Dietary carotenoid supplementation has long-term and community-wide effects on the amphibian skin microbiome.

The amphibian skin microbiome plays a crucial role in host immunity and pathogen defence, yet we know little about the environmental drivers of skin microbial variation across host individuals. Inter-individual variation in the availability of micro-nutrients such as dietary carotenoids, which are involved in amphibian immunity, may be one factor that influences skin microbial assembly across different life history stages. We compared the effect of four carotenoid supplementation regimes during different life stages on the adult skin microbiome using a captive population of the critically endangered southern corroboree frog, Pseudophryne corroboree. We applied 16S rRNA sequencing paired with joint-species distribution models to examine the effect of supplementation on taxon abundances. We found that carotenoid supplementation had subtle yet taxonomically widespread effects on the skin microbiome, even 4.5 years post supplementation. Supplementation during any life-history stage tended to have a positive effect on the number of bacterial taxa detected, although explanatory power was low. Some genera were sensitive to supplementation pre-metamorphosis, but most demonstrated either additive or dominant effects, whereby supplementation during one life history stage had intermediate or similar effects, respectively, to supplementation across life. Carotenoid supplementation increased abundances of taxa belonging to lactic acid bacteria, including Lactococcus and Enterococcus, a group of bacteria that have previously been linked to protection against the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). While the fitness benefits of these microbial shifts require further study, these results suggest a fundamental relationship between nutrition and the amphibian skin microbiome which may be critical to amphibian health and the development of novel conservation strategies.

The Increasing Role of Short-Term Sperm Storage and Cryopreservation in Conserving Threatened Amphibian Species.

Multidisciplinary approaches to conserve threatened species are required to curb biodiversity loss. Globally, amphibians are facing the most severe declines of any vertebrate class. In response, conservation breeding programs have been established in a growing number of amphibian species as a safeguard against further extinction. One of the main challenges to the long-term success of conservation breeding programs is the maintenance of genetic diversity, which, if lost, poses threats to the viability and adaptive potential of at-risk populations. Integrating reproductive technologies into conservation breeding programs can greatly assist genetic management and facilitate genetic exchange between captive and wild populations, as well as reinvigorate genetic diversity from expired genotypes. The generation of offspring produced via assisted fertilisation using frozen-thawed sperm has been achieved in a small but growing number of amphibian species and is poised to be a valuable tool for the genetic management of many more threatened species globally. This review discusses the role of sperm storage in amphibian conservation, presents the state of current technologies for the short-term cold storage and cryopreservation of amphibian sperm, and discusses the generation of cryo-derived offspring.

Application of Reproductive Technologies to the Critically Endangered Baw Baw Frog, Philoria frosti.

Reproductive technologies (RTs) can assist integrated conservation breeding programs to attain propagation targets and manage genetic diversity more effectively. While the application of RTs to enhance the conservation management of threatened amphibians has lagged behind that of other taxonomic groups, a recent surge in research is narrowing the divide. The present study reports on the first application of RTs (hormone-induced spawning, hormone-induced sperm-release, and sperm cryopreservation) to the critically endangered Baw Baw frog, Philoria frosti. To determine the effect of hormone therapy on spawning success, male-female pairs were administered either 0 µg/g gonadotropin-releasing hormone agonist (GnRHa), 0.5 µg/g GnRHa, or 0.5 µg/g GnRHa + 10 µg/g metoclopramide (MET) (n = 6-7 pairs/treatment), and the number of pairs ovipositing, total eggs, and percent fertilisation success were quantified. To determine the effect of hormone therapy on sperm-release and to establish the peak time to collect sperm post-hormone administration, males were administered 0 IU/g (n = 4), or 20 IU/g hCG (n = 16). Total sperm, sperm concentration, and percent viability were quantified at 0, 2, 4, 6, 8, 10, and 12 h post-hormone administration. Overall, the percentage of pairs ovipositing was highest in the GnRHa + MET treatment, with 71% of pairs ovipositing, compared to 57% and 33% of pairs in the GnRHa and control treatments, respectively. The quantity of sperm released from males in response to hCG peaked at 4 h post-hormone administration, though it remained high up to 12 h. The percent sperm viability also peaked at 4 h post-administration (94.5%), exhibiting a steady decline thereafter, though viability remained above 77% throughout the 12 h collection period. The remaining sperm samples (n = 22) were cryopreserved using established protocols and biobanked for long-term storage and future conservation applications. The mean post-thaw sperm viability was 59%, and the percent total motility was 17%. The results from this preliminary study will direct further applications of RTs to the critically endangered Baw Baw frog to assist with species recovery.

