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Outbreaks of emerging infectious diseases are trained by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Extinction of the Tanzanian Kihansi spray toad (Nectophrynoides asperginis) in 2004 was contemporaneous with the construction of a dam, implicating habitat modification in the loss of this species. However, high burdens of a globally emerging infection, Batrachochytrium dendrobatidis (Bd) were synchronously observed implicating infectious disease in this toads extinction. Here, by shotgun sequencing skin DNA from archived toad mortalities and assembling chytrid mitogenomes, we prove this outbreak was caused by the BdCAPE lineage and not the panzootic lineage BdGPL that is widely associated with global amphibian extinctions. Molecular dating showed an invasion of BdCAPE across Southern Africa overlapping with the timing of the extinction event. However, post-outbreak surveillance of conspecific species inhabiting this mountainous region showed widespread infection by BdCAPE yet no signs of amphibian ill-health or species decline. Our findings show that despite efforts to mitigate the environmental impact caused by dams construction, invasion of the pathogen ultimately led to the loss of the Kihansi spray toad; a synergism between emerging infectious disease and environmental change that likely heralds wider negative impacts on biodiversity in the Anthropocene.

The fruit from various pepper plants has been employed for the seasoning of food, as perfuming agents, and also as traditional medicines. Phytochemicals isolated from different pepper species have been found to modulate the pharmacokinetics of orally administered drugs. This study investigated the possibility to apply capsaicin and piperine (extracted alkaloids) as modulators for drug delivery across the nasal epithelium. Both a nasal epithelial cell line (RPMI 2650) and excised sheep nasal tissue were used as models to investigate the effects of the selected pepper compounds on drug permeation. FITC-dextran 4400 (MW 4400 Da) was used as a large molecular weight marker compound for paracellular transport, while rhodamine 123 was used as a marker compound that is a substrate for P-glycoprotein-mediated efflux. From the permeation results, it was clear that capsaicin inhibited P-glycoprotein efflux to a larger extent, while piperine showed drug permeation enhancement via other mechanisms. The cell cytotoxicity studies indicated that capsaicin was noncytotoxic up to a concentration of 200 µM and piperine up to a concentration of 500 µM as indicated by cell viability above 80%. The histological analysis of the excised nasal tissue and cultured RPMI 2650 cell layers indicated that some damage occurred after treatment with 200 µM capsaicin, but no changes were observed for piperine up to a concentration of 50 µM.

Accurate survey methods are required for any wildlife research to yield reliable population data. This constraint finds significance in amphibian research that involves a highly threatened group of animals with a large proportion of cryptic species not easily detected by conventional survey methods. Across a growing spectrum of zoology research, survey outcomes are benefitting from the efficacy of scent detection dogs in assisting with species detection. We investigated the ability of a scent detection dog to locate and identify traces of giant bullfrog, Pyxicephalus adspersus scent and investigate methods of preserving frog scent for use in subsequent conditioning training of dogs. The scent detection dog was able to detect 100,000 times diluted scent with 87% sensitivity and 84% efficacy. High specificity (98,6%) was also achieved while presented with the challenge of detecting P. adspersus scent amid that of other frog species. Detection sensitivity was negatively correlated with scent preservation time but yielded the highest sensitivity for samples that were preserved as skin swabs stored at 4 °C and diluted shortly before use. Conservationists, scientists, and customs officials alike can benefit from scent detection dog detection of amphibians through enhanced sample acquisition rates with reduced collection biases.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide., S.J.O., T.W.J.G., L.Br., A.Lo., A.A.C., D.S.S., E.A.C., C.M., J.B., D.M.A., F.C., and M.C.F. were supported through NERC (standard grant NE/K014455/1). S.J.O. acknowledges a Microsoft Azure for Research Sponsorship (subscription ID: ab7cd695-49cf-4a83-910a-ef71603e708b). T.W.J.G., A.Lo., A.A.C., D.S.S., E.A.C., C.M., J.B., D.M.A., F.C., and M.C.F. were also supported by the EU BiodivERsA scheme (RACE, funded through NERC directed grant NE/G002193/1 and ANR08-Biodiversa-002-03) and NERC (standard grant NE/K012509/1). M.C.F., E.A.C., and C.M. acknowledge the Nouragues Travel Grant Program 2014. R.A.F. was supported by an MIT/Wellcome Trust Fellowship. T.W.J.G. was supported by the People’s Trust for Endangered Species and the Morris Animal Foundation (D12ZO002). J.M.G.S. and M.C.F. were supported by the Leverhulme Trust (RPG-2014-273) and the Morris Animal Foundation (D16ZO-022). F.B. was supported by the ERC (grant ERC 260801–Big_Idea). D.M.A. was funded by Wellcome Trust grant 099202. J.V. was supported by the Hungarian Scientific Research Fund (OTKA K77841) and Bolyai János Research Scholarship, Hungarian Academy of Sciences (BO/00579/14/8). D.J.G. was supported by the Conservation Leadership Programme (grant 0134010) with additional assistance from F. Gebresenbet, R. Kassahun, and S. P. Loader. C.S.-A. was supported by Fondecyt Nº11140902 and 1181758. T.M.D.-B. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland with assistance from M. Hirschfeld and the Budongo Conservation Field Station. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282). L.F.T. was supported by FAPESP (#2016/25358-3) and CNPq (#300896/2016-6). L.Be., L.F.S., and R.J.W. were supported by the Australian Research Council (FT100100375, DP120100811). A.A.C. was supported by a Royal Society Wolfson Research Merit award. J.H., A.La., and S.M. were funded by the Swedish Research Council Formas (grant no. 2013- 1389-26445-20). C.W. was funded by the National Research Foundation, South Africa. T.Y.J. and T.S.J. acknowledge NSF grant DEB-1601259. W.E.H. was funded by the NSERC Strategic and Discovery grant programs

The ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21st century and is composed of six lineages which are display varying levels of virulence in their hosts. Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage‐specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: BdGPL and BdCAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates., see also the Perspective by Claudio Azat

Lambert et al . question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

Introduction: The nasal route of drug administration offers an alternative way for oral drug delivery and has the benefit of avoiding first-pass metabolism through drug delivery directly into the systemic circulation. The drug absorption enhancing effects of selected aloe leaf materials have been shown across various delivery routes, but their efficacies in this regard across nasal epithelia have not yet been investigated. The aim of this study was to determine the effects of gel and whole leaf extract materials from three selected aloe species (Aloe vera, Aloe ferox and Aloe muth-muth) on FITC-dextran 4400 permeation across two nasal epithelial models. Methods: Permeation of FITC-dextran 4400 and histological studies were conducted on both RPMI 2650 cell layers and excised sheep nasal mucosa, while toxicity studies were conducted using a neutral red assay on the RPMI 2650 cell model. Results: Significantly increased (P ≤ 0.05) apparent permeability coefficient (Papp) values of FITC-dextran 4400 in the presence of the aloe materials as compared to the control were found with all three aloe species at the highest concentrations (1.5% and 3% w/v) in the RPMI 2650 cell line, while only Aloe muth-muth at the highest concentration exhibited significantly (P ≤ 0.05) higher Papp values across the excised tissue model. Histological and neutral red analysis showed that Aloe vera materials exhibited detrimental effects, Aloe muth-muth only showed slight effects on cell viability and Aloe ferox exhibited no effect on the nasal epithelium. Conclusion: This in vitro study showed for the first time the potential of Aloe ferox and Aloe muth-muth leaf materials to enhance nasal drug delivery without causing damaging effects on the epithelium, while Aloe vera enhanced nasal drug delivery with detrimental effects as determined by means of cytotoxicity assays and histological analysis.

Nasal drug administration has been identified as a potential alternative to oral drug administration, especially for systemic delivery of large molecular weight compounds. Major advantages of nasal drug delivery include high vascularity and permeability of the epithelial membranes as well as circumvention of first-pass metabolism. RPMI 2650 cell layers (in vitro cell model) and excised sheep nasal mucosal tissues (ex vivo sheep model) were evaluated with regard to epithelial thickness, selected tight junction protein expression (i.e. claudin-1, F-actin chains, zonula occludin-1), extent of p-glycoprotein (P-gp) related efflux of a model compound (Rhodamine-123, R123) and paracellular permeation of a large molecular weight model compound (FITC-dextran 4400, FD4). The cell model grown under liquid cover conditions (LCC) was thinner (24 ± 4 μm) than the epithelial layer of the sheep model (53 ± 4 μm), whereas the thickness of cell model grown under air liquid interface (ALI) conditions (53 ± 8 μm) compared well with that of the sheep model. Although the location and distribution of tight junction proteins and F-actin differed to some extent between the cell model grown under ALI conditions and the sheep model, the extent of paracellular permeation of FD4 was similar (Papp = 0.48 × 10-6 cm.s-1 and 0.46 × 10-6 cm.s-1, respectively). Furthermore, the bi-directional permeation of R123 yielded the same efflux ratio (ER = 2.33) in both models. The permeation results from this exploratory study indicated similarity in terms of compound permeation between the RPMI 2650 nasal epithelial cell line and the excised sheep nasal epithelial tissue model.

Amphibian populations worldwide are at risk of extinction from infectious diseases, including chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Amphibian cutaneous microbiomes interact with Bd and can confer protective benefits to the host. The composition of the microbiome itself is influenced by many environment- and host-related factors. However, little is known about the interacting effects of host population structure, genetic variation and developmental stage on microbiome composition and Bd prevalence across multiple sites. Here we explore these questions in Amietia hymenopus, a disease-affected frog in southern Africa. We use microsatellite genotyping and 16S amplicon sequencing to show that the microbiome associated with tadpole mouthparts is structured spatially, and is influenced by host genotype and developmental stage. We observed strong genetic structure in host populations based on rivers and geographic distances, but this did not correspond to spatial patterns in microbiome composition. These results indicate that demographic and host genetic factors affect microbiome composition within sites, but different factors are responsible for host population structure and microbiome structure at the between-site level. Our results help to elucidate complex within- and among- population drivers of microbiome structure in amphibian populations. That there is a genetic basis to microbiome composition in amphibians could help to inform amphibian conservation efforts against infectious diseases.

