Simple SummaryThis study maps critical events of gonadogenesis in the live-bearing fishGambusia holbrooki. Significantly, it provides the first evidence for primary gonochorism, the transition of an embryonically bi-lobed gonad to a mono-lobed organ, as occurs in adults and the dimorphically programmed onset of sex differentiation in this species. The outcomes are significant in basic biology and are applicable for developing control options for this notorious pest fish.AbstractDespite their uniqueness, the ontogeny and differentiation of the single-lobed gonads in the poeciliids are very poorly understood. To address this, we employed both cellular and molecular approaches to systematically map the development of the testes and ovary inGambusia holbrookifrom pre-parturition to adulthood, encompassing well over 19 developmental stages. The results show that putative gonads form prior to the completion of somitogenesis in this species, a comparatively early occurrence among teleosts. Remarkably, the species recapitulates the typical bi-lobed origin of the gonads during early development that later undergoes steric metamorphosis to form a single-lobed organ. Thereafter, the germ cells undergo mitotic proliferation in a sex-dependent manner before the acquisition of the sexual phenotype. The differentiation of the ovary preceded that of the testes, which occurred before parturition, where the genetic females developed meiotic primary oocytes stage I, indicating ovarian differentiation. However, genetic males showed gonial stem cells in nests with slow mitotic proliferation at the same developmental stage. Indeed, the first signs of male differentiation were obvious only post-parturition. The expression pattern of the gonadosoma markersfoxl2,cyp19a1a,amhanddmrt1in pre- and post-natal developmental stages were consistent with morphological changes in early gonad; they were activated during embryogenesis, followed by the onset of gonad formation, and a sex-dimorphic expression pattern concurrent with sex differentiation of the ovary (foxl2,cyp19a1a) and testes (amhanddmrt1). In conclusion, this study documents for the first time the underlying events of gonad formation inG. holbrookiand shows that this occurs relatively earlier than those previously described for ovi- and viviparous fish species, which may contribute to its reproductive and invasive prowess.