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.

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.

Polyploidy breaks speciation barriers in Australian burrowing frogs Neobatrachus.

Polyploidy has played an important role in evolution across the tree of life but it is still unclear how polyploid lineages may persist after their initial formation. While both common and well-studied in plants, polyploidy is rare in animals and generally less understood. The Australian burrowing frog genus Neobatrachus is comprised of six diploid and three polyploid species and offers a powerful animal polyploid model system. We generated exome-capture sequence data from 87 individuals representing all nine species of Neobatrachus to investigate species-level relationships, the origin and inheritance mode of polyploid species, and the population genomic effects of polyploidy on genus-wide demography. We describe rapid speciation of diploid Neobatrachus species and show that the three independently originated polyploid species have tetrasomic or mixed inheritance. We document higher genetic diversity in tetraploids, resulting from widespread gene flow between the tetraploids, asymmetric inter-ploidy gene flow directed from sympatric diploids to tetraploids, and isolation of diploid species from each other. We also constructed models of ecologically suitable areas for each species to investigate the impact of climate on differing ploidy levels. These models suggest substantial change in suitable areas compared to past climate, which correspond to population genomic estimates of demographic histories. We propose that Neobatrachus diploids may be suffering the early genomic impacts of climate-induced habitat loss, while tetraploids appear to be avoiding this fate, possibly due to widespread gene flow. Finally, we demonstrate that Neobatrachus is an attractive model to study the effects of ploidy on the evolution of adaptation in animals.

Seasonal variation in the prevalence of a fungal pathogen and unexpected clearance from infection in a susceptible frog species.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is a primary driver for amphibian population declines and extinctions worldwide. For highly susceptible species, such as the green and golden bell frog Litoria aurea, large numbers of Bd-related mortalities are thought to occur during the colder season (winter), when low temperatures favour the growth of the pathogen. However, extant L. aurea populations are persisting with Bd. We measured Bd prevalence and infection levels of wild L. aurea using capture-mark-recapture and radio-tracking methods. Using this information, we sought to determine host and environmental correlates of Bd prevalence and infection load. Mean ± SE infection load was higher in frogs sampled in autumn (431.5 ± 310.4 genomic equivalents; GE) and winter (1147.5 ± 735.8 GE), compared to spring (21.8 ± 19.3 GE) and summer (0.9 ± 0.8 GE). Furthermore, prevalence of Bd infection in L. aurea was highest in winter (43.6%; 95% CI 33.1-54.7%) and lowest in summer (11.2%; 95% CI 6.8-17.9%). Both prevalence and infection load decreased with increasing temperature. Seven frogs cleared their fungal infection during the coolest months when Bd prevalence was highest; however, these clearances were not permanent, as 5 frogs became infected again. Understanding the factors that allow amphibians to clear their Bd infections when temperatures are optimal for Bd growth presents the potential for manipulating such factors and provides an important step in future research.

Genetic evidence for polyandry in the threatened green and golden bell frog.

Identifying which species exhibit polyandry may lead to further insights into evolutionary biology and social behaviour. However, confirming polyandry can be difficult. High-resolution genetics provides a useful means to gain evidence. Although the threatened Pelodryadid frog, the green and golden bell frog Litoria aurea, has been subject to numerous ecological studies, there is uncertainty surrounding its reproductive ecology. Polyandry has not been formally identified in L. aurea or any species within the Pelodryadidae family. We aimed to identify if there was genetic evidence of polyandry in a population occurring in a wetland complex on Kooragang Island, New South Wales. To accomplish this, we collected genetic samples of tadpoles within the same size cohort about 20-30 days after explosive breeding events. Genotypes of 14 females, nine males and 70 tadpoles were analysed with COLONY (1988 single nucleotide polymorphisms after filtering) to identify parentage, full-siblings and half-siblings. We found support for the hypothesis that L. aurea is polyandrous. Based on previous observations of multi-male matings and the narrow time periods that breeding occurred in, it is likely this species exhibits simultaneous polyandry. We discuss these results in regards to behavioural adaptive processes and avenues for further research.