A test for plasticity in sperm motility activation in response to osmotic environment in an anuran amphibian.

Evolutionary theory predicts that selection will favor phenotypic plasticity in sperm traits that maximize fertilization success in dynamic fertilization environments. In species with external fertilization, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but evidence for osmotic-induced sperm plasticity is limited to euryhaline fish and marine invertebrates. Whether this capacity extends to freshwater taxa remains unknown. Here, we provide the first test for plasticity in sperm-motility activation in response to osmotic environment in an anuran amphibian. Male common eastern froglets (Crinia signifera) were acclimated to either low (0 mOsmol kg-1) or high (50 mOsmol kg-1) environmental osmolality, and using a split-sample experimental design, sperm were activated across a range of osmolality treatments (0, 25, 50, 75, 100, and 200 ± 2 mOsmol kg-1). Unexpectedly, there was no detectable shift in the optimal osmolality for sperm-motility activation after approximately 13 weeks of acclimation (a period reflecting the duration of the winter breeding season). However, in both the low and high acclimation treatments, the optimal osmolality for sperm-motility activation mirrored the osmolality at the natural breeding site, indicating a phenotypic match to the local environment. Previously it has been shown that C. signifera display among-population covariation between environmental osmolality and sperm performance. Coupled with this finding, the results of the present study suggest that inter-population differences reflect genetic divergence and local adaptation. We discuss the need for experimental tests of osmotic-induced sperm plasticity in more freshwater taxa to better understand the environmental and evolutionary contexts favoring adaptive plasticity in sperm-motility activation.

Does dietary ß-carotene influence ontogenetic colour change in the southern corroboree frog?

Ontogenetic colour change occurs in a diversity of vertebrate taxa and may be closely linked to dietary changes throughout development. In various species, red, orange and yellow colouration can be enhanced by the consumption of carotenoids. However, a paucity of long-term dietary manipulation studies means that little is known of the role of individual carotenoid compounds in ontogenetic colour change. We know even less about the influence of individual compounds at different doses (dose effects). The present study aimed to use a large dietary manipulation experiment to investigate the effect of dietary ß-carotene supplementation on colouration in southern corroboree frogs (Pseudophryne corroboree) during early post-metamorphic development. Frogs were reared on four dietary treatments with four ß-carotene concentrations (0, 1, 2 and 3 mg g-1), with frog colour measured every 8 weeks for 32 weeks. ß-Carotene was not found to influence colouration at any dose. However, colouration was found to become more conspicuous over time, including in the control treatment. Moreover, all frogs expressed colour maximally at a similar point in development. These results imply that, for our study species, (1) ß-carotene may contribute little or nothing to colouration, (2) frogs can manufacture their own colour, (3) colour development is a continual process and (4) there may have been selection for synchronised development of colour expression. We discuss the potential adaptive benefit of ontogenetic colour change in P. corroboree. More broadly, we draw attention to the potential for adaptive developmental synchrony in the expression of colouration in aposematic species.

Hormone-induced ovulation and artificial fertilisation in four terrestrial-breeding anurans.

