Reproduction-associated and sex determining genes expressed differently in testes of genotypic and temperature-induced males of gibel carp (Carassius gibelio): the molecular mechanisms underlying the reproductive plasticity and invasiveness

Sex in vertebrates is commonly determined by genotype and in some cases by environmental conditions, such as temperature. A few vertebrate species display intermediate conditions of sex determination, combining sex chromosomes with an effect of temperature. This phenomenon is known for the gibel carp (Carassius gibelio), an invasive cyprinid fish species, whose abilities to adapt and colonize new environments is linked with the combination of sexual and asexual reproduction. In this study, the gonad transcriptomes were compared between genotypic males and temperature-induced males of C. gibelio. We specifically focused on the meiosis-related and reproduction-associated genes. Many genes and pathways of cell cycle control and meiosis, male differentiation and spermatogenesis were common to genotypic and temperature-induced males of gibel carp. However, the underrepresentation of several GO terms and KEGG pathways related to reproduction and spermatogenesis in genotypic males suggests lower reproductive abilities of temperature-induced males. Our study highlights the differential transcription regulation of key genes related to male sex differentiation, steroid hormone regulation, meiosis, spermatogenesis and sperm-egg interaction including the genes associated with sperm fertilization, motility and sperm flagellum function. Especially, induced males strongly overexpressed hsd17b2 and slightly overexpressed the meiotic gene mnd1, while genotypic males overexpressed sox8a and cyp19a1a, as well as the crucial fertilization gene izumo. These findings contribute to the understanding of the molecular mechanisms underlying the reproductive plasticity and invasiveness of C. gibelio in Europe. Our study highlighs the role of males, whose appearance was crucial for the transition from asexual to sexual reproduction in this species. Our findings contribute to the understanding of the molecular mechanisms underlying the reproductive plasticity and invasiveness of C. gibelio in Europe. Overall design: The specimens of cyprinid fish, gibel carp (Carassius gibelio), were obtained from artificial breeding of parental fish sampled in Dyje River (Czech Republic). We performed a set of sexual crossing (diploid females and diploid males) and a set of induced gynogenesis (using sperm of diploid male Carassius gibelio, Cyprinus carpio or Abramis brama for induction of gynogenesis). Fertilized eggs resulting from sexual crossing of diploid (2n) gibel carp were exposed to 22 °C. The eggs resulting from a gynogenesis were activated by the sperm of conspecific or heterospecific males and were exposed to one of the following temperatures 22, 25, 28 or 31 °C. The gibel carp offspring, specifically genotypic diploid males and temperature-induced triploid (3n) males were reared in aquarium conditions until the age of two years (i.e. until reaching maturity) before their testis's tissues were sampled. For DEGs associated with reproduction, we used the comparison of genotypic diploid males (male 2n) and temperature induced males (male 3n) males. For DEGs associated with sex determination, we used the comparison of genotypic diploid males (male 2n) and three groups of the temperature induced males representing (1) male 3n group 1 – male offspring sampled at 28 and 31 °C and produced by a gynogenetic female for which offspring showed no sex reversal at 22 °C and full sex reversal at 28 and 31 °C, (2) male 3n group 2 – male offspring sampled at 28-31 °C and produced by a gynogenetic female for which offspring showed a high proportion of sex reversal at 22 °C and full sex reversal at 28 and 31 °C, and (3) male 3n group 3 - male offspring sampled at 22 °C and produced by a gynogenetic female for which offspring showed a high proportion of sex reversal at 22 °C and full sex reversal at 28 and 31 °C.