Genomic Approaches to Study Genetic and Environmental Influences on Fish Sex Determination and Differentiation

Francesc Piferrer Laia Ribas Noelia Daz The embryonic gonad is the only organ that takes two mutually exclusive differentiating pathways and hence gives rise to two different adult organs: testes or ovaries. The recent application of genomic tools including microarrays, next-generation sequencing approaches, and epigenetics can significantly contribute to decipher the molecular mechanisms involved in the processes of sex determination and sex differentiation. However, in fish, these studies are complicated by the fact that these processes depend, perhaps to a larger extent when compared to other vertebrates, on the interplay of genetic and environmental influences. Here, we review the advances made so far, taking into account different experimental approaches, and illustrate some technical complications deriving from the fact that as development progresses it becomes more and more difficult to distinguish whether changes in gene expression or DNA methylation patterns are the cause or the consequence of such developmental events. Finally, we suggest some avenues for further research in both model fish species and fish species facing specific problems within an aquaculture context. - The main problems that fish may experience under captive conditions are skewed sex ratios, the existence of sexual growth dimorphism, the lack of sexual maturation or side effects of it on product quality, especially if maturation occurs before marketing, and the absence of final maturation or of gamete release (Fig. 1). Under culture conditions, some species either do not reproduce altogether or exhibit sex-related problems in reproduction. This is, for example, the case of the Senegalese sole (Solea senegalensis), where F1 males, which are normally oligospermic, do not produce or release sperm (Cerd et al. 2008), a situation that has significantly hampered the development of the aquaculture for this species. On the other hand, and as it is part of the biology of many fish (Breder and Rosen 1966; Parker 1992), in many aquacultured species one sex grows more than the other does. This sexual growth dimorphism can favor males (e.g., tilapias) or, more commonly, females (flatfishes, sea basses, etc.). In some cases, as for example in the turbot (Scophthalmus maximus), females can be up to 50 % larger than males (Imsland et al. 1997). In other cases, as in European sea bass (Dicentrarchus labrax), the rearing conditions result in highly male-biased stocks (Piferrer et al. 2005), and if males, as it happens also to be the case in this species, grow less than females, then the exploitation is ran at a suboptimal capacity. Thus, skewed sex ratios induced by the captive conditions in a particular species may have negative consequences because of the sex growth dimorphism stated earlier. It is then clear that knowing how sex ratios are established would contribute to the development of methods to achieve monosex populations consisting only of the desired sex. A great deal of research towards the development of such sex control methods has been carried out in fish (Piferrer 2001). Finally, many species of fish also mature precociously when subjected to the fast growing conditions Fig. 1 Reproduction-related problems in finfish aquaculture. The inner circle shows the different stages of the typical life cycle of finfish (not referred to any particular species), starting clockwise with the zygote, and indicating the approximate stage when marketing takes place. The middle circle shows events related to reproduction, starting with sex determination and finishing with spawning. The outer circle shows typical reproduction-related problems in aquaculture, linked to the former reproductive events (see text for further explanation). The shaded area represents the issues dealt with in this paper of modern aquaculture. This affects particularly males, and a well-known example also concerns the European sea bass (Felip et al. 2006). In other cases, such as in the rainbow trout (Onchorhynchus mykiss), monosex stocks are desired not as much because one sex grows more than the other, as is also the case in many salmonids, but because maturation affects the organoleptic properties of the edible parts more in one sex than in the other. Therefore, female monosexing in salmonids is especially useful when associated with triploidy because triploid females do not develop ovaries while triploid males still are able to develop testis (Piferrer et al. 2009). In addition, in species such as the sturgeons, the advantage of getting only females is due to the value of the ovaries as a source of caviar. Thus, knowing the underlying molecular mechanisms present along the brainhypophysisgonadal axis would also help in devising methods to alleviate maturation, including precocious maturation. In addition, in newly aquacultured species such as the bluefin tuna (Thynnus thunnus), many aspects of reproduction are still not known and therefore studies on the reproductive of this and other potential species for aquaculture are needed (e.g., Aranda et al. 2011) . The physiology, including the reproductive physiology, of an animal can be monitored largely not only by measuring key hormones but also by examining the expression of individual genes or a set of genes. Traditionally, this has been achieved in what is known as the candidate gene approach. Recently, with the advent of genomics, a range of new possibilities have emerged where one can examine the expression of thousands of genes and gene networks at once, contributing to a better understanding of key signaling and regulatory pathways related to important biological functions such as reproduction. The moment has arrived when this knowledge can be channeled to applied methods within an aquaculture production context. This paper briefly reviews the application of genomic approaches to the study of reproduction in fish of relevance for aquaculture, focusing on studies aimed at improving our understanding of the complexities of gene expression patterns underlying the establishment of sex ratios. Background on Fish Sex Determination and Differentiation Sex ratios are an important aspect of populations because these not only determine their reproductive potential but also directly influence growth dynamics, and this is very important in farmed animals and thus relevant in finfish aquaculture. The sex ratio is the product of sex determination, the genetic and/or environmental process that establishes the gender of an organism (Penman and Piferrer 2008), and of sex differentiation, the various genetic, physiological processes that transform an undifferentiated gonad into a testis or an ovary (Piferrer and Guiguen 2008) (Fig. 2a). Sex determination in fish can range from genotypic (genotypic sex determination, GSD) to environmental sex determination (ESD), with temperaturedependent sex determination (TSD) being the most common type of ESD (Fig. 2b). Controversy has surrounded the (...truncated)