Multi-species occupancy modeling provides novel insights into amphibian metacommunity structure and wetland restoration.

A fundamental goal of community ecology is to understand species-habitat relationships and how they shape metacommunity structure. Recent advances in occupancy modeling enable habitat relationships to be assessed for both common and rare species within metacommunities using multi-species occupancy models (MSOM). These models account for imperfect species detection and offer considerable advantages over other analytical tools commonly used for community analyses under the elements of metacommunity structure (EMS) framework. Here, we demonstrate that MSOM can be used to infer habitat relationships and test metacommunity theory, using amphibians. Repeated frog surveys were undertaken at 55 wetland sites in southeastern Australia. We detected 11 frog species from three families (Limnodynastidae, Myobatrachidae, and Pelodryadidae). The rarest species was detected at only one site whereas the most common species was detected at 42 sites (naive occupancy rate 0.02-0.76). Two models were assessed representing two competing hypotheses; the best-supported model included the covariates distance to the nearest site (connectivity), wetland area, presence of the non-native eastern mosquitofish (Gambusia holbrooki), proportion cover of emergent vegetation, an interaction term between Gambusia and emergent vegetation cover, and the proportion canopy cover over a site. Hydroperiod played no detectable role in metacommunity structure. We found species-habitat relationships that fit with current metacommunity theory: occupancy increased with wetland area and connectivity. There was a strong negative relationship between occupancy and the presence of predatory Gambusia, and a positive interaction between Gambusia and emergent vegetation. The presence of canopy cover strongly increased occupancy for several tree frog species, highlighting the importance of terrestrial habitat for amphibian community structure. We demonstrated how responses by amphibians to environmental covariates at the species level can be linked to occupancy patterns at the metacommunity scale. Our results have clear management implications: wetland restoration projects for amphibians and likely other taxa should maximize wetland area and connectivity, establish partial canopy cover, and eradicate Gambusia or provide aquatic vegetation to mitigate the impact of this non-native fish. We strongly advocate the use of MSOM to elucidate the habitat drivers behind animal occupancy patterns and to derive unbiased occupancy estimates for monitoring programs.

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.

Rapid population increase of the threatened Australian amphibian Litoria aurea in response to wetlands constructed as a refuge from chytrid-induced disease and introduced fish.

Amphibians have declined due to multiple impacts including invasive fish and the disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Wetland restoration can be used to increase amphibian populations. However the design of created wetlands must account for threats such as Bd and introduced fish. There have been no attempts on a landscape level to manage these threats with habitat design. Here we monitored the green and golden bell frog (Litoria aurea) in 2.6 ha of constructed wetlands designed to enhance breeding and increase survival through passive mitigation of Bd and exotic fish. We compared the fecundity, adult population sizes, introduced fish occupancy, Bd prevalence and survival rates of frogs in created wetlands (CW) to three control sites to determine if and why the habitat design was successful. Monitoring involved weekly capture-recapture during the austral spring and summer for three L. aurea breeding seasons. We hypothesised that (1) if the CWs were successful in passively limiting fish colonisation, a larger number of breeding events would be detected compared to control sites which are known to be widely colonised by introduced fish. (2) If the wetlands were successful in passively mitigating Bd, then we would observe an equal or greater survival rate and equal to or lower Bd prevalence compared to control wetlands. We observed a 3.3-fold increase in adult population size in CW from season 1 to 2, and the population increased further in season 3.We found strong support for hypothesis (1) and weak support for (2). Based on these results, we conclude that this design was beneficial shortly after their formation primarily due to fish exclusion, but further study is required to determine if these benefits extend long-term. Future amphibian restoration studies are needed to improve the design of wetlands to enhance suppression of Bd.

