Sex differentiation and determination in the invasive fish, Gambusia holbrooki

The eastern mosquitofish, Gambusia holbrooki, is a freshwater viviparous fish that originates from North America. This along with its sister species G. affinis, were introduced throughout the world (i.e., over 110 countries), including Australia as mosquito control agents. Due to their high adaptability and tolerance, they have rapidly established populations in new ecosystems and caused decline in native aquatic fauna through competition and predation. For example, in Australia, G. holbrooki has been implicated in the decline of nine native fish species (genera Galaxias, Chlamydogobius, Melanotaenia, Craterocephalus, Mogurnda, Pseudomugil, Ambassis, Scaturiginichthys and Retropinna) and 10 species of frog. Therefore, G. holbrooki is considered as one of the highly invasive species. Their impacts have become apparent, particularly in freshwater ecosystems, where invasive species constitute the single greatest threat to biodiversity. Hence, development of an effective 'threat abatement strategy' is vital. One of the most promising solutions for eradication, is the Trojan Y chromosome (TYC) approach. The strategy works by manipulating the sex ratio of the target population through the introduction of sex-reversed females carrying two Y chromosomes (Trojan Y) that produce only male offspring. However, for this to be successful, the chromosomal sex mechanism must be known. In addition, developing a phenotypic sex marker could facilitate the process of genetic screening of the embryos prior to and post-hormonal treatments (i.e., production of sex-reversed embryos) for rapid identification of sex reversed individuals. To investigate the relationship between genetic sex and phenotypic traits amongst various embryonic stages, a detailed developmental study is needed. However, little information was available on embryonic development in this species, with the exception of an earlier detailed staging system for Gambusia sp. Although less complete than the present study, the earlier study accurately describes stages of embryonic development, and includes useful sets of hand-drawn illustrations. More recently, another study established time-saving (i.e., without sacrificing the brood) approach to predict the developmental progress, by applying the gravid spot index of pregnant females as surrogates for five broad stages. Generally, proposed classifications are a guide but not precise for G. holbrooki developmental staging due to divergent traits and morphology. Regardless, several basic keys specific to different organ systems (e.g., circulation), detailed morphology and precise indices for each stage were virtually non-existent. The developmental studies are also essential to investigate the genotype-phenotype relationships as several sex-related events occur during the embryogenesis. However, these remain uninvestigated, particularly in G. holbrooki development. Low mitotic index is a common problem in cytogenetic studies when using direct (i.e., harvesting cells from tissue of a living animal instead of in vitro tissue culture) chromosome preparation methods. Moreover, to improve cytogenetic studies, physical stretching/relaxation of metaphase chromosomes is essential, primarily to enhance the resolution of the fine chromosomal region (i.e., precise detection). Intercalated agents such ethidium bromide (EtBr) is known as effective in altering the conformational and physical properties of DNA helix of the chromosomes. Although there are several reports confirming the key roles of EtBr that intercalates between DNA bases and prevents DNA folding and condensing in mammalian models like mice, there is no report of using these agents in any fish species including G. holbrooki. Several studies have suggested a XX/XY sex-determining system for G. holbrooki based on the melanic color pattern inheritance from father to son and its allelic linkage group associated to males. However, a sex determination mechanism has not been cytogenetically confirmed in this or any poecilids, with the exception of P. reticulata. Therefore, due to inconclusive evidence in the literature, multiple evidence, including cytogenetic are required to verify the chromosomal sex determining mechanism in G. holbrooki. In this context, the overarching objectives of this study were to discover early phenotypic markers of sex differentiation and ascertain the sex determining mechanism in G. holbrooki, specifically: 1) standardise developmental staging of G. holbrooki embryos for determining the timing and onset of sex determination and differentiation; 2) develop a phenotypic sex marker to assist distinguishing the sex of live embryos at very early developmental stages (e.g., prior to sex-reversal of embryos), and; 3) identify and confirm a sex determination mechanism (i.e. male or female heterogamety) using classical karyomorphology and comparative genomic hybridisation (CGH). Objectives were addressed by