The Kihansi spray toad, Nectophrynoides asperginis, became extinct in the wild despite population monitoring and conservation management of its habitat in the Kihansi gorge, Tanzania. Anecdotal evidence has indicated human induced habitat modification, predators, pesticides and disease as possible causes of a rapid population decline and the species extirpation. Here, we systematically investigate the role of disease in the extinction event of the wild toad population. The amphibian chytrid fungus, Batrachochytrium dendrobatidis, was detected in spray toads that died during the extinction event and subsequently in other amphibian species in Kihansi Gorge and the adjacent Udagaji Gorge, but not in any toads collected prior to this. Following the population decline, the remnant spray toad population gradually disappeared over a nine-month period. We demonstrate how demographic and behavioral attributes predisposed the spray toads to chytridiomycosis, due to B. dendrobatidis infection, and how epidemic disease could have been exacerbated by altered environmental conditions in the spray wetlands. Our results show that chytridiomycosis was the proximate cause of extinction in the wild of N. asperginis. This represents the first known case of extinction by disease of an amphibian species in Africa. A captive breeding program in the US and Tanzania ensures the survival of the species and a reintroduction program is underway. However, we caution that chytridiomycosis remains an existing threat that requires a comprehensive mitigation strategy before the desired conservation outcome of an established population of repatriated toads can be achieved.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), responsible for numerous amphibian declines and extinctions, was previously thought to originate from the African continent. This was based on infected museum specimens from early 20th century South Africa, Cameroon and Uganda. Further research on archived specimens from other continents eventually revealed early 20th century records also in Brazil and Japan. Recent robust analysis of genomic diversity and phylogeny of Bd has shown origin from Asia to be more plausible. This raises the issue that the threat of Bd for African amphibians as a novel pathogen has been underestimated. There are now cases where dramatic amphibian declines in disparate mountains on the continent could be attributed to Bd, and this necessitates an urgent realigning of hypotheses and the research agenda for amphibian conservation on the continent. Notably, hotspots of amphibian host naivety include West Africa where this pathogen has so far not been detected. We discuss research gaps that amphibian conservationists might focus on, notably more genomic sequencing of the BdCAPE (the less virulent) lineage to determine its date of emergence, and assessing the susceptibility of different amphibian species to infection, disease and decline to better prioritize conservation actions.

Summary According to the threat-sensitivity hypothesis, prey avoidance behaviour should reflect the magnitude of predation risk. Since predation can strongly affect reproduction success, ovipositing females are expected to adaptively adjust their predator avoidance response, or local breeding patch selectivity, in accordance with the perceived level of threat posed for their progeny by specific predators. However, association between avoidance and predation can be disrupted when the prey and the predator lack spatiotemporal opportunities to co-evolve, such as in cases of non-native predator introductions. We examined the interactions between mosquitoes (from the genus Culex) and three species of sympatric predaceous freshwater fish, a native cyprinid Barbus paludinosus, a cichlid Pseudocrenilabrus philander and an introduced poeciliid, the western mosquitofish Gambusia affinis. In an outdoor mesocosm experiment, we quantified patterns of Culex oviposition site selection across fish species using free-roaming, caged and fish-free treatments. In a complementary laboratory experiment, we tested the effectiveness of each fish species as predators of mosquito eggs and larvae. Synthesis and applications. We found evidence for: (i) mosquito egg raft predation by free-roaming fish; (ii) fish-specific avoidance by ovipositing Culex; and (iii) a positive association between fish-specific oviposition avoidance and fish-specific efficiency as an egg predator. These results contribute towards a better understanding of predator–prey co-evolution, predator-borne cue recognition, and suggest local native fish, the southern mouthbrooder Pseudocrenilabrus philander, as an alternative to Gambusia for the biocontrol of Culex mosquitoes.

The demise of amphibians? Rapid spread of disease is a hazard in our interconnected world. The chytrid fungus Batrachochytrium dendrobatidis was identified in amphibian populations about 20 years ago and has caused death and species extinction at a global scale. Scheele et al. found that the fungus has caused declines in amphibian populations everywhere except at its origin in Asia (see the Perspective by Greenberg and Palen). A majority of species and populations are still experiencing decline, but there is evidence of limited recovery in some species. The analysis also suggests some conditions that predict resilience. Science , this issue p. 1459 ; see also p. 1386

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into sterile culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to interested researchers worldwide as part of the BiodivERsA project RACE – here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been widely applied across at least 5 continents, 23 countries and in 62 amphibian species, and have been successfully used to isolate chytrids in remote field locations. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this important group of emerging fungal pathogens.

The fungal pathogen

The fungal pathogen Batrachochytrium dendrobatidis has caused declines and extinctions in hundreds of amphibian species across the world. Virulence varies among and within lineages; the Global Panzootic Lineage (GPL) is the most pathogenic, although there is also variation in lethality between GPL isolates. Amphibians have a number of defences against pathogens, and skin products including the microbiota and host peptides have been shown to have considerable influence over disease progression. Here we show the collective skin products (the mucosome) of two amphibian species show significant variation in their ability to inhibit different globally- distributed isolates of GPL. This may in part explain the variation in disease susceptibility of hosts to different strains of Batrachochytrium dendrobatidis. More work is required to identify particular traits associated with mucosomes that confer broad- spectrum inhibition across GPL in order to facilitate the development of prophylaxis and/or treatments for chytridiomycosis in situ.

