Hotspot shelters stimulate frog resistance to chytridiomycosis.

Many threats to biodiversity cannot be eliminated; for example, invasive pathogens may be ubiquitous. Chytridiomycosis is a fungal disease that has spread worldwide, driving at least 90 amphibian species to extinction, and severely affecting hundreds of others1-4. Once the disease spreads to a new environment, it is likely to become a permanent part of that ecosystem. To enable coexistence with chytridiomycosis in the field, we devised an intervention that exploits host defences and pathogen vulnerabilities. Here we show that sunlight-heated artificial refugia attract endangered frogs and enable body temperatures high enough to clear infections, and that having recovered in this way, frogs are subsequently resistant to chytridiomycosis even under cool conditions that are optimal for fungal growth. Our results provide a simple, inexpensive and widely applicable strategy to buffer frogs against chytridiomycosis in nature. The refugia are immediately useful for the endangered species we tested and will have broader utility for amphibian species with similar ecologies. Furthermore, our concept could be applied to other wildlife diseases in which differences in host and pathogen physiologies can be exploited. The refugia are made from cheap and readily available materials and therefore could be rapidly adopted by wildlife managers and the public. In summary, habitat protection alone cannot protect species that are affected by invasive diseases, but simple manipulations to microhabitat structure could spell the difference between the extinction and the persistence of endangered amphibians.

Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

A bird-like genome from a frog: Mechanisms of genome size reduction in the ornate burrowing frog, Platyplectrum ornatum.

The diversity of genome sizes across the tree of life is of key interest in evolutionary biology. Various correlates of variation in genome size, such as accumulation of transposable elements (TEs) or rate of DNA gain and loss, are well known, but the underlying molecular mechanisms driving or constraining genome size are poorly understood. Here, we study one of the smallest genomes among frogs characterized thus far, that of the ornate burrowing frog (Platyplectrum ornatum) from Australia, and compare it to other published frog and vertebrate genomes to examine the forces driving reduction in genome size. At ∼1.06 gigabases (Gb), the P. ornatum genome is like that of birds, revealing four major mechanisms underlying TE dynamics: reduced abundance of all major classes of TEs; increased net deletion bias in TEs; drastic reduction in intron lengths; and expansion via gene duplication of the repertoire of TE-suppressing Piwi genes, accompanied by increased expression of Piwi-interacting RNA (piRNA)-based TE-silencing pathway genes in germline cells. Transcriptomes from multiple tissues in both sexes corroborate these results and provide insight into sex-differentiation pathways in Platyplectrum Genome skimming of two closely related frog species (Lechriodus fletcheri and Limnodynastes fletcheri) confirms a reduction in TEs as a major driver of genome reduction in Platyplectrum and supports a macroevolutionary scenario of small genome size in frogs driven by convergence in life history, especially rapid tadpole development and tadpole diet. The P. ornatum genome offers a model for future comparative studies on mechanisms of genome size reduction in amphibians and vertebrates generally.

Paradigm shift in frog sperm cryopreservation: reduced role for non-penetrating cryoprotectants.

In brief: Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs. Abstract: Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.

Modeling genetic benefits and financial costs of integrating biobanking into the conservation breeding of managed marsupials.

Managed breeding programs are an important tool in marsupial conservation efforts but may be costly and have adverse genetic effects in unavoidably small captive colonies. Biobanking and assisted reproductive technologies (ARTs) could help overcome these challenges, but further demonstration of their potential is required to improve uptake. We used genetic and economic models to examine whether supplementing hypothetical captive populations of dibblers (Parantechinus apicalis) and numbats (Myrmecobius fasciatus) with biobanked founder sperm through ARTs could reduce inbreeding, lower required colony sizes, and reduce program costs. We also asked practitioners of the black-footed ferret (Mustela nigripes) captive recovery program to complete a questionnaire to examine the resources and model species research pathways required to develop an optimized biobanking protocol in the black-footed ferret. We used data from this questionnaire to devise similar costed research pathways for Australian marsupials. With biobanking and assisted reproduction, inbreeding was reduced on average by between 80% and 98%, colony sizes were on average 99% smaller, and program costs were 69- to 83-fold lower. Integrating biobanking made long-standing captive genetic retention targets possible in marsupials (90% source population heterozygosity for a minimum of 100 years) within realistic cost frameworks. Lessons from the use of biobanking technology that contributed to the recovery of the black-footed ferret include the importance of adequate research funding (US$4.2 million), extensive partnerships that provide access to facilities and equipment, colony animals, appropriate research model species, and professional and technical staff required to address knowledge gaps to deliver an optimized biobanking protocol. Applied research investment of A$133 million across marsupial research pathways could deliver biobanking protocols for 15 of Australia's most at-risk marsupial species and 7 model species. The technical expertise and ex situ facilities exist to emulate the success of the black-footed ferret recovery program in threatened marsupials using these research pathways. All that is needed now for significant and cost-effective conservation gains is greater investment by policy makers in marsupial ARTs.