Thermal processing of live bivalve molluscs for controlling viruses: On the need for a risk-based design.

Norovirus (NoV) and Hepatitis A virus (HAV) are the most important viral hazards associated with human illness following consumption of contaminated bivalve molluscs. The effectiveness of the current EU criteria for heat processing of bivalve molluscs (i.e. raising the temperature of the internal mollusc flesh to at least 90°C for a minimum of 90 seconds) was evaluated using predictive microbiology. A HAV thermal inactivation model was developed based on literature data in mollusc matrices during isothermal heat treatment. Application of the developed model demonstrated that the 90°C-90 s requirement may lead to significantly different virus inactivation depending on the commercial process design. This shows the need for the establishment of a Performance Criterion for bivalve molluscs heat processing which will assure a common specified level of consumer protection. A risk-based approach is described that allows for an effective processing design providing a more transparent and objective relation between the thermal processing targets and public health. Model simulations demonstrate that the F-value is a more appropriate Process Criterion than a single time-temperature combination since it enables the food business operators to design a process that is compliant with the safety requirements while at the same time achieving a desired product quality.

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.

A cross-cultural study of acceptability and food pairing for hot sauces.

This study was conducted to understand the acceptance levels of hot sauces among consumers from different culinary cultures. Two newly developed hot sauces [fermented red chili pepper with soybean-paste-based sauce(GS) and fermented red chili-pepper-based sauce(KS)] were compared with Tabasco sauce(TB) and Sriracha sauce(SR). Two separate cross-cultural home-use tests(HUTs) were conducted: pizza and cream soup were provided as food items in HUT 1, whereas grilled chicken wings and rice noodle soup were provided in HUT 2. Consumers residing in Denmark, South Korea, and US participated in each HUT (n≅100 per country). Acceptance levels and the reasons for (dis)liking particular hot sauces applied to food systems were assessed. The food items that paired well with different hot sauces when the sauces were applied freely to regular meals were also analyzed among the US and Korean subjects. When the hot-sauce samples were applied to pizza and cream soup, the preferred order of the samples exhibited a cross-cultural agreement (GS = KS > TB). In the case of grilled chicken and rice noodle soup, the acceptance rating was similar for the three types of hot sauces among Koreans, whereas the acceptance was higher for SR among the US subjects for both foodstuffs, while Danish subjects preferred GS and KS over SR. The US subjects did not like hot-sauce samples with sweet and weak spiciness, whereas the Korean and Danish subjects disliked the hot-sauce sample when it was too spicy and not sufficiently sweet. These findings indicate that the matching of particular sauces with specific food items is culture-dependent, and this needs to be considered when trying to export food products such as hot sauce to other countries.

Patterns of Disease in Patients with Middle-Lobe Predominant Bronchiectasis.

BACKGROUND: Middle-lobe predominant bronchiectasis affecting the right middle-lobe and/or lingula (RMLP) is classically described in asthenic, elderly females with skeletal abnormalities or associated nontuberculous mycobacterial (NTM) infection. OBJECTIVES: We aimed to evaluate the frequency and clinical characteristics of patients with an RMLP phenotype in a cohort of newly diagnosed bronchiectasis patients and determine associations with disease severity. METHODS: A retrospective observational cross-sectional cohort study of consecutive bronchiectasis patients in our institution was performed. Data were collected on baseline variables, microbiology status, lung function, and radiology according to the modified Bhalla score. Disease severity was assessed using bronchiectasis severity index (BSI) and FACED severity scores. RESULTS: Of 81 patients (mean age [SD] 62.6 [12.4], females 55 [67.9%], BMI 26.9 [5.7%]), 20 (24.7%) had RMLP disease. These patients were significantly younger, female, and with lower BMIs than patients with the classical bronchiectasis phenotype (p = 0.03, 0.01, and p <0.01, respectively). Fewer symptoms of cough and daily sputum (p = 0.01 and <0.01), prior exacerbation frequency (p = 0.03), and higher baseline forced expiratory volume (p = 0.04) were noted. A higher incidence of NTM at diagnosis was demonstrated (p = 0.01). BSI and FACED severity scores in RMLP patients were significantly lower than their counterparts (both p < 0.001). CONCLUSIONS: The RMLP phenotype is associated with younger patients than classically described in the literature. An increased rate of NTM infection in this phenotype was noted, particularly in females, but much lower than previously described. Lung function and disease severity scores in this patient group are relatively normal, suggesting a milder phenotype in patients with this form of the disease.