For decades, Amphibians have been globally threatened by the still expanding infectious disease, chytridiomycosis. Madagascar is an amphibian biodiversity hotspot where Batrachochytrium dendrobatidis (Bd) has only recently been detected. While no Bd-associated population declines have been reported, the risk of declines is high when invasive virulent lineages become involved. Cutaneous bacteria contribute to host innate immunity by providing defense against pathogens for numerous animals, including amphibians. Little is known, however, about the cutaneous bacterial residents of Malagasy amphibians and the functional capacity they have against Bd. We cultured 3179 skin bacterial isolates from over 90 frog species across Madagascar, identified them via Sanger sequencing of approximately 700 bp of the 16S rRNA gene, and characterized their functional capacity against Bd. A subset of isolates was also tested against multiple Bd genotypes. In addition, we applied the concept of herd immunity to estimate Bd-associated risk for amphibian communities across Madagascar based on bacterial antifungal activity. We found that multiple bacterial isolates (39% of all isolates) cultured from the skin of Malagasy frogs were able to inhibit Bd. Mean inhibition was weakly correlated with bacterial phylogeny, and certain taxonomic groups appear to have a high proportion of inhibitory isolates, such as the Enterobacteriaceae, Pseudomonadaceae, and Xanthamonadaceae (84, 80, and 75% respectively). Functional capacity of bacteria against Bd varied among Bd genotypes; however, there were some bacteria that showed broad spectrum inhibition against all tested Bd genotypes, suggesting that these bacteria would be good candidates for probiotic therapies. We estimated Bd-associated risk for sampled amphibian communities based on the concept of herd immunity. Multiple amphibian communities, including those in the amphibian diversity hotspots, Andasibe and Ranomafana, were estimated to be below the 80% herd immunity threshold, suggesting they may be at higher risk to chytridiomycosis if a lethal Bd genotype emerges in Madagascar. While this predictive approach rests on multiple assumptions, and incorporates only one component of hosts' defense against Bd, their culturable cutaneous bacterial defense, it can serve as a foundation for continued research on Bd-associated risk for the endemic frogs of Madagascar.

Amphibians are threatened globally by the chytridiomycete fungus Batrachochytrium dendrobatidis (Bd), which is still expanding in range. Madagascar, rich in amphibian diversity, remains one of the few places that the fungus has not invaded. Herein, we present results from a pilot survey for Bd in conjunction with a rapid amphibian survey conducted at Ivoloina Park, a forestry station near Toamasina, eastern Madagascar. This park is located on the south-western side of the Ivoloina River in a former lowland rainforest now predominantly covered by plantations of exotic trees. Our amphibian survey confirmed the presence of 12 anuran species identified by both morphology and molecular barcoding and revealed the presence of four candidate species. Real-time polymerase chain reaction screening for the presence of Bd showed that all 59 samples tested negative, thus confirming the absence of the pathogen in this area. Our survey of Ivoloina provides the first species list for a suburban park in Madagascar...

Unit for Environmental Science and Management, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa CIBIO, Centro de Investigacao em Biodiversidade e Recursos Geneticos, Campus Agrario de Vairao, Rua Padre Armando Quintas, 4485-661 Vairao, Portugal Madagascar Fauna Group, BP 442, Morafeno, 501 Toamasina, Madagascar Department of Animal Biology, University of Antananarivo, BP 906, 101 Antananarivo, Madagascar International Training Centre, Durrell Wildlife Conservation Trust, Les Augres Manor, La Profonde Rue, Trinity, Jersey JE3 5BP, UK North of England Zoological Society, Chester Zoo, Upton-by-Chester CH2 1LH, UK Museo Regionale di Scienze Naturali, Via G. Giolitti, 36, 10123 Turin, Italy

Amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been hypothe- sised to be an indigenous parasite of African amphibians. In Cameroon, however, previous sur- veys in one region (in the northwest) failed to detect this pathogen, despite the earliest African Bd having been recorded from a frog in eastern Cameroon, plus one recent record in the far south- east. To reconcile these contrasting results, we present survey data from 12 localities across 6 regions of Cameroon from anurans (n = 1052) and caecilians (n = 85) of ca. 108 species. Bd was detected in 124 amphibian hosts at 7 localities, including Mt. Oku, Mt. Cameroon, Mt. Manen- gouba and lowland localities in the centre and west of the country. None of the hosts were observed dead or dying. Infected amphibian hosts were not detected in other localities in the south and eastern rainforest belt. Infection occurred in both anurans and caecilians, making this the first reported case of infection in the latter order (Gymnophiona) of amphibians. There was no significant difference between prevalence and infection intensity in frogs and caecilians. We highlight the importance of taking into account the inhibition of diagnostic qPCR in studies on Bd, based on all Bd-positive hosts being undetected when screened without bovine serum albumin in the qPCR mix. The status of Bd as an indigenous, cosmopolitan amphibian parasite in Africa, including Cameroon, is supported by this work. Isolating and sequencing strains of Bd from Cameroon should now be a priority. Longitudinal host population monitoring will be required to determine the effects, if any, of the infection on amphibians in Cameroon.

The elygium is a darkly pigmented projection over the pupil of the tadpoles of certain species that live mostly at high altitudes. It has been suggested that this structure shades the retina, protecting it from high UV levels. In post-metamorphic individuals, the elygium is replaced by a structure known as an umbraculum. Confusion arose in the past from the inconsistent use of terminology when referring to these two structures. While they may serve the same function, these structures differ fundamentally in structure and origin. Our investigation of the ultrastructure of the eye in Amietia vertebralis tadpoles, using electron and light microscopy, revealed that the elygium consists of an accumulation of melanophores situated within the inner cornea, whereas the umbraculum is a dorsal extension of the iris pigment epithelium, analogous to the ventral pupillary nodule, which is present in most frog species. In A. vertebralis, the umbraculum starts to develop on the iris of older tadpoles (Gosner [1960] stage 37 Herpetologica 16:183-190), medial to the overlying elygium. A smaller, ventral elygium is also present in this species and is similar in structure to the dorsal elygium. The development of the elygium over a six-month period is also described.

