Heat-induced masculinization in domesticated zebrafish is family-specific and yields a set of different gonadal transcriptomes

Abstract

Understanding environmental influences on sex ratios is important for the study of the evolution of sex-determining mechanisms and for evaluating the effects of global warming and chemical pollution. Fishes exhibit sexual plasticity, but the underlying mechanisms of environmental effects on their reproduction are unclear even in the well-established teleost research model, the zebrafish. Here we established the conditions to study the effects of elevated temperature on zebrafish sex. We showed that sex ratio response to elevated temperature is family-specific and typically leads to masculinization (female-to-male sex reversal), resulting in neomales. These results uncovered genotype-by-environment interactions that support a polygenic sex determination system in domesticated (laboratory) zebrafish. We found that some heat-treated fish had gene expression profiles similar to untreated controls of the same sex, indicating that they were resistant to thermal effects. Further, most neomales had gonadal transcriptomes similar to that of regular males. Strikingly, we discovered heat-treated females that displayed a normal ovarian phenotype but with a "male-like" gonadal transcriptome. Such major transcriptomic reprogramming with preserved organ structure has never been reported. Juveniles were also found to have a male-like transcriptome shortly after exposure to heat. These findings were validated by analyzing the expression of genes and signaling pathways associated with sex differentiation. Our results revealed a lasting thermal effect on zebrafish gonads, suggesting new avenues for detection of functional consequences of elevated temperature in natural fish populations in a global warming scenario.A major paradox in developmental biology is the lack of conservation of sex-determining mechanisms even in closely related taxa (1). Thus, in mammals and birds, sex is genetically canalized by a chromosomal sex determination (CSD) system of male (XX/XY in mammals) or female (ZW/ZZ in birds) heterogamety, driven by the action of a master sex-determining gene: sry in mammals and dmrt1 in birds (1). In contrast, fish sex can be very plastic, with the combination of genetic and environmental influences (2). In addition to fish species with CSD, there are species with polygenic sex determination (PSD), in which sex depends on the combined action of several promale and profemale genetic factors, and species with temperature-dependent sex determination (TSD) (3). Understanding how the environment influences sex ratios in vertebrates is of fundamental importance in the study of the evolution of sex-determining mechanisms (4, 5). It is also of practical relevance in evaluating the effects of climate change and chemical pollution (6, 7).The effects of temperature on gene expression during sex differentiation have been investigated in different teleost species, including the African catfish, Clarias gariepinus (8), European sea bass, Dicentrarchus labrax (9, 10), Japanese flounder, Paralichthys olivaceus (11), pejerrey, Odontesthes...

On the proper rearing density in domesticated zebrafish to avoid unwanted masculinization. Links with the stress response

“…Because of this loss, the TU and AB strain sexual development is more sensitive to environmental cues. This concurs with the fact that domesticated zebrafish show a wide range of inter-family variation in sex ratios and that a polygenic system of sex determination has been proposed for domesticated zebrafish (Liew et al, 2012;Ribas et al, 2017; see Table 1). Taken together, it can be concluded that the effects of density found in this study with the AB strain would also apply to the TU strain, while experiments with the WIK strain are needed in order to determine whether the presence of the WZ/ZZ system confers greater resistance to the influence of environmental cues on sex ratios.…”

“…Studies looking at genetic polymorphisms and using a variety of screening methods on domesticated zebrafish strains have identified putative sex-linked loci in different chromosomes: 3 and 4 (Anderson et al, 2012), 5 (Bradley et al, 2011) and 16 (Howe et al, 2013). Moreover, using different families and several crosses, family-dependent sex ratios were obtained, which led to the proposal that domesticated zebrafish have a polygenetic sex-determining system in which genetic factors and environment determine the sex (Liew et al, 2012;Ribas et al, 2017). Recently, in wild zebrafish populations it has been found that a locus at the telomeric region of chromosome 4 is strongly linked with sex and compatible with a WZ/ZZ sex determination system (Wilson et al, 2014).…”

Response to “The importance of controlling genetic variation – remarks on ‘Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response’”

“…This was reflected by different susceptibilities of elevated temperature among different families (see fig. 1C of Ribas et al, 2017b). Furthermore, in Ribas et al (2017a) we clearly stated that ongoing experiments in our laboratory confirm the masculinizing effects of elevated densities.…”

“…In that study, pairs 2, 3 and 4 showed an increase in the number of males, while pair 1 showed the opposite effect but, again, the masculinizing effect of elevated density was seen when the sex ratios obtained at each density were compared with the sex ratios of the lowest density, as also stated. Genotype-by-environment (G×E) interactions in the sex ratio response to external factors in zebrafish have been recently described by Ribas et al (2017b), in accordance with the existence of the sex ratio variation among, but not within, families of domesticated zebrafish, as previously proposed by Liew et al (2012). This was reflected by different susceptibilities of elevated temperature among different families (see fig.…”

Stress and sex: does cortisol mediate sex change in fish?

“…temperature, social changes, pH, hypoxia, relative density, pathogens, etc., have been reported to act as environmental cues for ESR (reviewed by Devlin & Nagahama 2002, Stelkens & Wedekind 2010. Of these, water temperature and social factors have been the most extensively studied (Warner 1984, Ospina-Alvarez & Piferrer 2008, Godwin 2009, Kato et al 2011, Ribas et al 2017. Studies on temperature sex determination (a type of ESD) in fish constitute a great example of the interaction and blurred lines between the false GSD-ESD dichotomy, showing that the production of cortisol as a result of thermal stress can lead to masculinization of female fish under artificial conditions, in a transformation known as TIM.…”