system, with no genetically distinct sex chromosomes (Traut and Winkling, 2001). In those species where *Corresponding author. addresses: mh@ceh.ac.uk (M.A. Hurley), pmatt@ceh.ac.uk (P. Matthiessen). heteromorphic chromosomes occur, the system may be XX female XY male (Patino et al., 1996) or ZZ male ZW female (Campos-Ramos et al., 2001). Most work on environmental sex determination in fish has focused on the effects of temperature. In many thermosensitive species of fish, elevated temperature during early embryonic development increases the proportion of males, but in a few species high temperatures may produce female-biased sex ratios (Baroiller and D Cotta, 2001). Moreover, in Paralichthys olivaceus both high and low temperatures induce monosex male populations whilst intermediate temperatures yield a 1:1 sex ratio (Baroiller and D Cotta, 2001). Conover and Heins (1987) reported that the strength of the genetic, compared with the environmental (temperature), component of sex determination in the Atlantic silverside Menidia menidia varies with latitude and the evolutionary significance of this finding is discussed by Bulmer (1987). Other factors that can influence sex determination in fish include: social environment (Degani and Kushnirov, 1992; Fishelson, 1998), ph (Rubin, 1985) and stocking density (Olivier and Kaiser, 1997). It is now well established that sex determination can be modified by exposure of fish to exogenous hormones /$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi: /j.jtbi

2 160 M.A. Hurley et al. / Journal of Theoretical Biology 227 (2004) or hormone-like chemicals during the sensitive, early juvenile stage. Fish may be masculinized by exposure to androgens (Katsiadaki et al., 2002) or feminized by exposure to estrogens (Matthiessen, 1998). The aquaculture industry has exploited this plasticity to develop single sex populations of fish with the desired growth characteristics. For the salmonid industry this has resulted in the development of all-female stocks (Hunter and Donaldson, 1983), whereas all-male stock are the preferred option for the tilapia industry (Beardmore et al., 2001). Moreover, there is evidence of skewed sex ratios in some natural fish populations, particularly in those exposed to potential endocrine disrupting chemicals. Thus Larsson et al. (2000) observed a 1:1 sex ratio in normal populations of eel pout Zoarces viviparus fry but a significant bias towards males in the androgenic effluent gradient from a large Swedish pulp mill and Nagler et al. (2001) found evidence of sex reversal of male Chinook salmon Oncorhynchus tshawytcha in a sub-catchment of the Columbia River, USA although, in the latter case, no link was made to endocrine disrupting substances. We also know that many fish populations exposed to estrogen-containing discharges contain a high proportion of partially feminized males (e.g. with oocytes in their testes and yolk protein in their blood), but do not know whether any fish recorded as normal females are actually completely sex-reversed genetic males (Matthiessen, 2003; Jobling and Tyler, 2003). The consequences for the population of environmental sex reversal in fish have barely been studied experimentally or theoretically. The present investigation examines the potential impacts of sex reversal on the sex ratios of future generations, using a modelling approach for gonochoristic species where heteromorphic chromosomes occur and the system is XX female XY male. A wholly analogous approach can be developed for the ZZ male ZW female system. 2. The generic model The generic model developed here assumes that the system of sex determination is equivalent to the XX female XY male quantitative characteristic model. It is also presumed that mating is random and that any sexreversed fish have a similar reproductive capability to normal fish of the same phenotypic sex. This may or may not be true and the implications of this assumption are discussed later. A general model was developed to predict the impacts of simultaneous environmental masculinization and feminization on the phenotypic and genotypic sex ratios of sequential generations. Thus the model allows for both the sex change of genetic males to females and of genetic females to males. Suppose the initial generation, F 0 ; has a proportion f 0 