The Infectious disease chytridiomycosis, which is caused by the fungal pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of amphibian declines and extinction. In vitro B. dendrobatidis is susceptible to a range of disinfectants, but not all have been tested on animals and some that have been proven effective have harmful side effects on the surrounding environment or the animals being treated. We tested the efficacy of F10SC veterinary disinfectant to treat B. dendrobatidis in experimentally infected tadpole and juvenile Sclerophrys gutturalis and tadpoles of Sclerophrys poweri and Amietia hymenopus. The minimum inhibitory concentration for F10SC on in vitro B. dendrobatidis ranged between 1:7000 for 5-min contact time and 1:10000 for 10-min contact time. Based on the survival data of test animals the no observed effect concentration for 15-min contact time was estimated to be 1:2000 dilution for juveniles, and 1:10000 for tadpoles. In S. gutturalis juveniles an 86% infection clearance rate was achieved after five 15-min doses of 1:3000 dilution. A 100% clearance was achieved in A. hymenopus tadpoles after seven 15-min doses of 1:10000 dilution, and after nine doses of the same treatment in S. poweri tadpoles. F10SC has the benefit of being a concentrated compound that provides a treatment protocol which is nontoxic to tadpoles and post-metamorphic individuals, has a short half-life and is effective against B. dendrobatidis during short contact times, but further testing on different species of amphibians is advised.

Morphological similarities between the tadpoles of Amietia umbraculata and A. vertebralis have led to confusion and incorrect descriptions and identifications in the literature. Based on 33 body measurements and ratios we revised the morphological descriptions of the tadpoles of the two species. Tadpole identification was verified through DNA sequencing using mitochondrial (16S) gene fragments. A combination of four morphological characters proved to be informative and consistent in distinguishing between tadpoles of the two species. Tadpoles of A. umbraculata are characterized by having four labial tooth rows in the lower jaw, extensive tail mottling, a dorsal fin that originates well behind the body, reaching a maximum depth at 50% of the tail length, and an average tail length of 1.9 times body length. Amietia vertebralis tadpoles on the other hand are characterized by having five or more labial tooth rows in the lower jaw, tail mottling that is confined to the upper half of the tail musculature, a dor...

Environmental parameters such as temperature and rainfall influence the biology of amphibians and are likely to similarly influence the growth and prevalence of associated pathogens. Amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), causes an infectious disease, chytridiomycosis, in amphibians worldwide. Field studies on post-metamorphic anurans from tropical Australia have correlated increased prevalence with cool winter temperatures, but similar studies are lacking from Africa. We monitored the seasonality of amphibian chytrid in the Highveld of South Africa through microscopic examination of common river frog (Amietia angolensis) tadpoles over 12 months. Within the study area Bd was found to be widespread, but largely limited to riverine systems. The seasonal infection pattern was inconsistent with the findings of past studies, which showed that prevalence usually peaks during the cooler months of the year. This study indicates that infection levels increased during spring in the Grassland...

We comment on the results of a previous study which evaluate the accuracy of mouthpart depigmentation as an indicator of Batrachochytrium dendrobatidis (Bd) infection in anuran larvae. Macroscopic mouthpart inspection is a useful technique for Bd identification since it is practical and nonlethal; however, this method is also expected to result in increased rates of false negatives and false positives when compared to other methods. We therefore suggest that there are scenarios in which the use of mouthpart depigmentation is not appropriate, and we present a conceptual framework for deciding when mouthpart depigmentation is an appropriate indicator of oral chytridiomycosis in tadpoles. We conclude that more sensitive techniques are preferable when false positives or false negatives could result in the incorrect classification of populations as infected or uninfected, namely when Bd is absent or present at low prevalence. In contrast, mouthpart depigmentation is an appropriate indicator of infecti...

Harvesting wild amphibians for animal trade and consequent introductions of exotic species are considered threats to biodiversity. For this study, we evaluated the literature and unpublished data on Xenopus laevis exports from the Western Cape, South Africa, since the onset of the trade in the early 1930s. Exports for medical science have changed from the use of both captive‐bred and wild‐caught animals to the export of wild‐caught animals only. More than 10 000 frogs were exported annually during 1998–2004 to 132 facilities situated in 30 countries. Uncontrolled harvesting, feral populations, and the spread of parasites and disease associated with X. laevis trade, highlight the relevance of this trade to the conservation of amphibian biodiversity.

The fungal pathogen Batrachochytrium dendrobatidis contributes to the global decline of amphibians. Although mortality from B. dendrobatidis infections occurs primarily in postmetamorphic individuals, infected tadpoles may suffer reduced growth and developmental rates as a result of oral chytridiomycosis, possibly affecting adult fitness. We conducted a field study in which we examined South African tadpoles for oral chytridiomycosis and compared the body sizes of infected and uninfected individuals of 2 species, Heleophryne natalensis and Strongylopus hymenopus. Presence of B. dendrobatidis was determined by microscopic inspection of mouthparts. Infection prevalence was high in both species, 62.5 and 38.6%, respectively, and infected individuals were significantly larger in both species. The inclusion of developmental stage in the analysis of S. hymenopus body size eliminated the relationship between body size and infection status, suggesting that differences in body size were not due to differences in growth, but to differences in developmental stage of infected larvae. These results suggest that larvae at more advanced developmental stages are more likely to be infected with B. dendrobatidis and that infection in larval amphibians may be dependent on time or developmental status of larvae. Contrary to the results of past studies, there was no evidence that oral chytridiomycosis resulted in decreased growth of tadpoles, despite the occurrence of oral abnormalities in infected individuals of 1 species. Because tadpole performance can subsequently affect the health of anuran populations and because tadpoles can act as reservoirs of infection, the study of B. dendrobatidis in larval amphibians is important to understanding the effects of this emerging disease.