Los programas de reproducción controlada son una herramienta importante para los esfuerzos de conservación de marsupiales, aunque pueden resultar costosos y tener efectos genéticos adversos en las colonias cautivas incapaces de aumentar en tamaño. Los biobancos y las tecnologías de reproducción asistida (TRA) podrían ayudar a superar estos problemas, pero es necesario seguir demostrando su potencial para mejorar su adopción. Utilizamos modelos genéticos y económicos para analizar si la introducción de esperma fundador proveniente de biobancos mediante tecnologías de reproducción asistida a poblaciones cautivas hipotéticas de los marsupiales Parantechinus apicalis y Myrmecobius fasciatus podría reducir la endogamia, disminuir el tamaño efectivo de las colonias y reducir el costo de los programas. También pedimos a los profesionales del programa de recuperación en cautiverio del hurón de patas negras (Mustella nigripes) que respondieran un cuestionario para analizar los recursos y los métodos de investigación de las especies modelo necesarias para desarrollar un protocolo de biobanco optimizado para el hurón de patas negras. Utilizamos los datos de este cuestionario para diseñar métodos de investigación con costos similares para los marsupiales australianos. Con el biobanco y la reproducción asistida, la endogamia se redujo en promedio entre un 80 y un 98%, el tamaño de las colonias fue en promedio un 99% más pequeño y los costos del programa entre 69 y 83 veces menores. La integración del biobanco posibilitó los objetivos de retención genética en cautiverio a largo plazo en marsupiales (90% de heterocigosidad de la población de origen durante un mínimo de 100 años) dentro de un marco realista de costos. Entre el aprendizaje extraído del uso de la tecnología de biobancos que contribuyó a la recuperación del hurón de patas negras figuran la importancia de una financiación adecuada de la investigación (4.2 millones de dólares), colaboraciones profundas que faciliten el acceso a instalaciones y equipos, colonias de animales, especies modelo adecuadas para la investigación y el personal profesional y técnico necesario para abordar las lagunas de conocimiento y ofrecer un protocolo optimizado para los biobancos. Una inversión en investigación aplicada de 133 millones de dólares australianos para la investigación de los marsupiales podría proporcionar protocolos de biobancos para 15 de las especies de marsupiales australianos en mayor riesgo y 7 especies modelo. Existen los conocimientos técnicos y las instalaciones ex situ para emular el éxito del programa de recuperación del hurón de patas negras en marsupiales amenazados utilizando estas vías de investigación. Ahora sólo se necesita una mayor inversión por parte de los responsables políticos de las TRA para marsupiales para obtener beneficios de conservación significativos y rentables.

High clutch failure rate due to unpredictable rainfall for an ephemeral pool-breeding frog.