The development and application of reproductive technologies has great potential to enhance the conservation management of threatened amphibians globally. The present study quantified the efficacy of protocols previously developed for Pseudophryne guentheri for hormonally inducing egg release and artificial fertilisation in three additional terrestrial-breeding species of Australian ground frog; namely Pseudophryne bibronii, Pseudophryne coriacea and Heleioporus eyrei. Females of each species were administered a priming dose of 0.4µg g-1 of gonadotropin-releasing hormone analogue (GnRHa), followed by an ovulatory dose of 2µg g-1 GnRHa. Egg release was achieved in all species (response 94.7-100%), but early embryo survival differed significantly, ranging from 23.8% to 89.3%. Overall, the protocols were considered effective in P. guentheri, P. bibronii and P. coriacea, but further protocol refinement is required for H. eyrei.

Evidence that genetic compatibility underpins female mate choice in a monandrous amphibian.

Mate choice for genetic benefits remains controversial, largely because few studies have estimated the relative contributions of additive and non-additive sources of genetic variation to offspring fitness. Moreover, there remains a deficit of these estimates for species where female-mate preferences have been quantified in the wild, especially species characterized by monandry or monogamy. Here, we use artificial fertilization techniques combined with a cross-classified breeding design to simultaneously test for "good genes" and "compatible genes" benefits of mate choice in the monandrous red backed toadlet (Pseudophryne coriacea). In addition, we used a genomic approach to estimate effects of parental-genetic relatedness (assessed using 27, 768 single nucleotide polymorphisms) on offspring fitness. Our results revealed no significant additive genetic effects (sire effects), but highly significant non-additive genetic effects (sire × dam interaction effects), on fertilization success, survival during embryonic development, and hatching success. We also found significant associations between parental genetic similarity and offspring survival (whereby survival was higher when parents were more related), and significant positive relationships between fertilization success and embryo survival through to hatching. These results indicate that offspring viability is significantly influenced by the genetic compatibility of parental genotypes, that more related parents are more genetically compatible, and that gametes with greater compatibility at fertilization produce more viable offspring. More broadly, our findings provide new quantitative genetic evidence that genetic incompatibility underpins female mate preferences. Continued quantitative genetic assessment of the relative importance of good genes versus compatible genes is needed to ascertain the general importance of genetic benefits as a driver of female mate choice.

Love at first flight: wing interference patterns are species-specific and sexually dimorphic in blowflies (Diptera: Calliphoridae).

Wing interference patterns (WIPs) are stable structural colours displayed on insect wings which are only visible at specific viewing geometries and against certain backgrounds. These patterns are widespread among flies and wasps, and growing evidence suggests that they may function as species- and sex-specific mating cues in a range of taxa. As such, it is expected that WIPs should differ between species and show clear sexual dimorphisms. However, the true extent to which WIPs vary between species, sexes and individuals is currently unclear, as previous studies have only taken a qualitative approach, without considering how WIPs might be perceived by the insect. Here, we perform the first quantitative analysis of inter- and intra-specific variation in WIPs across seven Australian species of the blowfly genus Chrysomya. Using multispectral digital imaging and a tentative model of blowfly colour vision, we provide quantitative evidence that WIPs are species-specific, highlight that the extent of divergence is greater in males than in females and demonstrate sexual dimorphisms in several species. These data suggest that WIPs have diversified substantially in blowflies as a result of either sexual or ecological selection.

The effect of injection and topical application of hCG and GnRH agonist to induce sperm-release in the roseate frog, Geocrinia rosea.

