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Fish show extraordinary sexual plasticity, changing sex naturally as part of their life cycle or reversing sex because of environmental stressors. This plasticity shows that sexual fate is not an irreversible process but the result of an ongoing tug-of-war for supremacy between male and female signaling networks. The behavioral, gonadal, and morphological changes involved in this process are well described, yet the molecular events that underpin those changes remain poorly understood. Epigenetic modifications emerge as a critical link between environmental stimuli, the onset of sex change, and subsequent maintenance of sexual phenotype. Here we synthesize current knowledge of sex change, focusing on the genetic and epigenetic processes that are likely involved in the initiation and regulation of sex change. We anticipate that better understanding of sex change in fish will shed new light on sex determination and development in vertebrates and on how environmental perturbations affect sexual fate.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings. Here we have established the protogynous New Zealand spotty wrasse, Notolabruscelidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using aromatase inhibition or manipulation of social groups. Complete female-to-male transition occurred over 60 days in both cases and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy. Early-stage decreases in plasma 17β-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

Many teleost fishes undergo natural sex change, and elucidating the physiological and molecular controls of this process offers unique opportunities not only to develop methods of controlling sex in aquaculture settings, but to better understand vertebrate sexual development more broadly. Induction of sex change in some sequentially hermaphroditic or gonochoristic fish can be achievedin vivothrough social manipulation, inhibition of aromatase activity, and steroid treatment. However, the induction of sex changein vitrohas been largely unexplored. In this study, we established anin vitroculture system for ovarian explants in serum-free medium for a model sequential hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). This culture technique enabled evaluating the effect of various treatments with 17β-estradiol (E2), 11-ketotestosterone (11KT) or cortisol (CORT) on spotty wrasse ovarian architecture for 21 days. A quantitative approach to measuring the degree of ovarian atresia within histological images was also developed, using pixel-based machine learning software. Ovarian atresia likely due to culture was observed across all treatments including no-hormone controls, but was minimised with treatment of at least 10 ng/mL E2. Neither 11KT nor CORT administration induced proliferation of spermatogonia (i.e. sex change) in the cultured ovaries indicating culture beyond 21 days may be needed to induce sex changein vitro. Thein vitrogonadal culture and analysis systems established here enable future studies investigating the paracrine role of sex steroids, glucocorticoids and a variety of other factors during gonadal sex change in fish.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings because sex change has been challenging to achieve in captivity. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using either aromatase inhibition or manipulation of social groups. Complete transition from female to male occurred over 60 days and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy using radioimmunoassay, nanoString nCounter mRNA and histological analyses, respectively. Early-stage decreases in plasma 17β-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Expression of male-related genes responsible for androgen production cyp11c1 and hsd11b2 increased from mid sex change. Gonadal expression of the glucocorticoid and mineralocorticoid receptors nr3c1 and nr3c2, putative mediators of the stress hormone cortisol, increased in late stages of sex change. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.Summary statementThe spotty wrasse, Notolabrus celidotus, is a new temperate model for the study of vertebrate sex change, this work characterises endocrine and genetic markers based on laboratory induced sex change.

Mollusc shells are an abundant resource that have been long used to predict the structures of ancient ecological communities, examine evolutionary processes, reconstruct paleoenvironmental conditions, track and predict responses to climatic change, and explore the movement of hominids across the globe. Despite the ubiquity of mollusc shell in many environments, it remains relatively unexplored as a substrate for molecular genetic analysis. Here we undertook a series of experiments using the New Zealand endemic greenshell mussel, Perna canaliculus, to explore the utility of fresh, aged, beach-cast and cooked mollusc shell for molecular genetic analyses. We find that reasonable quantities of DNA (0.002–21.48 ng/mg shell) can be derived from aged, beach-cast and cooked mussel shell and that this can routinely provide enough material to undertake PCR analyses of mitochondrial and nuclear gene fragments. Mitochondrial PCR amplification had an average success rate of 96.5% from shell tissue extracted thirteen months after the animal’s death. A success rate of 93.75% was obtained for cooked shells. Amplification of nuclear DNA (chitin synthase gene) was less successful (80% success from fresh shells, decreasing to 10% with time, and 75% from cooked shells). Our results demonstrate the promise of mollusc shell as a substrate for genetic analyses targeting both mitochondrial and nuclear genes.