Animals that reproduce in temporary aquatic systems expose their offspring to a heightened risk of desiccation, as they must race to complete development and escape before water levels recede. Adults must therefore synchronise reproduction with the changing availability of water, yet the conditions they experience to trigger such an event may not relate to those offspring face throughout development, potentially leading to clutch failure. The sandpaper frog (Lechriodus fletcheri) breeds in ephemeral pools that dry within days to weeks after rainfall has ceased. We examined whether spawning frequency and offspring survival differed across two consecutive breeding seasons based on (1) rainfall at the moment of oviposition and throughout offspring development, and (2) pool volume, given their combined effect on hydroperiod. Reproduction was triggered by rainfall, with more spawn laid during periods of greater rainfall and in larger pools. While pool size was a predictor of offspring survival, rainfall during oviposition was not. Rather, follow-up rain events were required to prevent pools drying prior to metamorphosis, with rainfall evenness during development the strongest predictor of reproductive success. High clutch failure rates recorded in both seasons suggest that adults do not have the capability to predict rainfall frequency post-oviposition. We thus conclude that unpredictable rainfall leading to premature desiccation of spawning sites is the primary source of pre-metamorphic mortality for this species. Understanding the influence of rainfall predictability on offspring survival could be critical in predicting the effects of altered hydroperiod regimes due to climate change for species that exploit temporary waters.

Common goals, different stages: the state of the ARTs for reptile and amphibian conservation.

Amphibians and reptiles are highly threatened vertebrate taxa with large numbers of species threatened with extinction. With so many species at risk, conservation requires the efficient and cost-effective application of all the tools available so that as many species as possible are assisted. Biobanking of genetic material in genetic resource banks (GRBs) in combination with assisted reproductive technologies (ARTs) to retrieve live animals from stored materials are two powerful, complementary tools in the conservation toolbox for arresting and reversing biodiversity decline for both amphibians and reptiles. However, the degree of development of the ARTs and cryopreservation technologies differ markedly between these two groups. These differences are explained in part by different perceptions of the taxa, but also to differing reproductive anatomy and biology between the amphibians and reptiles. Artificial fertilisation with cryopreserved sperm is becoming a more widely developed and utilised technology for amphibians. However, in contrast, artificial insemination with production of live progeny has been reported in few reptiles, and while sperm have been successfully cryopreserved, there are still no reports of the production of live offspring generated from cryopreserved sperm. In both amphibians and reptiles, a focus on sperm cryopreservation and artificial fertilisation or artificial insemination has been at the expense of the development and application of more advanced technologies such as cryopreservation of the female germline and embryonic genome, or the use of sophisticated stem cell/primordial germ cell cryopreservation and transplantation approaches. This review accompanies the publication of ten papers on amphibians and twelve papers on reptiles reporting advances in ARTs and biobanking for the herpetological taxa.

Efficacy of short-term cold storage prior to cryopreservation of spermatozoa in a threatened lizard.

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 25°C or 4°C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 25°C, but did not decrease significantly until 4 days after collection at 4°C, with >30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 4°C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles.

Generation of reproductively mature offspring from the endangered green and golden bell frog Litoria aurea using cryopreserved spermatozoa.

Amphibians are becoming increasingly reliant on captive breeding programs for continued survival. Assisted reproductive technologies including gamete cryopreservation and IVF can help reduce costs of breeding programs, provide insurance against extinction and assist genetic rescue in wild populations. However, the use of these technologies to produce reproductively mature offspring has only been demonstrated in a few non-model species. We aimed to optimise sperm cryopreservation in the threatened frog Litoria aurea and generate mature offspring from frozen-thawed spermatozoa by IVF. We tested three concentrations (1.4, 2.1 and 2.8M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3M sucrose. Using DMSO was more likely to result in recovery of sperm motility, vitality and acrosome integrity than glycerol, regardless of concentration, with forward progressive motility being most sensitive to damage. The lowest concentrations of 1.4 and 2.1M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1M DMSO outperformed spermatozoa cryopreserved in equivalent concentrations of glycerol in terms of their ability to fertilise ova, resulting in higher rates of embryos hatching and several individuals reaching sexual maturity. We have demonstrated that sperm cryopreservation and subsequent offspring generation via IVF is a feasible conservation tool for L. aurea and other threatened amphibians.

Optimal cooling rates for sperm cryopreservation in a threatened lizard conform to two-factor hypothesis of cryo-injury.