Reproductive technologies may assist amphibian conservation breeding programs (CBPs) to achieve propagation targets and genetic management goals. However, a trial-and-error approach to protocol refinement has led to few amphibian CBPs routinely employing reproductive technologies with predictable outcomes. Additionally, while injections can be safely administered to amphibians, perceived animal welfare risks, such as injury and disease transmission, warrant the development of alternative hormone administration protocols. The present study investigated the spermiation response of roseate frogs, Geocrinia rosea, administered various doses of human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone agonist (GnRH-a) via subcutaneous injection. This study also quantified the spermiation response of frogs administered both hormones via topical application. Total sperm, sperm concentration and sperm viability were assessed over a 12-h period post hormone administration. Males released sperm in response to the injection of hCG (88-100% response; 5, 10 or 20 IU), but all samples collected from males administered hCG topically (100, 100 + DMSO or 200 IU hCG) were aspermic. In contrast, males consistently released sperm in response to both the injection (100% response; 1, 5 or 10 µg), or topical application (80-100% response; 50, 50 + DMSO or 100 µg) of GnRH-a. Overall, the administration of GnRH-a was more effective at inducing spermiation than hCG. Mean total sperm and sperm concentration were highest in response to the optimal topically applied dose of 100 µg GnRH-a (mean total sperm = 2.44 × 103, sperm concentration = 1.48 × 105 sperm/ml). We provide novel evidence that topical application provides a viable alternative to injection for the administration of GnRH-a to induce spermiation in amphibians.

Major Transitions in Cuticular Hydrocarbon Expression Coincide with Sexual Maturity in a Blowfly (Diptera: Calliphoridae).

In many animals, there is a prolonged pre-reproductive period prior to sexual maturity. To avoid premature mating attempts, it is common for phenotypic changes to occur during this period that signal the onset of reproductive viability. Among the insects, pre-reproductive phases can last for up to 50% of the adult lifespan, but little is known about the accompanying phenotypic changes that signal sexual maturity. Contact pheromones such as cuticular hydrocarbons (CHCs) may fulfil this role, as they are known to change rapidly with age in many insects. Despite this, few studies have investigated CHC development in the context of sexual maturity or considered differences in CHC development between sexes. The blowflies (Diptera: Calliphoridae) provide an ideal system for such studies because CHCs are known to change rapidly with age and likely play an important role in sexual behaviour. As such, using the small hairy maggot blowfly Chrysomya varipes, we investigate whether there are age- and sex-specific changes in CHCs over the course of adult blowfly maturation. We show that: (1) major qualitative transitions in CHC expression coincide with the onset of sexual maturity and (2) these changes occur more slowly in females - in line with their extended pre-reproductive phase. We suggest that CHCs may play an important role in signalling sexual maturity in the small hairy maggot blowfly and that this species will likely serve as a useful model for understanding the complex ontogeny of cuticular hydrocarbons in insects.

The evolution of sexually dimorphic cuticular hydrocarbons in blowflies (Diptera: Calliphoridae).

Cuticular hydrocarbons (CHCs) are organic compounds found on the cuticles of all insects which can act as close-contact pheromones, while also providing a hydrophobic barrier to water loss. Given their widespread importance in sexual behaviour and survival, CHCs have likely contributed heavily to the adaptation and speciation of insects. Despite this, the patterns and mechanisms of their diversification have been studied in very few taxa. Here, we perform the first study of CHC diversification in blowflies, focussing on wild populations of the ecologically diverse genus Chrysomya. We convert CHC profiles into qualitative and quantitative traits and assess their inter- and intra-specific variation across 10 species. We also construct a global phylogeny of Chrysomya, onto which CHCs were mapped to explore the patterns of their diversification. For the first time, we demonstrate that blowflies express an exceptional diversity of CHCs, which have diversified in a nonphylogenetic and punctuated manner, are species-specific and sexually dimorphic. It is likely that both ecological and sexual selection have shaped these patterns of CHC diversification, and our study now provides a comprehensive framework for testing such hypotheses.

Warmer temperature and provision of natural substrate enable earlier metamorphosis in the critically endangered Baw Baw frog.

