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Background: Divergent morphology and placentation of Poeciliids make them suitable model for investigating how evolutionary selection has altered and conserved the developmental mechanisms. However, there is limited description of their embryonic staging, despite representing a key evolutionary node that shares developmental strategy with placental vertebrates. Here, we describe the embryonic developmental stages of Gambusia holbrooki from zygote to parturition using freshly harvested embryos. Results: We defined 40 embryonic stages using a numbered (stages 0‐39; zygote to parturition, respectively) and named (grouped into seven periods, ie, zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and parturition) staging system. Two sets of quantitative (ie, egg diameter, embryonic total length, otic vesicle closure index, heart rates, the number of caudal fin rays and elements) and qualitative (ie, three‐dimensional analysis of images and key morphological criteria) data were acquired and used in combination to describe each stage. All 40 stages are separated by well‐defined morphological traits, revealing developmental novelties that are influenced by narrow perivitelline space, placentation, internal gestation, and sex differentiation. Conclusions: The principal diagnostic features described are quick, reliable, and easy to apply. This system will benefit researchers investigating molecular ontogeny, particularly sexual differentiation mechanisms in G. holbrooki. Key Findings: Comprehensive developmental staging system.Well‐defined morphological traits.Introduced novel indices in stage identification.Chronological description of different systems.Presented staging system is quick, reliable, and easy to apply. [ABSTRACT FROM AUTHOR]

Hormonal sex reversal can produce monosex fish stocks and provide insights into their gamity and reproductive physiology. However, paradoxical effects have been reported in several fish species that remain largely ignored as anomalies, particularly those of masculinisation. As a first step, this study examined reproductive viability of paradoxically masculinised Gambusia holbrooki produced following oral administration (20-100 mg/kg feed) of a feminizing hormone diethylstilbestrol (DES). Contrary to expectation, all treatment groups produced 100% male populations. Survival, mating behaviour, gamete production, breeding output as well as expression of anti-Mullerian hormone (amh), ovarian (cyp19a1a) and brain (cyp19a1b) aromatase of masculinised fish were also examined. Survival (≤ 54.1 ± 7.3%) at termination of DES treatment was significantly lower compared with controls (88.6 ± 4.3%) but remained unaffected post treatment. Gonopodium thrusting frequency (33 ± 9.8 per 10 min) was not significantly different to untreated males just as sperm abundance (3.9 ± 1.5 × 10 8 /male) and their motility (88.6 ± 29.1%). Importantly, paradoxically masculinised fish mated with virgin females and produced clutch sizes (22 ± 4) and progeny survival (87.0 ± %) that were comparable to that of untreated males. Masculinised testes showed high amh and low cyp19a1a expression, a pattern resembling those of untreated males. Production of paradoxically sex-reversed males with a capability to produce viable offspring has not been reported previously in this or other fish species. The outcomes support a feed-back regulation of oestrogenic pathways in this viviparous fish and could be useful for ecological applications such as controlling invasive fish populations., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)