International audience; Amphibian chytridiomycosis, an emerging infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), has been a significant driver of amphibian declines. While globally widespread, Bd had not yet been reported from within Madagascar. We document surveys conducted across the country between 2005 and 2014, showing Bd's first record in 2010. Subsequently, Bd was detected in multiple areas, with prevalence reaching up to 100%. Detection of Bd appears to be associated with mid to high elevation sites and to have a seasonal pattern, with greater detectability during the dry season. Lineage-based PCR was performed on a subset of samples. While some did not amplify with any lineage probe, when a positive signal was observed, samples were most similar to the Global Panzootic Lineage (BdGPL). These results may suggest that Bd arrived recently, but do not exclude the existence of a previously undetected endemic Bd genotype. Representatives of all native anuran families have tested Bd-positive and exposure trials confirm infection by Bd is possible. Bd's presence could pose significant threats to Madagascar's unique “megadiverse” amphibians.

The ability to quantify infections provides a tool with which to perform comparative pathological research. The need exists for a simplistic standard method to compare infection levels of Batrachochytrium dendrobatidis, a major cause of global amphibian declines. Through examination of skin sloughs of the Cape river frog Afrana fuscigula, we present an accessible method that not only provides quantitative measurements of B. dendrobatidis, but also provides information that increases the confidence of detection through histological surveys. The method relies on the availability of live animals that are actively shedding skin. By employing a direct microscopic count of sporangia, it is possible to express infection in terms of density. Micro-spatial infection in the skin of A. fuscigula is characterised by significant differences in sporangium density among the different components of the foot, and by similar differences in site infection frequency. Notably, toe tips and tubercles contain higher infection densities and are more often infected than webbing or the base of the foot. This pattern of infection might facilitate disease transmission due to the increased exposure of these components to abrasion. Density data can be used with the Poisson frequency function to approximate binomial probabilities of detecting B. dendrobatidis through histology. The probability matrix produced for A. fuscigula indicated that foot-site selection for histology markedly influenced the number of sections required to detect B. dendrobatidis at a specific level of probability. Thus, examination of a test sample of skin tissue with direct-count quantification can help in planning the sampling of tissues for histological surveys.

The herbicide atrazine has been suggested to cause gonadal deformities in frogs and could possibly impact on reproduction. Since the early 1960s, atrazine has been used in large amounts in maize production areas of South Africa. These areas overlap with populations of the African Clawed Frog (Xenopus laevis) that has a wide distribution in southern Africa and is found in most water-bodies includ- ing those where atrazine residues are detected. The aim of this study was to compare various attributes of individual- and population-level responses of X. laevis from maize-growing and non-maize-growing areas. Xenopus laevis were studied in three reference and five maize-growing sites. Sex ratio, snout-vent length, body-mass and age profiles were found to be similar for populations in maize-growing and non- maize-growing areas. Our mark-recapture data indicated that all sites had robust populations. There were no significant relationships between exposure to atrazine and any of the parameters investigated in pop- ulations of X. laevis.

Amphibians across the planet face the threat of population decline and extirpation caused by the disease chytridiomycosis. Despite consensus that the fungal pathogens responsible for the disease are conservation issues, strategies to mitigate their impacts in the natural world are, at best, nascent. Reducing risk associated with the movement of amphibians, non-amphibian vectors and other sources of infection remains the first line of defence and a primary objective when mitigating the threat of disease in wildlife. Amphibian-associated chytridiomycete fungi and chytridiomycosis are already widespread, though, and we therefore focus on discussing options for mitigating the threats once disease emergence has occurred in wild amphibian populations. All strategies have shortcomings that need to be overcome before implementation, including stronger efforts towards understanding and addressing ethical and legal considerations. Even if these issues can be dealt with, all currently available approaches, or those under discussion, are unlikely to yield the desired conservation outcome of disease mitigation. The decision process for establishing mitigation strategies requires integrated thinking that assesses disease mitigation options critically and embeds them within more comprehensive strategies for the conservation of amphibian populations, communities and ecosystems.

A putative driver of global amphibian decline is the panzootic chytrid fungus Batrachochytrium dendrobatidis (Bd). While Bd has been documented across continental Africa, its distribution in West Africa remains ambiguous. We tested 793 West African amphibians (one caecilian and 61 anuran species) for the presence of Bd. The samples originated from seven West African countries - Bénin, Burkina Faso, Côte d'Ivoire, Ghana, Guinea, Liberia, Sierra Leone - and were collected from a variety of habitats, ranging from lowland rainforests to montane forests, montane grasslands to humid and dry lowland savannahs. The species investigated comprised various life-history strategies, but we focused particularly on aquatic and riparian species. We used diagnostic PCR to screen 656 specimen swabs and histology to analyse 137 specimen toe tips. All samples tested negative for Bd, including a widespread habitat generalist Hoplobatrachus occipitalis which is intensively traded on the West African food market and thus could be a potential dispersal agent for Bd. Continental fine-grained (30 arc seconds) environmental niche models suggest that Bd should have a broad distribution across West Africa that includes most of the regions and habitats that we surveyed. The surprising apparent absence of Bd in West Africa indicates that the Dahomey Gap may have acted as a natural barrier. Herein we highlight the importance of this Bd-free region of the African continent - especially for the long-term conservation of several threatened species depending on fast flowing forest streams (Conraua alleni ("Vulnerable") and Petropedetes natator ("Near Threatened")) as well as the "Critically Endangered" viviparous toad endemic to the montane grasslands of Mount Nimba (Nimbaphrynoides occidentalis).