Temperature and food availability are known to independently trigger phenotypic change in ectotherms, but the interactive effects between these factors have rarely been considered. This study investigates the independent and interactive effects of water temperature and food availability on larval growth and development of the critically endangered Baw Baw frog, Philoria frosti. Larvae were reared at low (12°C) or high (17°C) water temperature in the absence or presence of substrate that controlled food availability, and body size and time to metamorphosis were quantified. Growth and development of larvae was influenced by the individual effects of temperature and food availability; time to metamorphosis was shorter in warm water treatment groups and in the presence of substrate and increased food. Unexpectedly, however, water temperature and food availability did not have an interactive effect on either time to metamorphose or body size at metamorphosis. Under all treatment groups, metamorphic onset occurred once a developmental size threshold was reached, indicating that growth rate and body size are key factors controlling the metamorphic process in Baw Baw frogs (consistent with the Wilbur-Collins model for ectotherm development). From an applied perspective, our findings have implications for amphibian conservation because they indicate that simple manipulations of temperature and food availability can be used to increase the rate of frog production in conservation breeding programs.

Genetic benefits of extreme sequential polyandry in a terrestrial-breeding frog.

Sequential polyandry may evolve as an insurance mechanism to reduce the risk of females choosing mates who are genetically inferior (intrinsic male quality hypothesis) or genetically incompatible (genetic incompatibility hypothesis). The prevalence of such indirect benefits remains controversial, however, because studies estimating the contributions of additive and nonadditive sources of genetic variation to offspring fitness have been limited to a small number of taxonomic groups. Here, we used artificial fertilization techniques combined with a crossclassified breeding design (North Carolina Type II) to simultaneously test the "good genes hypothesis" and the "genetic incompatibility hypothesis" in the brown toadlet (Pseudophryne bibronii); a terrestrial-breeding species with extreme sequential polyandry. Our results revealed no significant additive or nonadditive genetic effects on fertilization success. Moreover, they revealed no significant additive genetic effects, but highly significant nonadditive genetic effects (sire by dam interaction effects), on hatching success and larval survival to initial and complete metamorphosis. Taken together, these results indicate that offspring viability is significantly influenced by the combination of parental genotypes, and that negative interactions between parental genetic elements manifest during embryonic and larval development. More broadly, our findings provide quantitative genetic evidence that insurance against genetic incompatibility favors the evolution and maintenance of sequential polyandry.

Effect of carotenoid class and dose on the larval growth and development of the critically endangered southern corroboree frog.

Dietary carotenoids are expected to improve vertebrate growth and development, though evidence for beneficial effects remains limited. One reason for this might be that few studies have directly compared the effects of carotenoids from different classes (carotenes versus xanthophylls) at more than one dose. Here, we tested the effect of two doses of dietary ß-carotene and lutein (representing two different carotenoid classes) on the growth and development of larval southern corroboree frogs (Pseudophryne corroboree). Individuals were supplemented with either ß-carotene or lutein at one of two doses (0.1 mg g-1, 1 mg g-1), or given a diet without carotenoids (control). Each dietary treatment included 36 replicate individuals, and individuals remained on the same diet until metamorphosis (25-39 weeks). We measured larval survival, larval growth (body length), time to metamorphosis, metamorphic body size (mass and SVL), and body condition. Lutein had no detectable effect on larval growth and development. However, larvae receiving a high dose (1 mg g-1) of ß-carotene metamorphosed significantly faster than all other dietary treatments, despite no significant differences in growth rate. This result indicates that ß-carotene supplementation in P. corroboree has positive effects on development independent of growth effects. Our study provides new evidence for differential effects of carotenoid class and dose on vertebrate development. From a conservation perspective, our findings are expected to assist with the recovery of P. corroboree by expediting the generation of frogs required for the maintenance of captive insurance colonies, or the provision of frogs for release. More broadly, our study highlights the potential for dietary manipulation to assist with the ex situ management of threatened amphibian species worldwide.

Hormone-induced sperm-release in the critically endangered Booroolong frog (Litoria booroolongensis): effects of gonadotropin-releasing hormone and human chorionic gonadotropin.