of genetic females (XX) and g 0 of genetic males (XY), then f 0 þ g 0 ¼ 1: With sex reversal, subsequent generations may contain supermales (YY), which could arise from a successful cross of a normal male (XY) with a feminized male (also XY). Suppose generation F i has a proportion f i of genetic females (XX), g i of genetic males (XY) and h i of genetic supermales (YY), then f i þ g i þ h i ¼ 1: Following exposure to an endocrine disrupting substance/effluent, let p i be the probability that a genetic female (XX) in the F i generation is sex-reversed to a phenotypic male, q i the probability that a genetic male (XY) is sex-reversed to a phenotypic female and r i the probability that a genetic supermale (YY) is feminized to a phenotypic female. Individuals then belong to one of two phenotypes; m i is the proportion of phenotypic males, n i is the proportion of phenotypic females and m i þ n i ¼ 1: In generation F i ; the male to female genotypic sex ratio s i is given by s i ¼ðg i þ h i Þ=f i and the phenotypic sex ratio t i is given by t i ¼ m i =n i where m i ¼ p i f i þð1 q i Þg i þð1 r i Þh i and n i ¼ð1 p i Þf i þ q i g i þ r i h i : Table 1 summarizes the relative frequency of each phenotype and genotype combination and the potential offspring of each cross between phenotypic males and phenotypic females. Assuming random mating and viability of all crosses, the frequencies f iþ1 ; g iþ1 and h iþ1 of the three genotypes XX, XY and YY in the F iþ1 generation are obtained by summing and simplifying the probability of occurrence of each cross and each genotype within each cross using the nine offspring cells of Table 1, thus f iþ1 ¼ð2p i f i þð1 q i Þg i Þð2ð1 p i Þf i þ q i g i Þ=ð4m i n i Þ; ð2:1þ h iþ1 ¼ð2ð1 r i Þh i þð1 q i Þg i Þð2r i h i þ q i g i Þ=ð4m i n i Þ; ð2:2þ g iþ1 ¼ 1 f iþ1 h iþ1 : ð2:3þ From f iþ1 ; g iþ1 and h iþ1 the number of phenotypic males and females, m iþ1 and n iþ1 ; and the genotypic and phenotypic sex ratios, s iþ1 and t iþ1 in the F iþ1 generation can be obtained. A range of sex reversal scenarios can be modelled using the above formulation by fixing model parameters at set values. Environmental masculinization occurs when q i ¼ r i ¼ 0 and feminization when p i ¼ 0: Chronic and constant feminization is modelled when p i is fixed at the same value in all generations, while feminization in a single generation is modelled when p 0 ¼ 0; p 1 > 0 and p i ¼ 0 for i > 1: If certain crosses in Table 1 are not viable then the offspring genotype proportions can be set to zero. The relative proportions of the surviving genotypes can be calculated from Table 1, but the offspring genotype proportions must be rescaled to sum to unity.

3 M.A. Hurley et al. / Journal of Theoretical Biology 227 (2004) Table 1 All crosses between phenotypic males and phenotypic females and the resultant genotype and relative frequency of offspring within each cross Parent phenotype: population frequency Female: n i Parent genotype: XX: (1 p i )f i XY: q i g i YY: r i h i population frequency Offspring genotype: frequency within cross Male: m i XX: p i f i XX: 1.00 XX: 0.50 XY: 1.00 XY: 0.50 XY: ð1 q i Þg i XX: 0.50 XX: 0.25 XY: 0.50 XY: 0.50 XY: 0.50 YY: 0.50 YY: 0.25 YY: ð1 r i Þh i XY: 1.00 XY: 0.50 YY: 1.00 YY: Special cases of the generic model t iþ1 ¼ðp iþ1 þ s iþ1 Þ=ð1 p iþ1 Þ: ð3:4þ Three special cases of the generic model will be considered here. These are where sex reversal is in one direction only, masculinization or feminization, and where the pressure to sex reverse is chronic and constant in each generation. Equally well, the generic model can be used to look at sex reversal as a perturbation in a single generation or as a chronic influence over several sequential generations Environmental masculinization Suppose the initial generation, F 0 ; has a proportion f 0 of genetic females (XX) and g 0 of genetic males (XY), then f 0 þ g 0 ¼ 1: With environmental masculinization, subsequent generations cannot contain supermales (YY) since this requires a XY XY cross which cannot occur. If, during development, a proportion, p i ; of the genotypic females in the F i generation are converted to phenotypic males by exposure to some masculinizing influence, then the proportions at sexual maturity will be ð1 p i Þf i genetic females, p i f i phenotypic males with