Assisted reproductive technologies provide important tools for wildlife conservation but have rarely been developed for reptiles. A critical step in developing cryopreservation protocols is establishing optimal cooling rates for cell survival. The two-factor hypothesis explaining cryoinjury to cells originates from an inverted 'U' shape of recovery curves generated in many cell types thawed after cryopreservation, due to cell recovery declining at cooling rates either side of a single optimum. We generated such a curve for the yellow-spotted monitor lizard Varanus panoptes, the first for any reptile. We cryopreserved sperm using two cooling devices (LN2 dry shipper; LN2 bath vapour) and two sperm-holding vessels (Cassou sperm straws; Nunc CryoTubes) to generate four different cooling-rate curves during freezing. Sperm motility and viability (47.3% and 76.5% respectively) were highest when frozen in straws suspended in a LN2 bath at an intermediate cooling rate of 73 °C/min between 0 and -50 °C, whereas sperm frozen in straws suspended in a dry shipper at the fastest cooling rate (231 °C/min between 0 and -50 °C) produced the lowest recovery (10.4% and 36.4% motility and viability, respectively). Sperm frozen in cryotubes at the lowest cooling rates in either LN2 bath vapour or dry shipper produced intermediate recovery. The shape of the optimal cooling curve conformed to the two-factor hypothesis of cryoinjury, the first such evidence in reptile sperm. This in turn led to the identification of simple cryopreservation setups (LN2 vapour with straws and cryotubes; dry shipper with cryotubes but not straws) suitable for cryopreserving lizard sperm in the field.

Deconstructing compassionate conservation.

Compassionate conservation focuses on 4 tenets: first, do no harm; individuals matter; inclusivity of individual animals; and peaceful coexistence between humans and animals. Recently, compassionate conservation has been promoted as an alternative to conventional conservation philosophy. We believe examples presented by compassionate conservationists are deliberately or arbitrarily chosen to focus on mammals; inherently not compassionate; and offer ineffective conservation solutions. Compassionate conservation arbitrarily focuses on charismatic species, notably large predators and megaherbivores. The philosophy is not compassionate when it leaves invasive predators in the environment to cause harm to vastly more individuals of native species or uses the fear of harm by apex predators to terrorize mesopredators. Hindering the control of exotic species (megafauna, predators) in situ will not improve the conservation condition of the majority of biodiversity. The positions taken by so-called compassionate conservationists on particular species and on conservation actions could be extended to hinder other forms of conservation, including translocations, conservation fencing, and fertility control. Animal welfare is incredibly important to conservation, but ironically compassionate conservation does not offer the best welfare outcomes to animals and is often ineffective in achieving conservation goals. Consequently, compassionate conservation may threaten public and governmental support for conservation because of the limited understanding of conservation problems by the general public.

Deconstrucción de la Conservación Compasiva Resumen La conservación compasiva se enfoca en cuatro principios: no causar daño; los individuos importan; la integración de los animales individualmente; y la coexistencia pacífica entre los humanos u los animales. Recientemente, la conservación compasiva ha sido promovida como una alternativa a la filosofía convencional de la conservación. Creemos que los ejemplos presentados por los conservacionistas compasivos han sido elegidos arbitraria o deliberadamente por estar enfocados en los mamíferos; por ser inherentes y no compasivos; y por ofrecer soluciones de conservación poco efectivas. La conservación compasiva se enfoca arbitrariamente en las especies carismáticas, principalmente los grandes depredadores y los megaherbívoros. La filosofía no es compasiva cuando deja que los depredadores invasores dentro del ambiente causen daño a un vasto número de individuos nativos o usa el miedo al daño por superdepredadores para aterrorizar a los mesodepredadores. El entorpecimiento del control de especies exóticas (megafauna, depredadores) in situ no mejorará las condiciones de conservación de la mayoría de la biodiversidad, incluso si los conservacionistas compasivos no dañan a los individuos exóticos. Las posiciones que toman los llamados conservacionistas compasivos sobre especies particulares y sobre las acciones de conservación podrían extenderse para entorpecer otros tipos de conservación, incluyendo las reubicaciones, el encercado para la conservación y el control de la fertilidad. El bienestar animal es increíblemente importante para la conservación e irónicamente, la conservación compasiva no ofrece los mejores resultados de bienestar para los animales y comúnmente es poco efectiva en el logro de los objetivos de conservación. Como consecuencia, la conservación compasiva puede poner en peligro el apoyo público y del gobierno que tiene la conservación debido al entendimiento poco limitado que tiene el público general sobre los problemas de conservación.