The elygium is a darkly pigmented projection over the pupil of the tadpoles of certain species that live mostly at high altitudes. It has been suggested that this structure shades the retina, protecting it from high UV levels. In post-metamorphic individuals, the elygium is replaced by a structure known as an umbraculum. Confusion arose in the past from the inconsistent use of terminology when referring to these two structures. While they may serve the same function, these structures differ fundamentally in structure and origin. Our investigation of the ultrastructure of the eye in Amietia vertebralis tadpoles, using electron and light microscopy, revealed that the elygium consists of an accumulation of melanophores situated within the inner cornea, whereas the umbraculum is a dorsal extension of the iris pigment epithelium, analogous to the ventral pupillary nodule, which is present in most frog species. In A. vertebralis, the umbraculum starts to develop on the iris of older tadpoles (Gosner [1960] stage 37 Herpetologica 16:183-190), medial to the overlying elygium. A smaller, ventral elygium is also present in this species and is similar in structure to the dorsal elygium. The development of the elygium over a six-month period is also described.

Batrachochytriumdendrobatidis (Bd) is a globally ubiquitous fungal infection that has emerged to become a primary driver of amphibian biodiversity loss. Despite widespread effort to understand the emergence of this panzootic, the origins of the infection, its patterns of global spread, and principle mode of evolution remain largely unknown. Using comparative population genomics, we discovered three deeply diverged lineages of Bd associated with amphibians. Two of these lineages were found in multiple continents and are associated with known introductions by the amphibian trade.We found that isolates belonging to one clade, the global panzootic lineage (BdGPL) have emerged across at least five continents during the 20th century and are associated with the onset of epizootics in North America, Central America, the Caribbean, Australia, and Europe. The two newly identified divergent lineages, Cape lineage (BdCAPE) and Swiss lineage (BdCH), were found to differ in morphological traits when compared against one another and BdGPL, and we show that BdGPL is hypervirulent. BdGPL uniquely bears the hallmarks of genomic recombination, manifested as extensive intergenomic phylogenetic conflict and patchily distributed heterozygosity. Wepostulate that contact between previously genetically isolated allopatric populations of Bd may have allowed recombination to occur, resulting in the generation, spread, and invasion of the hypervirulent BdGPL leading to contemporary disease-driven losses in amphibian biodiversity.

4 pages, 2 figures.-- PMID: 18462021 [PubMed].-- PMCID: PMC2365978., Frogs from Madagascar constitute one of the richest groups of amphibian fauna in the world, with currently 238 described species; caecilians and salamanders are absent. Several frog radiations of the island are species-rich and parallel lemurs and tenrecs in their astonishing morphological and ecological diversity. According to the Global Amphibian Assessment (GAA), Madagascar ranks as the country with the 12th highest amphibian species richness (see also http://www.globalamphibians.org), but this is likely an underestimate, because an additional 182 candidate species have been identified since. Diversity is concentrated in rainforests and can locally reach over 100 species. Impressively, 100% of the autochthonous species and 88% of the genera are strictly endemic to Madagascar and its inshore islands. Most of these species belong to two radiations of astonishing ecomorphological and reproductive diversity, the mantellids and the scaphiophrynine plus cophyline microhylids.

