Genotype by temperature interaction effects on sex determination in zebrafish (Danio rerio)

Abstract

The mechanism of sex determination and gonad differentiation in zebrafish is one of the more challenging research questions and complex puzzles in biology. The regulation of sexual dimorphism in this species is not yet fully understood. According to current knowledge, the sex of zebrafish is determined genetically on the basis of a polygenic sex determination system. In this system the interaction of genetic and environmental factors contributes to determine the sexual fate of an organism. The main objective of this thesis was to investigate the effects of high ambient temperature, as one of the major environmental factors, in interaction with the genome on sexual plasticity and phenotypic traits of zebrafish. Chapter 1 introduces a general overview of sexual diversity in teleost fish and explains the main features of sex determination and gonad differentiation mechanisms in zebrafish. The sexual selection theory in relation to the reproductive success and the effect of environment on secondary sexual traits is generally described. In chapter 2, the phenotypic plasticity of zebrafish in response to increased water temperature is investigated. The study was designed to test the influence of transient temperature perturbations during the thermosensitive embryonic developmental period (from gastrula to pharyngula) in a high number of families (69 families) on various biological functions. The physiological response to the high ambient temperature revealed a lower hatchability and survival ability during embryonic and post-embryonic development. The results of survival trajectories until adulthood demonstrated that in zebrafish the life stages most sensitive to thermal changes are the first day after fertilization and the first two weeks after hatching. Consideration of the effect of temperature on morphometric traits (weight and length) in order to investigate the temperature × sex interaction effect indicated a higher growth performance in temperature-treated animals at different time points of adulthood development. A heat-induced masculinization was clearly observed across all families, while a wide range of interfamily sex ratio variations was detected. These observations emphasize a genetic × environment (G×E) interaction of sex determination and constitute a strong confirmation for a polygenic sex determination system in zebrafish. In chapter 3, a fully automated phenotypic sex classification in zebrafish was established for the first time using two different machine learning methods: Deep Convolutional Neural Networks (DCNNs) and Support Vector Machine (SVM). Based on phenotypic characteristics, a high accuracy of sex differentiation was obtained using these two methods in non-heat-treated groups. However, in treated animals, some males were misclassified using SVM due to reduced pigmentation intensity, suggesting these animals were probably masculinized females. Investigation of the colour intensity of the caudal fin using SVM shows that males exhibited a higher pigmentation intensity compared to females. Furthermore, a positive association of male caudal fin colouration with the morphometric traits (weight and length) was determined. These results imply the effect of temperature on secondary phenotypic sexual characteristics, which in turn may influence the sexual attractiveness for mating and reproductive success. Phenotypic modifications in response to different environmental conditions indicate the changes in the expression of genes responsible for those traits. Hence chapter 4 examines, whether the sex and colour genes are differentially expressed in the two sexes with respect to the observed sexually dimorphic in the caudal fin colouration. This hypothesis is derived from chapter 3, where the sexually differing colour intensity in the caudal fin of the two sexes was detected. Therefore, a transcriptome analysis of caudal fins and gonads was performed in chapter 4 in order to identify the differentially expressed genes and pathways regulating sexual dimorphism in interaction with high water temperature. In addition, the mechanism of gene expression in the masculinization process, as one of the most important consequences of increased temperature, was studied in this chapter. A significantly differentiated expression of sex determination and colour pattern genes was identified in the gonad. In the caudal fin, a high expression magnitude of a set of colour pattern genes was observed, although they were not differentially expressed in two sexes of adult fish. The enrichment of a subset of pathways containing sex and colour genes provides an evidence of the involvement of those genes in the regulation of phenotypic sexual dimorphism in zebrafish. These results led to additional analyses in chapter 5 examining the validity of this hypothesis. In chapter 5, the topics of previous chapters are first discussed in more detail, and then the hypothesis of chapter 4 is further investigated by pathway analysis, gene interaction networks and transcription factor analysis. All analyses in this context supported the association between sex determination and colour pattern genes in zebrafish, as proposed in chapter 4 of this thesis. The conclusion of this chapter highlights the perspective for future studies answering the open questions derived from this thesis.