Molecular investigations on sex determination and differentiation pathways in the common carp, Cyprinus carpio

Common carp is a highly important species, with significant aquaculture production and frequent use for fish biology and aquaculture research. In Australia, New Zealand and the US, common carp are declared as an invasive pest causing damage to endemic ecosystems. This thesis aims to advance our understanding of the molecular pathway of sex determination and differentiation in common carp with a view to facilitate the development of genetic control mechanisms in this species. The objective was addressed by the molecular cloning of six key genes involved in sexual development, 3 each critical for female and male development. Spatial expression of these genes was analysed in adult tissues and the onset and timing of expression was determined during early ontogeny and through larval development at two temperatures (20 and 25 °C) allowing investigation of the effect of temperature on expression and final sex ratios. The study confirms that there are two isoforms of the cytochrome P450 aromatase gene in the species, namely the ovarian (cyp19a) and brain (cyp19b). Based on the level of CYP19 expression, the brain was found to be the main site of aromatase synthesis, contributed predominantly by the brain isoform. Also expressed highly in the brain were both isoforms of SRY (sex-determining region Y)-box 9 (sox9) and forkhead box L2 (foxL2). Within the gonad, cyp19a and foxL2 were predominantly expressed in the ovary while doublesex and mab-3 related transcription factor 1 (dmrt1) was primarily expressed in the testis. Ontogenic expression indicated that cyp19a and sox9a were maternally inherited. Female critical cyp19a showed sexually dimorphic expression only in fish larger than 20 mm, with warmer conditions (25 °C) suppressing expression and suggesting a male-skewed final sex ratio. This indicates that differential expression maybe a result of sex differentiation rather than a cause. Conversely, expression of cyp19b peaked prior to hatching possibly indicating that sexual differentiation occurs first in the brain, before the gonads are present. Expression of dmrt1, critical for male development, peaked soon after fertilisation in the 25 °C treatment indicating a role early in the sex-determining pathway. Peak expression of sox9 genes and foxL2 occurred prior to hatch, with consequent expression failing to show any sexually dimorphic expression, suggesting that these genes play a role in early larval development in the species, but not sex differentiation. This thesis found cyp19a and dmrt1 to be accurate markers of either ovarian or testis differentiation respectively. The ability to influence the expression of these genes may result in manipulation of sex ratios of common carp, and other fish. This would be of benefit to both pest control, where population sex bias can result in extinction, and also aquaculture, where monosex populations can improve production efficiency. By developing a greater understanding of sex determination and differentiation teleost fish it is possible to gain further insights into the evolution of sex determination in all vertebrate species.