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


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Norazmi, LNH 2016 , 'Hormonal feminization and associated reproductive impacts in the eastern mosquitofish Gambusia holbrooki', PhD thesis, University of Tasmania.

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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.

Item Type: Thesis - PhD
Authors/Creators:Norazmi, LNH
Keywords: Invasive alien fish, Live bearing fish, Development, Endocrine disrupting compound, Estradiol, Diethylstilbestrol, Reproduction
Copyright Information:

Copyright 2016 the Author

Additional Information:

Chapter 2 has been published as: Norazmi-Lokman, N. H., Purser, G. J., Patil, J. G., 2016. Gravid spot predicts developmental progress and reproductive output in a livebearing fish, Gambusia holbrooki, PLoS one, 11(1), e0147711, 1-18 Copyright: © 2016 Norazmi-Lokman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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