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.

Effects of pond salinization on survival rate of amphibian hosts infected with the chytrid fungus.

The chytrid fungus Batrachochytrium dendrobatidis has been implicated in the decline and extinction of amphibian populations worldwide, but management options are limited. Recent studies show that sodium chloride (NaCl) has fungicidal properties that reduce the mortality rates of infected hosts in captivity. We investigated whether similar results can be obtained by adding salt to water bodies in the field. We increased the salinity of 8 water bodies to 2 or 4 ppt and left an additional 4 water bodies with close to 0 ppt and monitored salinity for 18 months. Captively bred tadpoles of green and golden bell frog (Litoria aurea) were released into each water body and their development, levels of B. dendrobatidis infection, and survival were monitored at 1, 4, and 12 months. The effect of salt on the abundance of nontarget organisms was also investigated in before and after style analyses. Salinities remained constant over time with little intervention. Hosts in water bodies with 4 ppt salt had a significantly lower prevalence of chytrid infection and higher survival, following metamorphosis, than hosts in 0 ppt salt. Tadpoles in the 4 ppt group were smaller in length after 1 month in the release site than those in the 0 and 2 ppt groups, but after metamorphosis body size in all water bodies was similar . In water bodies with 4 ppt salt, the abundance of dwarf tree frogs (Litoria fallax), dragonfly larvae, and damselfly larvae was lower than in water bodies with 0 and 2 ppt salt, which could have knock-on effects for community structure. Based on our results, salt may be an effective field-based B. dendrobatidis mitigation tool for lentic amphibians that could contribute to the conservation of numerous susceptible species. However, as in all conservation efforts, these benefits need to be weighed against negative effects on both target and nontarget organisms.

The 9-point hedonic scale and hedonic ranking in food science: some reappraisals and alternatives.

The 9-point hedonic scale has been used routinely in food science, the same way for 60 years. Now, with advances in technology, data from the scale are being used for more and more complex programs for statistical analysis and modeling. Accordingly, it is worth reconsidering the presentation protocols and the analyses associated with the scale, as well as some alternatives. How the brain generates numbers and the types of numbers it generates has relevance for the choice of measurement protocols. There are alternatives to the generally used serial monadic protocol, which can be more suitable. Traditionally, the 'words' on the 9-point hedonic scale are reassigned as 'numbers', while other '9-point hedonic scales' are purely numerical; the two are not interchangeable. Parametric statistical analysis of scaling data is examined critically and alternatives discussed. The potential of a promising alternative to scaling itself, simple ranking with a hedonic R-Index signal detection analysis, is explored in comparison with the 9-point hedonic scale.

Identifying the drivers of liking by investigating the reasons for (dis)liking using CATA in cross-cultural context: a case study on barbecue sauce.