XX genotype ( pseudomales ) and g i ¼ 1 f i true males with XY genotype. The genotypic sex ratio is s i ¼ g i =f i and the phenotypic sex ratio is t i ¼ðp i f i þ g i Þ=ð1 p i Þf i ; that is t i ¼ðp i þ s i Þ=ð1 p i Þ: If random mating then takes place, only the true males can produce male offspring in the next F iþ1 generation and, therefore, the proportion of genetic males in the next generation will be directly related to the proportion of genetic males within the phenotypic males of generation i: Thus, if generation i þ 1 hatches to give f iþ1 genetic females and g iþ1 genetic males, then f iþ1 ¼ð2p i f i þ g i Þ=ð2p i f i þ 2g i Þ; ð3:1þ g iþ1 ¼ g i =ð2p i f i þ 2g i Þ: The genotype and phenotype sex ratios are s iþ1 ¼ s i =ð2p i þ s i Þ; ð3:2þ ð3:3þ If the pressure to masculinize is chronic and constant in each generation, that is p i ¼ p; then after a number of generations the genotype sex ratio will settle down to s ¼ 1 2p at which point t ¼ 1: The between generation change in the population frequency of genotypic and phenotypic males under constant and chronic pressure to reverse sex from female to male is illustrated in Fig. 1. If p exceeds one-half, the genotypic sex ratio will fall to zero and genotypic males (XY) will die out. The phenotypic male population will be sustained only by the masculinized females, the population frequency of phenotypic males (all of which are masculinized genetic females) will become equal to p. If p is less than one-half (Fig. 1), the sex ratio will decline to a non-zero value of s ¼ 1 2p: Genotypic males (XY) will not die out but the population frequency will be sustained at a stable value smaller than one-half of the total population, equal to ð1 2pÞ= ð2 2pÞ: The population of phenotypic males will settle to one-half, at which point it will not be possible to detect the environmental pressure to masculinize from the phenotypic individuals alone Environmental feminization with all offspring viable Again we suppose the initial generation, F 0 ; has a proportion f 0 of genetic females (XX) and g 0 of genetic males (XY) and f 0 þ g 0 ¼ 1: With environmental feminization, subsequent generations can contain supermales (YY) which arise when true males (XY) cross with sex-reversed males (also XY) and the genetic YY offspring are viable. Eggs of generation F i hatch to give a proportions f i of females (XX), g i of males (XY) and h i of super-males (YY) where f i þ g i þ h i ¼ 1: Suppose that a proportion q i of genetic males and supermales are then converted to phenotypic females following exposure to a feminizing chemical or effluent (thus assuming that the proportion r i in the general model is

4 162 M.A. Hurley et al. / Journal of Theoretical Biology 227 (2004) h iþ1 ¼ð1 q i Þq i ðg i þ 2h i Þ=ð4m i n i Þ; ð3:6þ g iþ1 ¼ 1 f iþ1 h iþ1 : ð3:7þ The between generation change in the population frequency of genotypic and phenotypic males under constant and chronic pressure to reverse sex from male to female is illustrated in Fig. 2. Whatever the pressure to feminize, the population frequency of the male genotype rises from one-half and settles to a stable value less than unity; the female genotype does not die out. The pressure to sex reverse from male to female can be detected by the magnitude of the population frequency of phenotypic males. The pressure is least when the frequency is close to one-half and, as pressure increases, the population frequency of phenotypic males declines. In contrast an increasing pressure to sex reverse from male to female results in an increasing population frequency of genetic males. Fig. 1. The proportion of males in each generation when a population is under chronic and constant pressure, p, to masculinize for p ¼ 0:25; 0.35, 0.45, 0.55, 0.65 and The solid line ( ) shows the genotypic proportion and the dashed line (- - -) shows the phenotypic proportion of males. equal to q i ), then the population will consist of five groups of fish in the following proportions: * ð1 q i Þg i genotypic males (XY), phenotypic males, * ð1 q i Þh i genotypic super-males (YY), phenotypic males, * f i genotypic females (XX), phenotypic females, * q i g i genotypic males (XY), phenotypic females, and * q i h i genotypic super-males (YY), phenotypic females. Thus the total proportion of phenotypic males is m i ¼ ð1 q i Þðg i þ h i Þ and that of phenotypic females is n i ¼ f i þ q i ðg i þ h i Þ: The genotypic sex ratio is s i ¼ðg i þ h i Þ=f i and the phenotypic sex ratio is t i ¼ð1 q i Þðg i þ h i Þ= ðf i þ q i ðg i þ h i ÞÞ; that is t i ¼ð1 q i Þs i =ð1 þ q i s i Þ: Assuming random mating between phenotypic males and phenotypic females, the next generation, F iþ1 ; will have f iþ1 genotypic females (XX), g iþ1 genotypic males (XY) and h iþ1 genotypic super-males (YY) where f iþ1 ¼ð1 q i Þg i ð2f i þ q i g i Þ=ð4m i n i Þ; ð3:5þ Fig. 2. The proportion of males in each generation when a population is under chronic and constant pressure, q, to feminize for q ¼ 0:25; 0.35, 0.45, 0.55, 0.65 and 0.75 and with all offspring eggs viable. The solid line ( ) shows the genotypic proportion and the dashed line (- - -) shows the phenotypic proportion of males.

5 3.3. Environmental feminization when YY-genotype offspring non-viable If the fertilized eggs with the YY genotype are not viable then h i ¼ 0 in all generations. In generation F i the proportion of phenotypic males is m i ¼ð1 q i Þg i and that of phenotypic females is n i ¼ f i þ q i g i : The genotypic sex ratio is s i ¼ g i =f i and the phenotypic sex ratio is t i ¼ð1 q i Þg i =ðf i þ q i g i Þ; that is t i ¼ð1 q i Þs i =ð1 þ q i s i Þ: The next generation, F iþ1 ; will have f iþ1 genotypic females (XX), and g iþ1 genotypic males (XY) where f iþ1 ¼ð2f i þ q i g i Þ=ð4f i þ 3q i g i Þ; ð3:8þ M.A. Hurley et al. / Journal of Theoretical Biology 227 (2004) g iþ1 ¼ð2f i þ 2q i g i Þ=ð4f i þ 3q i g i Þ: The genotype and phenotype sex ratios are s iþ1 ¼ð2þ2q i s i Þ=ð2 þ q i s i Þ; ð3:9þ ð3:10þ t iþ1 ¼ð1 q iþ1 Þs iþ1 =ð1 þ q iþ1 s iþ1 Þ: ð3:11þ If the pressure to feminize is chronic and constant in each generation, that is q i ¼ q; then after a number of generations the genotype sex ratio will settle down to s ¼fð1þq 2 Þ 1=2 2ð12qÞg=q: Since q is between zero and unity, the sex ratio is between unity and The between generation change in the population frequency of genotypic and phenotypic males under constant and chronic pressure to reverse sex from male to female when the YY eggs are not viable is illustrated in Fig. 3. The population frequency of the male genotype and phenotype stabilizes within a few generations. The male genotype settles to a frequency of between 0.5 and and thus remains close to 50% whatever the pressure to reverse sex from male to female. 4. Discussion The foregoing has potential management implications for fisheries where the genetic XX-female/XY-male model of sex determination is appropriate to the species. Where chronic environmental pressure to masculinize is known to have impacted on a population for a number of generations, any population exhibiting circa 50% phenotypic males is likely to contain genotypic males at a frequency less than 50%, how much less being undetectable from the phenotypic frequency (see Fig. 4(a)). If more than 50% phenotypic males are recorded, it is possible than the genotypic males have died out altogether; the phenotypic males present being the result of masculinization of genetic females. If environmental masculinization ceases, for example when an androgen-containing effluent is cleaned up, the population will die out in one generation. Where environmental pressure to masculinize is suspected rather than proved, a population estimate of circa Fig. 3. The proportion of males in each generation when a population is under chronic and constant pressure, q, to feminize for q ¼ 0:25; 0.35, 0.45, 0.55, 0.65 and 0.75 and with all offspring eggs viable except the YY offspring. The solid line ( ) shows the genotypic proportion and the dashed line (- - -) shows the phenotypic proportion of males. 50% phenotypic males will not be sufficient evidence to discount the influence of environmental masculinization on a fish population. With chronic environmental pressure to feminize, the outcome for the fish population is not as serious (see Fig. 4(b)). Feminization can largely be detected by a decline in the proportion of phenotypic males to a level below 50%, this being accompanied by an increase in the proportion of genotypic males above 50%. However both genotypes will survive and the sex ratio of genotypic males to females will stabilize after several generations. If the YY eggs are not viable, the sex ratios of genotypic males to females will deviate little from unity (see Fig. 4(c)). If environmental feminization ceases, for example when an estrogen-containing effluent is cleaned up, the genetic sex ratio of the population will return to unity. Thus the consequences of this model run somewhat counter-intuitive; masculinization depresses the

