Hormonal feminization and associated reproductive impacts in the eastern mosquitofish Gambusia holbrooki

The eastern mosquitofish Gambusia holbrooki has been listed in Australia as a noxious invasive species that requires control and eradication solutions on a large spatial scale. The Trojan Sex Chromosome (TSC) strategy has been proposed in recent years as a new genetic solution to control invasive aquatic species. The TSC approach involves the release of Trojan chromosome carriers, individuals produced via hormone sex reversal into the wild to skew the sex ratio of the population. As a first step towards applying the TSC strategy in G. holbrooki, this study systematically investigated and documented the relationship of the gravid spot with gestation, clutch size and timing of parturition – knowledge that are essential for efficient administration of hormones in this species. Importantly, by utilizing this new knowledge, the efficacy of two feminizing hormones (Diethylstilbestrol-DES and Estradiol-E2) was tested and the reproductive fitness of treated fish assessed. The study identified the gravid spot of females as an excellent marker to predict embryonic developmental progress and reproductive output in G. holbrooki by means of quantifying its visual attributes (intensity and size). An equation to predict clutch size using the relationship between gravid spot intensity and size together with fish length was ascertained [CS = 1.835 − (0.85 X SS) + (0.196 X SI) + (3.543 X FL), where SS and SI are gravid spot size and intensity respectively, and FL is fish length]. The reliability of these findings was confirmed when it facilitated design and accurate observation on G. holbrooki gestation period and parturition behaviour. The gestation was significantly longer (F=364.58; df=1,48; P˃0.05) when reared at 23°C (39±1.91 days) compared to 25 °C (28.6±1.94 days). However, temperature did not have significant impact (P>0.05) on clutch size or diel timing of parturition which occurs predominantly in the morning (0900-1100h). The first-ever description on the posture of G. holbrooki fry (progenies) during parturition was also reported where the tail of the fry emerged first with a few exceptions of head-first, twin and premature births. All this information especially the utility of the gravid spot as an external marker of embryonic development was used to structure the sex reversal experiments. Sex reversal experiments administering both DES and E2 through food targeted two life stages separately: (i) embryonic stage through gravid females and (ii) newborn juveniles. The concentrations of DES tested at both life stages ranged between 20 to 100 mg/kg feed. Two control groups were set for each experiment: (C1) normal feed (no chemical exposure) and (C2) feed mixed with 70% ethanol (vehicle control). In the first experiment, DES treatment did not affect the duration of gestation period in parturating females but as the treatment continued, DES was found responsible for gonadal atrophy in all the treated females after 30 days of exposure. The mean survival rate (MSR) of juveniles was significantly lower (F=16.79; df: 6, 15; P<0.05) compared to the controls (C1: 74.76±15.37%; C2: 71.26±18.5%) where the highest MSR was only 30.15±6.05% in fish treated at the concentration of 40 mg/kg feed for the duration of 11-23 days. Similarly, the MSR of juveniles fed with DES supplemented diet (second experiment) was also significantly lower (F=3.216; df: 6, 28; P<0.05) than controls (C1: 78.42 ±22.0%; C2: 73.8 ±20.22%) with the highest MSR shown by fish treated with DES at 20 mg/kg feed for 30 days (45.6 ±17.93%). Unexpectedly, masculinization was observed in all treated fish (in both experiments 1 and 2) where all the juveniles were phenotypically (morphologically) male based on the elongation of the anal fin. Observation of treated fish at 365 days after parturition (DAP) old revealed that all individuals possessed an under-developed gonopodium compared to normal males, suggesting a paradoxical masculinising and an incomplete sex-reversal effect on genetic females and males respectively in all the treated groups. This condition also seems to have affected the reproductive viability of the fish since none managed to breed with unexposed fish. A paradoxical effect even at relatively low doses of treatment suggests that DES is not a suitable feminising agent for G. holbrooki sex reversal. Molecular and cellular experimentation is warranted for further understanding of the mechanism underpinning this rare observation. Similar to DES treatment, E2 was administered at concentration ranging between 50-400 mg/kg of feed in two life stages —embryos and juveniles. Two control groups were also set as those in the DES experiment. The MSR of controls were found to be slightly higher (F=4.38; df: 6, 27; P<0.05) compared to the treatment groups (C1: 79.96±20.327%; C2: 77.09±10.32%). Treatment of E2 at 200-400 mg/kg feed between 12-21 days successfully produced a 100% female population in the embryo administered group, with the highest MSR of 59.33±12.54% shown by those treated at 200 mg/kg feed. The gestation period of the treated females was not altered by the exposure to E2. In the second group (juvenile administration), the MSR of controls were also significantly higher (F=7.27; df: 5, 24; P<0.05) compared to the treatment groups (C1: 71.73±22.86%; C2: 70.02±18.26%). A 100% feminization was achieved at all administered doses with juveniles treated with an E2 concentration of 50 mg/kg feed displaying the highest MSR at 66.38±12.34%. The survival rates observed in this study are substantially low compared to other E2 treated livebearers such as guppies and black mollies. High stocking densities which lead to aggression by dominant females is proposed as one of the reasons for the low survival rates in E2 treated G. holbrooki. Reproductive fitness assessment was conducted on two groups of G. holbrooki that showed the best performance (highest MSR and feminization percentage) in the sex reversal experiment namely: (i) fish that were treated with E2 at 200 mg/kg of feed (first experimental group); (ii) those exposed to E2 concentration of 50 mg/kg of feed (second experimental group). In parallel a control group (unexposed fish) was also assessed. All juveniles were reared to maturity and bred with normal males. In general the assessment shows that E2 treatments at optimum dose did not compromise the reproductive fitness of the treated fish. There was no significant difference (P>0.05) in terms of ability to breed, gestation period, clutch size and the MSR of progenies produced between treated fish and controls. These observations demonstrate that the reproductive fitness of E2 treated fish is on par with controls. Nevertheless, the number of progeny produced by females in all three groups was low (1-5 fish) warranting continued longterm observations of subsequent clutches. In conclusion, feminization of G. holbrooki was successfully achieved by using E2 as a feminizing agent. The study established a protocol to successfully feminize this species at optimum dose of E2 through oral administration either during the embryonic stage via female brood (200 mg/kg) or to newborn juveniles (50 mg/kg). The protocols and information generated in this study provide a basis for further refinement of hormone treatment and for developing the TSC strategy to control and eradicate this noxious pest. Other studies on fish reproductive biology and ecology especially in livebearing species as well as research on ecotoxicology and pest fish management will benefit from the outcomes presented in this thesis.