A recent paper by Kolby et al. [1], surveying for Batrachochytrium dendrobatidis (Bd) and ranavirus in Madagascar, presents results for 508 amphibian specimens and 68 water bodies sampled during a 2-month period of the 2013–14 wet season. Kolby et al. [1] did not detect Bd in any of the samples, presenting evidence that add to our understanding of Bd dynamics in Madagascar. Earlier in 2015, we published “Widespread presence of the pathogenic fungus Batrachochytrium dendrobatidis in wild amphibian communities in Madagascar” in the journal Scientific Reports [2]. We presented rigorous spatial and temporal surveillance data for 4,155 amphibians sampled across a 10-year period, and used two independent molecular diagnostics to demonstrate the occurrence of a molecular signature of Bd infection at multiple locations across the island. We focus here on solely the Bd results, which directly relate to our published study. While the conclusions of Bd’s occurrence and prevalence in Madagascar may appear to conflict between these papers, upon closer investigation the data sets actually complement each other. Our evidence for Bd’s presence and its widespread incidence is based on multi-year monitoring data carried out through the National Monitoring Program [3] and allied survey efforts, occurring in both the wet and dry season. Our data collected during the same time as Kolby et al.’s sampling (2013–14 wet season), is consistent with their recently published results [1,2] (summarized in Table 1). Therefore, Kolby et al.’s conclusion that our data “highly contradict” those reported in their study is inaccurate. In the 2013–14 wet season, we sampled 569 frogs from 8 locations, of which only 3 samples showed a positive signal for Bd. The positive samples were collected from one individual at each of three sites: Antoetra, Ranomafana, and Ankaratra. While both datasets (the sampling reported in Kolby et al. 2015 and the wet season 2013–2014 sampling reported in Bletz et al. 2015) surveyed numerous individuals and locations across the island, there are some differences in sampling locations. More specifically, one of our positive occurrences came from Antoetra, which was not surveyed by Kolby et al [1]. Two of our positives do come from locations surveyed by both groups: Ranomafana and Ankaratra; if we combine the sub-sites within these locations, the prevalence is 0.0043 and 0.0062 respectively, which falls within the prevalence confidence intervals presented in Kolby et al. [1] (Table 1). This same logic is used by Kolby et al. [1] to show the complementarity of their field survey data and Kolby’s previous work showing Bd’s presence in amphibians imported into the US from Madagascar [4]. Both datasets are consistent with the conclusion that Bd had a very low prevalence during the 2013–2014 wet season. Table 1 Summary of published Bd survey data for the 2013–2014 wet season. The low prevalence detected in the wet season may likely be explained by seasonality of Bd. Both papers discuss the possibility of seasonal patterns, where Bd prevalence decreases in the warmer, wetter season and increases in the cooler, dryer season due to climatic or so-far un-described environmental factors. This phenomenon is not unusual and several studies have noted a high degree of seasonal variation in the prevalence of Bd (e.g. [5, 6, 7, 8]). In our published study [2] we present preliminary evidence of a seasonal pattern of Bd, showing that prevalence and/or detection was greater in the dryer, cooler season (May-Oct) than the wetter, warmer season (Nov-April) [2]. This seasonal pattern we documented likely explains the lack of detection by Kolby et al. since they sampled only in the wet (and warmer) season. Kolby et al. [1] supplement their individual sampling with the analysis of filtered water from natural habitats. In this case, the lack of Bd detection might be associated with increased water flow due to increased rainfall during the wet season, which could lower the concentration of Bd zoospores to undetectable levels. Additionally, water-filters of natural water bodies have also been found to be less sensitive than direct sampling of amphibians [9,10]. To better understand seasonal variation as well as other factors such as geographic distribution and host species variation, additional sampling across wet and dry season in a uniform and standardize manner will be important. The major difference between these papers is that we draw from data collected in multiple years and seasons and from a much larger sample of amphibians with further validation using chytrid lineage-based PCR amplification, making our study more comprehensive in nature. Kolby et al. [1] surveyed for a 2-month period, which makes it difficult to make general assumptions about pathogen occurrence from such a small snap shot in time. Our data set thus allows for the conclusion of the “widespread presence of Bd” as we document repeated detections of Bd at geographically distant locations in Madagascar albeit with varying degree of prevalence among sites and seasons. We also have secondary confirmation and validation of Bd’s presence from an independent non-nuclear lineage specific qPCR designed to the Bd mtDNA locus, which is unique to our study. Kolby et al. [1] suggest that Bd in Madagascar cannot yet be described with certainty in part due to the variability of sampling and detection methods. We acknowledge ourselves that our use of various methods may confound some of our findings, such as the seasonal pattern of Bd; however, and importantly, this does not negate or question the evidence for Bd-positive samples collected from Madagascar. While the data presented by both studies indicate a low prevalence of Bd in Madagascar in 2014, we argue that the additional multi-year data we have collected strongly supports the occurrence of one or more Bd lineage(s) in the samples collected from wild Madagascar amphibians. A similar conclusion was also made by Kolby et al. [4] based on their observations of Bd in wild-caught frogs from Madagascar that were imported into the USA. Definitive and final confirmation of Bd in Madagascar awaits histopathology, isolation of a Bd culture, and/or genome sequencing. These additional analyses can clarify whether Madagascar is facing the panzootic, hypervirulent Bd-GPL or a different (possibly endemic) Bd-lineage. More importantly, our results may have serious conservation implications. We presented strong evidence that at least one lineage of Bd exists in Madagascar, with increased prevalence at some locations during the dry season. It remains to be understood if this genotype is virulent with respect to the resident anuran fauna and capable of causing population declines. Using the ‘precautionary principle’ in reacting to suspected introductions of novel emerging infectious diseases infecting wildlife [11] it is essential to initiate conservation actions. Continuing ongoing population monitoring of Madagascar's amphibians and pathogen surveillance through the NMP are therefore essential and are a priority of the national amphibian conservation strategy for the country known as ‘A Conservation Strategy for the Amphibians of Madagascar (ACSAM) [12,13]. If it is relatively hypovirulent, it gives conservationists time to engage in mitigation strategies and to plan for the possible (and likely inevitable) arrival of a virulent genotype, which could threaten the diverse, endemic frog communities.

Histologic evidence indicates southern Africa as the origin of the amphibian chytrid fungus., The sudden appearance of chytridiomycosis, the cause of amphibian deaths and population declines in several continents, suggests that its etiologic agent, the amphibian chytrid Batrachochytrium dendrobatidis, was introduced into the affected regions. However, the origin of this virulent pathogen is unknown. A survey was conducted of 697 archived specimens of 3 species of Xenopus collected from 1879 to 1999 in southern Africa in which the histologic features of the interdigital webbing were analyzed. The earliest case of chytridiomycosis found was in a Xenopus laevis frog in 1938, and overall prevalence was 2.7%. The prevalence showed no significant differences between species, regions, season, or time period. Chytridiomycosis was a stable endemic infection in southern Africa for 23 years before any positive specimen was found outside Africa. We propose that Africa is the origin of the amphibian chytrid and that the international trade in X. laevis that began in the mid-1930s was the means of dissemination.