BACKGROUND: The present study cross-culturally investigated the drivers of liking for traditional and ethnic chicken marinades using descriptive analysis and consumer taste tests incorporating the check-all-that-apply (CATA) method. Seventy-three Koreans and 86 US consumers participated. The tested sauces comprised three tomato-based sauces, a teriyaki-based sauce and a Korean spicy seasoning-based sauce. Chicken breasts were marinated with each of the five barbecue sauces, grilled and served for evaluation. Descriptive analysis and consumer taste tests were conducted. Consumers rated the acceptance on a hedonic scale and checked the reasons for (dis)liking by the CATA method for each sauce. A general linear model, multiple factor analysis and chi-square analysis were conducted using the data. RESULTS: The results showed that the preference orders of the samples between Koreans and US consumers were strikingly similar to each other. However, the reasons for (dis)liking the samples differed cross-culturally. The drivers of liking of two sauces sharing relatively similar sensory profiles but differing significantly in hedonic ratings were effectively delineated by reasons of (dis)liking CATA results. CONCLUSION: Reasons for (dis)liking CATA proved to be a powerful supporting method to understand the internal drivers of liking which can be overlooked by generic descriptive analysis.

Identification and genetic characterization of a novel picornavirus from chickens.

A novel picornavirus from commercial broiler chickens (Gallus gallus domesticus) has been identified and genetically characterized. The viral genome consists of a single-stranded, positive-sense RNA genome of >9243 nt excluding the poly(A) tail and as such represents one of the largest picornavirus genomes reported to date. The virus genome is GC-rich with a G+C content of 54.5 %. The genomic organization is similar to other picornaviruses: 5' UTR-L-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3' UTR. The partially characterized 5' UTR of >373 nt appears to possess a type II internal ribosomal entry site (IRES), which is also found in members of the genera Aphthovirus and Cardiovirus. This IRES exhibits significant sequence similarity to turkey 'gallivirus A'. The 3' UTR of 278 nt contains the conserved 48 nt 'barbell-like' structure identified in 'passerivirus', 'gallivirus', Avihepatovirus and some Kobuvirus genus members. A predicted large open reading frame (ORF) of 8592 nt encodes a potential polyprotein precursor of 2864 amino acids. In addition, the virus contains a predicted large L protein of 462 amino acids. Pairwise sequence comparisons, along with phylogenetic analysis revealed the highest percentage identity to 'Passerivirus A' (formerly called turdivirus 1), forming a monophyletic group across the P1, P2 and P3 regions, with <40, <40 and <50 % amino acid identity respectively. Reduced identity was observed against 'gallivirus A' and members of the Kobuvirus genus. Quantitative PCR analysis estimated a range of 4×10(5) to 5×10(8) viral genome copies g(-1) in 22 (73 %) of 30 PCR-positive faeces. Based on sequence and phylogenetic analysis, we propose that this virus is the first member of a potential novel genus within the family Picornaviridae. Further studies are required to investigate the pathogenic potential of this virus within the avian host.

Detecting extinction risk from climate change by IUCN Red List criteria.

Anthropogenic climate change is a key threat to global biodiversity. To inform strategic actions aimed at conserving biodiversity as climate changes, conservation planners need early warning of the risks faced by different species. The IUCN Red List criteria for threatened species are widely acknowledged as useful risk assessment tools for informing conservation under constraints imposed by limited data. However, doubts have been expressed about the ability of the criteria to detect risks imposed by potentially slow-acting threats such as climate change, particularly because criteria addressing rates of population decline are assessed over time scales as short as 10 years. We used spatially explicit stochastic population models and dynamic species distribution models projected to future climates to determine how long before extinction a species would become eligible for listing as threatened based on the IUCN Red List criteria. We focused on a short-lived frog species (Assa darlingtoni) chosen specifically to represent potential weaknesses in the criteria to allow detailed consideration of the analytical issues and to develop an approach for wider application. The criteria were more sensitive to climate change than previously anticipated; lead times between initial listing in a threatened category and predicted extinction varied from 40 to 80 years, depending on data availability. We attributed this sensitivity primarily to the ensemble properties of the criteria that assess contrasting symptoms of extinction risk. Nevertheless, we recommend the robustness of the criteria warrants further investigation across species with contrasting life histories and patterns of decline. The adequacy of these lead times for early warning depends on practicalities of environmental policy and management, bureaucratic or political inertia, and the anticipated species response times to management actions.

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.