6 164 M.A. Hurley et al. / Journal of Theoretical Biology 227 (2004) (a) (b) (c) Fig. 4. The proportion of genotypic males after many generations when a population is under chronic and constant pressure to sex reverse with (a) masculinization, (b) feminization with all offspring viable and (c) feminization with all offspring viable except YY eggs. The solid line ( ) shows the genotypic proportion and the dashed line (- - -) shows the phenotypic proportion of males. proportion of genetic males whereas feminization elevates the proportion of genetic males. In both cases the outcome is a deviation from the one to one sex ratio. Similar biased sex ratios are predicted from the models of Charnov(1993) and Charnovand Bull (1989a, b) in species which naturally change sex during their reproductive life or in species for which sex is wholly determined by environment. The model described here makes certain implicit assumptions such as a large population size, random mate selection and that all fish have the same reproductive capacity. In this context model predictions are interpreted as expected values for large populations. The model can be extended into the stochastic realm by introducing probability distributions for the elements in Table 1 and modelling successive generations by computer simulation. By this means factors such as variable population size, including small populations, differential reproductive capacity or non-random mate selection can be studied and bounds on expectation can be obtained. It would also be desirable to extend the model to allow for the fact that many fish in an exposed population, while not experiencing total phenotypic sex reversal, become partially sex reversed. This has as yet unknown consequences for the sex ratios of future generations. There is great scope for further theoretical, empirical and experimental work in this whole area. The model is equally applicable to the genetic ZZmale/ZW-female system of sex determination in fish with the outcomes for masculinization and feminization reversed. Where the genetic basis for the sex determination of a species is poorly understood, caution is clearly advisable. The model throws some light on the evolutionary biology of gender determination in fish. If a species is vulnerable to environmental pressure to reverse sex from that prescribed by genes then one genotype is at risk of dying out. The aquatic environment readily imposes such pressures on fish species and this may partly explain why such complex systems of sex determination have evolved. It is clearly desirable to validate this model with experimental data before it is used for managerial or scientific purposes. Unfortunately, the available published information is unsuitable because multigeneration exposures of fish species with XX/XY sex determination to estrogens or androgens have hardly ever been conducted for more than two generations. Thus, for example, two-generation reproduction tests with the Japanese medaka (Oryzias latipes) have been made during continuous exposure to estrogen mimics

7 M.A. Hurley et al. / Journal of Theoretical Biology 227 (2004) (Yokota et al., 2001; Seki et al., in press), and femalebiased phenotypic sex ratios observed in both the F 0 and F 1 generations. However, these data are clearly insufficient for validation purposes. It is therefore desirable to conduct purpose-designed validation experiments which are continued for at least 5 generations, and which record both genotypic and phenotypic sex. Choice of suitable test species will be crucial, and essential features will include XX/XY sex determination, relatively short generation time, amenability to laboratory-based experimentation, and availability of a simple test for genetic sex. Two species which fit this profile reasonably well include the Japanese medaka O. latipes, and the three-spine stickleback Gasterosteus aculeatus. 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