Teleost fish are the only vertebrate lineage that includes sequentially hermaphroditic species, which begin life as one sex and change sometime later to the opposite sex as a natural part of their life cycle. Sequential hermaphrodites may be protandrous (male to female), protogynous (female to male) or bidirectional (serial sex change in both directions) sex changers. Sex change in fish is a remarkable example of sexual plasticity and involves cross-talk between multiple biological systems, including neuroendocrine, hormonal and molecular systems, to coordinate dramatic changes at a whole-body level.

The ornate spiny lobster Panulirus ornatus is a commercially important fishery and aquaculture species throughout south-east Asia and Australia. Thirty microsatellite markers were developed for P. ornatus of which 15 loci were consistently amplified and exhibited adequate levels of polymorphism for parentage and population genetic applications. Number of alleles observed per locus ranged from 2 to 23 (mean NA = 9.1 ± 1.4). Observed and expected heterozygosities ranged from 0.03–0.98 (HO mean = 0.61 ± 0.06) to 0.03–0.94 (HE mean = 0.66 ± 0.06), respectively. Microsatellite markers were also tested in three related Panulirus species, P. homarus, P. versicolor, and P. argus, with varying levels of amplification success and polymorphism exhibited.

454 shotgun sequencing was used to identify thousands of in silico microsatellite loci from two Australian freshwater turtle species (Pleurodira, Chelidae), Elseya albagula and Emydura macquarii krefftii. Twenty-nine polymorphic microsatellites were subsequently developed and tested as markers, 15 for E. m. krefftii and 14 for E. albagula. The former marker set showed greater allelic richness (mean N A = 7.7, range 3–15) than the latter (mean N A = 4.4, range 2–8). Levels of expected heterozygosity were high for both marker suites (mean H E = 0.718, range 0.177–0.875; and mean H E = 0.567, range 0.317–0.827, respectively), indicating both are suitable for studies of population genetic structure, connectivity and mating systems in these taxa. Cross-species amplification in 12 other short-necked freshwater turtle taxa highlighted the potential utility of these markers in other species, notably Emydura spp., Rheodytes leukops and Elusor macrurus.

Litoria nannotis is an endangered waterfall frog from the wet tropics region in north Queensland that has suffered significant population declines due to the emerging fungal disease known as chytridiomycosis. The species has two deeply divergent lineages, and we used 454 shotgun sequencing of DNA extracted from one individual of the northern lineage to identify and design PCR primers for 576 microsatellite loci. Thirty markers were tested for amplification success and variability in a population sample from each lineage. Of these, 17 were found to be polymorphic in the northern lineage and 10 loci were polymorphic in the southern lineage. Numbers of alleles per locus ranged from 2 to 14 (mean = 6.47, s.d. = 4.02) for the northern lineage (17 polymorphic loci), and from 2 to 8 (mean = 5.40, s.d. = 2.55) in the southern lineage (10 polymorphic loci). Levels of heterozygosity were high in both lineages (northern mean HE = 0.63, s.d. = 0.21, range = 0.27–0.89; southern mean HE = 0.57, s.d. = 0.25, range = 0.18–0.81). These loci will be useful in understanding the genetic variation and connectivity amongst populations of this species recovering from mass population declines due to disease.

Genetic parentage studies can provide detailed insights into the mating system dynamics of wild populations, including the prevalence and patterns of multiple paternity. Multiple paternity is assumed to be common among turtles, though its prevalence varies widely between species and populations. Several important groups remain to be investigated, including the family Chelidae, which dominate the freshwater turtle fauna of the Southern Hemisphere. We used seven polymorphic microsatellite markers to investigate the presence of multiple fathers within clutches from the white-throated snapping turtle (Elseya albagula), an Australian species of conservation concern. We uncovered a high incidence of multiple paternity, with 83% of clutches showing evidence of multiple fathers and up to three males contributing to single clutches. We confirm a largely promiscuous mating system for this species in the Burnett River, Queensland, although a lone incidence of single paternity indicates it is not the only strategy employed. These data provide the first example of multiple paternity in the Chelidae and extend our knowledge of the taxonomic breadth of multiple paternity in turtles of the Southern Hemisphere.