Differential success in obtaining gametes between male and female Australian temperate frogs by hormonal induction: A review.

Most Australian frogs fall into two deeply split lineages, conveniently referred to as ground frogs (Myobatrachidae and Limnodynastidae) and tree frogs (Pelodryadidae). Species of both lineages are endangered because of the global chytrid pandemic, and there is increasing interest and research on the endocrine manipulation of reproduction to support the use of assisted reproductive technologies in conservation. Hormonal induction of gamete release in males and females is one such manipulation of the reproductive process. This paper reviews progress in temperate ground and tree frogs towards developing simple and efficient hormonal protocols for induction of spermiation and ovulation, and presents some new data, that together build towards an understanding of advances and obstacles towards progress in this area. We report that protocols for the non-invasive induction of sperm release, relying on single doses of gonadotropin-releasing hormone (GnRH) or human chorionic gonadotropin are very effective in both ground and tree frog species investigated to date. However, we find that, while protocols based on GnRH, and GnRH and dopamine antagonists, are moderately efficient in inducing ovulation in ground frogs, the same cannot be said for the use of such protocols in tree frogs. Although induced ovulation in the pelodryadid tree frogs has not been successfully implemented, and is difficult to explain in terms of the underlying endocrinology, we propose future avenues of investigation to address this problem, particularly the need for a source of purified or recombinant follicle-stimulating hormone and luteinising hormone for species from this group.

Chronic effects of an invasive species on an animal community.

Invasive species can trigger trophic cascades in animal communities, but published cases involving their removal of top predators are extremely rare. An exception is the invasive cane toad (Rhinella marina) in Australia, which has caused severe population declines in monitor lizards, triggering trophic cascades that facilitated dramatic and sometimes unexpected increases in several prey of the predators, including smaller lizards, snakes, turtles, crocodiles, and birds. Persistence of isolated populations of these predators with a decades-long sympatry with toads suggests the possibility of recovery, but alternative explanations are possible. Confirming predator recovery requires longer-term study of populations with both baseline and immediate post-invasion densities. Previously, we quantified short-term impacts of invasive cane toads on animal communities over seven years at two sites in tropical Australia. Herein, we test the hypothesis that predators have begun to recover by repeating the study 12 yr after the initial toad invasion. The three predatory lizards that experienced 71-97% declines in the short-term study showed no sign of recovery, and indeed a worse fate: two of the three species were no longer detectable in 630 km of river surveys, suggesting local extirpation. Two mesopredators that had increased markedly in the short term due to these predator losses showed diverse responses in the medium term; a small lizard species increased by ~500%, while populations of a snake species showed little change. Our results indicate a system still in ecological turmoil, having not yet reached a "new equilibrium" more than a decade after the initial invasion; predator losses due to this toxic invasive species, and thus downstream effects, were not transient. Given that cane toads have proven too prolific to eradicate or control, we suggest that recovery of impacted predators must occur unassisted by evolutionary means: dispersal into extinction sites from surviving populations with alleles for toxin resistance or toad avoidance. Evolution and subsequent dispersal may be the only solution for a number of species or communities affected by invasive species for which control is either prohibitively expensive, or not possible.

Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian.

Ontogenetic changes in disease susceptibility have been demonstrated in many vertebrate taxa, as immature immune systems and limited prior exposure to pathogens can place less developed juveniles at a greater disease risk. By causing the disease chytridiomycosis, Batrachochytrium dendrobatidis (Bd) infection has led to the decline of many amphibian species. Despite increasing knowledge on how Bd varies in its effects among species, little is known on the interaction between susceptibility and development within host species. We compared the ontogenetic susceptibility of post-metamorphic green and golden bell frogs Litoria aurea to chytridiomycosis by simultaneously measuring three host-pathogen responses as indicators of the development of the fungus-infection load, survival rate, and host immunocompetence-following Bd exposure in three life stages (recently metamorphosed juveniles, subadults, adults) over 95 days. Frogs exposed to Bd as recently metamorphosed juveniles acquired higher infection loads and experienced lower immune function and lower survivorship than subadults and adults, indicating an ontogenetic decline in chytridiomycosis susceptibility. By corresponding with an intrinsic developmental maturation in immunocompetence seen in uninfected frogs, we suggest these developmental changes in host susceptibility in L. aurea may be immune mediated. Consequently, the physiological relationship between ontogeny and immunity may affect host population structure and demography through variation in life stage survival, and understanding this can shape management targets for effective amphibian conservation.