morphological studies of embryos during development from zygote to parturition (chapter 2), with emphasis on heart development (chapter 3) and cytogenetics (chapters 4 and 5). First (chapter 2), a comprehensive developmental staging of embryos that document sequential events of development from zygote to parturition (30 days post fertilization) at 25 ¬± 0.5 ¬∞C was defined. To overcome limitations associated with superfetation and obtaining an adequate number of embryos, two approaches were simultaneously employed; first, embryos were harvested from wild caught gravid females, with varying intensity of gravid spot, during peak reproductive season to ensure availability of sufficient embryos at multiple developmental stages. Second, to calibrate developmental timing, genetically sexed females (i.e., at parturition) were grown up to maturity (2‚-3 months) and then individually mated with males to record time of mating (i.e., first copulation behaviour) as proxy for fertilisation. Post-copulation females (n = 1 or 2) were sampled, and embryos harvested every 6 h for ten days (n = 35), then every 12 h for the remaining 20 days (n = 55). Live embryos were photographed. Morphological diagnostics were used for preliminary staging of embryos according to indices described for developmental staging in Gambusia sp. and zebrafish. For stage identification, a combination of key morphometric measurements (egg size, egg diameter and embryo total length), indices (i.e., otic vesicle closure, heart rates); and meristic counts (e.g., the number of caudal fin rays, and number of caudal fin ray elements) were also employed, as necessary. The development of nervous, circulation, musculoskeletal, visual, and digestive systems along with craniofacial and external body features, were also employed for staging. This was complimented by quantitative pixel analysis of embryonic photographs (i.e., three-dimensional pixel distribution analysis of images). The data were collectively used to describe and define each embryonic stage. The developmental process was defined into seven broad developmental phases (i.e., zygote, cleavage, blastula, gastrula, segmentation, pharyngula and parturition) and 40 stages numerically (stages 0‚-39; zygote to parturition, respectively). The superfetation, inadequacy of embryos, time/age-controlled during embryogenesis, non-transparency of the eggs and very limited developmental information amongst livebearers for detailed comparison posed some challenges. However, these were overcome by employing multiple strategies including the pixel intensity analysis to assist with identification of stages during early development. Traits found during development could be sex-related, for example, both pigmentation and skeletal traits (i.e., teeth) are sometimes linked to sex chromosome in teleost (e.g., Malawi cichlids). Therefore, the outputs and knowledge from the developmental staging allowed investigating the relationship between developmental phenotypic traits and their genetic sex. This was further validated using a genetic sex marker. Second (chapter 3), the heart rates (HRs) during embryogenesis of G. holbrooki, from onset of heartbeat to just prior to parturition was investigated. Genetic sex of the embryos was postverified using a sex-specific genetic marker. Heartbeat of embryos (n = 10 each sex/stage) at early organogenesis, mid-organogenesis, late organogenesis, early pharyngula, late pharyngula, just prior to parturition and adults (n = 10 each sex) were videographed for 60 s at 25 ¬± 0.5 ¬∞C. The HR and its frequencies were determined using a non-invasive light-cardiography (LCG) method previously described for adult zebrafish. LCG profiles for embryos corresponded to contraction of the ventricle (increased average brightness) conversely, relaxation (decreased average brightness) within the prescribed region of interests (ROI). LCG profiles for adults constitute an opposite signal intensity compared to embryos. The peaks for each LCG oscillation were found using Gauss model and peak analyser function (Origin Pro 2020). The time duration (s) between ventricular systolic (contraction) and atrial diastolic (dilation) phases, constituted a complete cardiac cycle. The time delay between the atrioventricular (A-V delay) peak values of the extracted synchronous chronologies within the same cardiac cycles were measured using time history values for ventricle and atrium peaks. A-V delay was defined as the time duration (s) between the onset of atrium peak and the onset of subsequent ventricular peak, which represents the resting phase of the heart. The average resting time per minute for all developmental stages was calculated by multiplying the total A-V delay/sec by 60. For quantification of ventricle size in adults, both ventricular surface area (mm2 ) and volume of the ventricle (mm3 ) were calculated using ImageJ. Ventricle volume measurements were normalised by condition factor (a) derived separately for each sex (male = 0.01; female ...