Masculinization of Adult Gambusia holbrooki: A Case of Recapitulation of Protogyny in a Gonochorist?

17α-Methyltestosterone (MT) is a synthetic steroid that has been widely used to masculinize many fish species when administered early during larval development, however, reports on its efficacy on adults is limited. To this end, this study investigated the efficacy of MT in the masculinization of the eastern mosquitofish (G. holbrooki) at two adult stages (maiden and repeat gravid females). The treated females were fed control or respective MT incorporated feed (0-200 mg/kg diet) for 50 days. Effects of the hormone on secondary sexual characteristics, internal gonad morphology, expression of the Anti-Müllerian Hormone (amh) gene and sexual behavior of the treated females were investigated. The results showed that MT at the dose of 50 mg/kg feed stimulated secondary sexual character development, upregulated expression of amh, formation of testicular tissue and a shift in the behavior similar to those of normal males, prominently so in treated maiden gravid females. Post-treatment, long-term observations indicated that only two masculinized females reverted back to being females and gave birth to young. Induction of masculinizing effects in most individuals suggests that the sexual phenotype of this species appears to be highly plastic with potential to sex reverse at adulthood. This in combination with its small size and short reproductive cycle could provide an ideal system to explore the mechanisms of sequential hermaphroditism in fish and contribute to genetic control of this pest fish.

Reproductive viability of paradoxically masculinised Gambusia holbrooki generated following diethylstilbestrol (DES) treatment.

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 × 108/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.

Sex biased expression of anti-Mullerian hormone (amh) gene in a live bearing fish, Gambusia holbrooki: Evolutionary implications and potential role in sex differentiation.

The amh, a member of transforming growth factor-ß (TGF-ß) family, is known to play a critical role in vertebrate male sex differentiation, with its paralogue/s evolving to determine sex in few heterogametic (XX/XY) teleosts. However, it remains relatively unexplored in the reproductively unique live bearing teleosts. Therefore, this study comparatively examined the structure and content of G. holbrooki amh as well as characterised its expression. A paralogous Y-specific amh (amhy) was not detected, suggesting an unlikely role in sex determination. Two transcripts (1.4 and 1.5 kb) were detected in adults: the larger (1.5 kb) retaining intron 5, coding for a truncated AMH-N and no TGF-ß domain. The small (1.4 kb) transcript, had both domains intact and clustered with members of Poeciliidae. In contrast to other vertebrates, a higher conservation between the N- rather than the C- terminus of amh in Poeciliidae was observed, suggesting an adaptation that may be unique to live bearing teleosts. The amh expression was 6 times higher in brain of both sexes and testis compared with ovaries (p = .001). Intriguingly, female splenic tissues showed 10 times higher expression (p = .006) and such female bias splenic expression has not been reported in any teleosts. Ontogenic expression was 25 times higher in male embryos at gastrulation stage (p = .001), much earlier than those reported in egg-laying teleosts. Such heightened expression in male embryos suggests a repressive role associated with proliferation and migration of primordial germ cells (PGCs) that are known to occur earlier at blastulation in teleosts-potentially influencing gonadal fate.

Genetic diversity of culturable Vibrio in an Australian blue mussel Mytilus galloprovincialis hatchery.

Bacillary necrosis associated with Vibrio species is the common cause of larval and spat mortality during commercial production of Australian blue mussel Mytilus galloprovincialis. A total of 87 randomly selected Vibrio isolates from various stages of rearing in a commercial mussel hatchery were characterised using partial sequences of the ATP synthase alpha subunit gene (atpA). The sequenced isolates represented 40 unique atpA genotypes, overwhelmingly dominated (98%) by V. splendidus group genotypes, with 1 V. harveyi group genotype also detected. The V. splendidus group sequences formed 5 moderately supported clusters allied with V. splendidus/V. lentus, V. atlanticus, V. tasmaniensis, V. cyclitrophicus and V. toranzoniae. All water sources showed considerable atpA gene diversity among Vibrio isolates, with 30 to 60% of unique isolates recovered from each source. Over half (53%) of Vibrio atpA genotypes were detected only once, and only 7 genotypes were recovered from multiple sources. Comparisons of phylogenetic diversity using UniFrac analysis showed that the culturable Vibrio community from intake, header, broodstock and larval tanks were phylogenetically similar, while spat tank communities were different. Culturable Vibrio associated with spat tank seawater differed in being dominated by V. toranzoniae-affiliated genotypes. The high diversity of V. splendidus group genotypes detected in this study reinforces the dynamic nature of microbial communities associated with hatchery culture and complicates our efforts to elucidate the role of V. splendidus group bacteria in vibriosis.