Research into the development of reproductive technologies for amphibians has increased in recent years due to the rapid decline of amphibian species globally. Reproductive technologies have great potential to overcome captive breeding failure and improve the propagation and genetic management of threatened species. However, the incorporation of these technologies into conservation breeding programs has been protracted, primarily as a result of trial-and-error approaches to the refinement of hormone therapies. The present study investigated the effects of: (1) GnRH-a dose (0, 0.5, 1, 2, 4, 8 or 16 µg g-1), and (2) hCG dose (0, 2.5, 5, 10, 20 or 40 IU g-1), on the sperm-release response of the critically endangered Booroolong frog. Administration of GnRH-a at a dose of 0.5 µg g-1 resulted in the greatest number of sperm released (mean total sperm = 3.5 ×106, n = 11). Overall, hCG was more effective at eliciting spermiation in Booroolong frogs, with peak sperm release (mean total sperm = 25.1 ×106, n = 10) occurring in response to a dose of 40 IU g-1. Sperm output in response to 40 IU g-1 hCG was greatest between 1 and 6 h and steadily declined between 8 and 24 h post-hormone administration. Percent sperm motility peaked between 4 and 10 h (58.1-62.7%), and sperm velocity between 4 and 12 h (24.3-27.2 µm s-1). Booroolong frogs join a small, but growing number of amphibian species that exhibit improved spermiation in response to hCG. Further research is required to identify optimal hormone-induction protocols for threatened amphibians and expedite the incorporation of reproductive technologies into CBPs.

The Role of Reproductive Technologies in Amphibian Conservation Breeding Programs.

Anthropogenic environmental change has led to unprecedented rates of species extinction, presenting a major threat to global biodiversity. Among vertebrates, amphibians have been most severely impacted, with an estimated 41% of species now threatened with extinction. In response to this biodiversity crisis, a moral and ethical obligation exists to implement proactive interventionist conservation actions to assist species recovery and decelerate declines. Conservation breeding programs have been successfully established for several threatened amphibian species globally, aiming to prevent species' extinction by maintaining genetically representative assurance colonies ex situ while providing individuals for population augmentation, translocation, and reestablishment in situ. Reproductive technologies have enormous potential to enhance the propagation and genetic management of threatened species. In this review, we discuss the role of reproductive technologies in amphibian conservation breeding programs and summarize technological advancements in amphibian hormone therapies, gamete storage, and artificial fertilization.

Dose and life stage-dependent effects of dietary beta-carotene supplementation on the growth and development of the Booroolong frog.

Carotenoids are known for their antioxidant capacity and are considered to play an important role in vertebrate growth and development. However, evidence for their beneficial effects remains limited, possibly because very few studies have tested for dose effects across different life stages. The present study investigated the effect of various doses of dietary beta-carotene supplements on the growth and development of larval and post-metamorphic Booroolong frogs (Litoria booroolongensis). Larval and post-metamorphic basal diets (containing 0.015 and 0.005 mg g-1 total carotenoids, respectively) were supplemented with beta-carotene at one of four concentrations: 0 mg g-1, 0.1 mg g-1, 1 mg g-1 and 10 mg g-1. Each treatment included 72 replicate individuals, and individuals remained on the same diet treatment over both life stages (spanning 53 experimental weeks). Our results show that larvae receiving an intermediate (1 mg g-1) beta-carotene supplement dose grew faster than unsupplemented larvae (0 mg g-1), and metamorphosed earlier. After metamorphosis, there was no effect of the lowest supplement dose (0.1 mg g-1) on growth and development. However, juveniles fed the highest supplement dose (10 mg g-1) displayed significantly smaller body mass and lower body condition, compared to all other supplement doses, from 4-months through to sexual maturity (7-months). These findings indicate that beta-carotene supplementation has positive effects on growth and development, but only at intermediate doses, and only in the larval life stage. This knowledge may assist with amphibian conservation by expediting the rate that metamorphs can be generated in captive breeding programmes. More broadly, this is the first study to demonstrate both dose and life stage-dependent effects of dietary beta-carotene supplementation on vertebrate growth and development.