Cryopreservation and other assisted reproductive technologies for the conservation of threatened amphibians and reptiles: bringing the ARTs up to speed.

Amphibians and reptiles are experiencing serious declines, with the number of threatened species and extinctions growing rapidly as the modern biodiversity crisis unfolds. For amphibians, the panzootic of chytridiomycosis is a major driver. For reptiles, habitat loss and harvesting from the wild are key threats. Cryopreservation and other assisted reproductive technologies (ARTs) could play a role in slowing the loss of amphibian and reptile biodiversity and managing threatened populations through genome storage and the production of live animals from stored material. These vertebrate classes are at different stages of development in cryopreservation and other ARTs, and each class faces different technical challenges arising from the separate evolutionary end-points of their reproductive biology. For amphibians, the generation of live offspring from cryopreserved spermatozoa has been achieved, but the cryopreservation of oocytes and embryos remains elusive. With reptiles, spermatozoa have been cryopreserved in a few species, but no offspring from cryopreserved spermatozoa have been reported, and the generation of live young from AI has only occurred in a small number of species. Cryopreservation and ARTs are more developed and advanced for amphibians than reptiles. Future work on both groups needs to concentrate on achieving proof of concept examples that demonstrate the use of genome storage and ARTs in successfully recovering threatened species to increase awareness and support for this approach to conservation.

Invasive toads shift predator-prey densities in animal communities by removing top predators.

Although invasive species can have substantial impacts on animal communities, cases of invasive species facilitating native species by removing their predators have rarely been demonstrated across vertebrate trophic linkages. The predictable spread of the invasive cane toad (Rhinella marina), however, offered a unique opportunity to quantify cascading effects. In northern Australia, three species of predatory monitor lizards suffered severe population declines due to toad-induced lethal toxic ingestion (yellow-spotted monitor (Varanus panoptes), Mertens' water monitor (V. mertensi), Mitchell's water monitor (V. mitchelli). We, thus, predicted subsequent increases in the abundance and recruitment of prey species due to the reduction of those predators. Toad-induced population-level declines in the water monitor species approached 50% over a five-year period spanning the toad invasion, apparently causing fledging success of the Crimson Finch (Neochmia.phaeton) to increase from 55% to 81%. The consensus of our original and published long-term data is that invasive cane toads are causing predators to lose a foothold on top-down regulation of their prey, triggering shifts in the relative densities of predator and prey in the Australian tropical savannah ecosystem.

Refrigerated storage and cryopreservation of hormonally induced sperm in the threatened frog, Litoria aurea.

As sperm cryopreservation and other assisted reproductive technologies (ARTs) advance in common amphibian species, focus on applying non-lethal sperm collection methods to the conservation and genetic management of threatened species is imperative. The goal of this study was to examine the application of logistically practical ART protocols in a threatened frog (Litoria aurea). First, we tested the efficacy of various concentrations of human chorionic gonadotropin (hCG) (20, 40 IU/g bodyweight) and Gonadotropin releasing hormone antagonist (0.25 µg/g and 0.5 µg/g body weight GnRH-a) on the induction of spermatozoa. Using the samples obtained from the previous trials, we tested the effect of cold storage and cryopreservation protocols on long-term refrigerated storage and post-thaw sperm recovery. Our major findings include: (1) high quality sperm were induced with 20 and 40 IU/g bodyweight of (hCG); (2) proportions of live, motile sperm post-thaw, were recovered at higher levels than previously reported for L. aurea (>50%) when preserved with 15% v/v DMSO and 1% w/v sucrose; and (3) spermic urine stored at 5 °C retained motility for up to 14 days. Our findings demonstrate that the protocols developed in this study allowed for successful induction and recovery of high-quality spermatozoa from a threatened Australian anuran, L. aurea, providing a prime example of how ARTs can contribute to the conservation of rare and threatened species.