The unexpected genetic mating system of the red-backed toadlet (Pseudophryne coriacea): A species with prolonged terrestrial breeding and cryptic reproductive behaviour.

Molecular technologies have revolutionized our classification of animal mating systems, yet we still know very little about the genetic mating systems of many vertebrate groups. It is widely believed that anuran amphibians have the highest reproductive diversity of all vertebrates, yet genetic mating systems have been studied in <1% of all described species. Here, we use single nucleotide polymorphisms to quantify the genetic mating system of the terrestrial breeding red-backed toadlet Pseudophryne coriacea. In this species, breeding is prolonged (approximately 5 months), and males construct subterranean nests in which females deposit eggs. We predicted that females would display extreme sequential polyandry because this mating system has been reported in a closely related species (P. bibronii). Parentage analysis revealed that mating success was heavily skewed towards a subset of males (30.6% of potential sires) and that nearly all females (92.6%) mated with one male. In a high percentage of occupied nests (37.1%), the resident male was not the genetic sire, and very few nests (4.3%) contained clutches with multiple paternity. Unexpectedly, these results show that sequential polyandry is rare. They also show that there is a high frequency of nest takeover and extreme competition between males for nest sites, but that males rarely sneak matings. Genetic analysis also revealed introgressive hybridization between P. coriacea and the red-crowned toadlet (Pseudophryne australis). Our study demonstrates a high level of mating system complexity, and it shows that closely related anurans can vary dramatically in their genetic mating system.

Effect of captivity on morphology: negligible changes in external morphology mask significant changes in internal morphology.

Captive breeding programmes are increasingly relied upon for threatened species management. Changes in morphology can occur in captivity, often with unknown consequences for reintroductions. Few studies have examined the morphological changes that occur in captive animals compared with wild animals. Further, the effect of multiple generations being maintained in captivity, and the potential effects of captivity on sexual dimorphism remain poorly understood. We compared external and internal morphology of captive and wild animals using house mouse (Mus musculus) as a model species. In addition, we looked at morphology across two captive generations, and compared morphology between sexes. We found no statistically significant differences in external morphology, but after one generation in captivity there was evidence for a shift in the internal morphology of captive-reared mice; captive-reared mice (two generations bred) had lighter combined kidney and spleen masses compared with wild-caught mice. Sexual dimorphism was maintained in captivity. Our findings demonstrate that captive breeding can alter internal morphology. Given that these morphological changes may impact organismal functioning and viability following release, further investigation is warranted. If the morphological change is shown to be maladaptive, these changes would have significant implications for captive-source populations that are used for reintroduction, including reduced survivorship.

Hormone-induced spawning of the critically endangered northern corroboree frog Pseudophryne pengilleyi.

Fundamental knowledge of the optimal hormone concentrations required to stimulate amplexus and spawning in breeding pairs of amphibians is currently lacking, hindering our understanding of the proximate mechanisms underpinning mating behaviour. The present study investigated the effects of: (1) the dose of a gonadotropin-releasing hormone analogue (GnRH-a) administered; (2) male-female hormone administration interval; and (3) topical application of GnRH-a, on spawning success in the northern corroboree frog. Administration of GnRH-a at doses of 0.5, 1.0 and 2.0µgg-1 were highly successful, with a significantly greater proportion of hormone-treated pairs ovipositing (89-100%) compared with the 0µgg-1 treatment (22%). Of the hormone-treated pairs, those receiving 0.5µgg-1 GnRH-a exhibited the highest fertilisation success (61%). Administration of GnRH-a to males and females simultaneously (0h) was more effective than injecting males either 48 or 24h before the injection of females. Overall, administration of GnRH-a was highly successful at inducing spawning in northern corroboree frogs. For the first time, we also effectively induced spawning following the topical application of GnRH-a to the ventral pelvic region. Topical application of GnRH-a eliminates the need for specialised training in amphibian injection, and will allow assisted reproductive technologies to be adopted by a greater number of captive facilities globally.