The effect of salt dosing for chytrid mitigation on tadpoles of a threatened frog, Litoria aurea.

The novel fungal pathogen Batrachochytrium dendrobatidis (chytrid) is one of the greatest threats to amphibians worldwide. Small increases in water salinity (up to ca. 4 ppt) have been shown to limit chytrid transmission between frogs, potentially providing a way to create environmental refugia to reduce its impact at a landscape scale. However, the effect of increasing water salinity on tadpoles, a life stage confined to water, is highly variable. Increased water salinity can lead to reduced size and altered growth patterns in some species, with flow-on effects to vital rates such as survival and reproduction. It is thus important to assess potential trade-offs caused by increasing salinity as a tool to mitigate chytrid in susceptible frogs. We conducted laboratory experiments to examine the effects of salinity on the survival and development of tadpoles of a threatened frog (Litoria aurea), previously demonstrated as a suitable candidate for trialling landscape manipulations to mitigate chytrid. We exposed tadpoles to salinity ranging from 1 to 6 ppt and measured survival, time to metamorphosis, body mass and locomotor performance of post-metamorphic frogs as a measure of fitness. Survival and time to metamorphosis did not differ between salinity treatments or controls reared in rainwater. Body mass was positively associated with increasing salinity in the first 14 days. Juvenile frogs from three salinity treatments also showed the same or better locomotor performance compared to rainwater controls, confirming that environmental salinity may influence life history traits in the larval stage, potentially as a hormetic response. Our research suggests that salt concentrations in the range previously shown to improve survival of frogs in the presence of chytrid are unlikely to impact larval development of our candidate threatened species. Our study lends support to the idea of manipulating salinity to create environmental refugia from chytrid for at least some salt-tolerant species.

Optimisation of an oviposition protocol employing human chorionic and pregnant mare serum gonadotropins in the barred frog Mixophyes fasciolatus (Myobatrachidae).

BACKGROUND: Protocols for the hormonal induction of ovulation and oviposition are essential tools for managing threatened amphibians with assisted reproduction, but responses vary greatly between species and even broad taxon groups. Consequently, it is necessary to assess effectiveness of such protocols in representative species when new taxa become targets for induction. The threatened genus Mixophyes (family Myobatrachidae) has amongst the highest proportion of endangered species of all the Australian amphibians. This study developed and optimised the induction of oviposition in a non-threatened member of this taxon, the great barred frog (Mixophyes fasciolatus). METHODS: Gravid female M. fasciolatus were induced to oviposit on one or more occasions by administration of human chorionic gonadotropin (hCG) with or without priming with pregnant mare serum gonadotropin (PMSG). Treatments involved variations in hormone doses and combinations (administered via injection into the dorsal lymph sacs), and timing of administration. Pituitary homogenates from an unrelated bufonid species (Rhinella marina) were also examined with hCG. RESULTS: When injected alone, hCG (900 to 1400 IU) induced oviposition. However, priming with two time dependent doses of PMSG (50 IU, 25 IU) increased responses, with lower doses of hCG (200 IU). Priming increased response rates in females from around 30% (hCG alone) to more than 50% (p = 0.035), and up to 67%. Increasing the interval between the first PMSG dose and first hCG dose from 3 to 6 days also produced significant improvement (p<0.001). Heterologous pituitary extracts administered with hCG were no more effective than hCG alone (p = 0.628). CONCLUSIONS: This study found that M. fasciolatus is amongst the few amphibian species (including Xenopus (Silurana) and some bufonids) that respond well to the induction of ovulation utilising mammalian gonadotropins (hCG). The optimal protocol for M. fasciolatus involved two priming doses of PMSG (50 IU and 25 IU) administered at 6 and 4 days respectively, prior to two doses of hCG (100 IU), 24 hours apart. This study is also the first to demonstrate in an amphibian species that responds to mammalian gonadotropins that an increase in the ovulation rate occurs after priming with a gonadotropin (PMSG) with FSH activity.