Estrogen contamination increases vulnerability of amphibians to the deadly chytrid fungus.

Aquatic contaminants and infectious diseases are among the major drivers of global amphibian declines. However, the interaction of these factors is poorly explored and could better explain the amphibian crisis. We exposed males and females of the Brazilian Cururu Toad, Rhinella icterica, to an environmentally relevant concentration of the estrogen 17-alpha-ethinylestradiol (an emerging contaminant) and to the chytrid infection (Batrachochytrium dendrobatidis), in their combined and isolated forms, and the ecotoxicity was determined by multiple biomarkers: cutaneous, hematological, cardiac, hepatic, and gonadal analysis. Our results showed that Cururu toads had many physiological alterations in response to the chytrid infection, including the appearance of cutaneous Langerhans's cells, increased blood leukocytes, increased heart contraction force and tachycardia, increased hepatic melanomacrophage cells, which in turn led to gonadal atrophy. The estrogen, in turn, increased the susceptibility of the toads to the chytrid infection (higher Bd loads) and maximized the deleterious effects of the pathogen: reducing leukocytes, decreasing the contraction force, and causing greater tachycardia, increasing hepatic melanomacrophage cells, and leading to greater gonadal atrophy, which were more extreme in females. The exposure to estrogen also revealed important toxicodynamic pathways of this toxicant, as shown by the immunosuppression of exposed animals, and the induction of the first stages of feminization in males, which corroborates that the synthetic estrogen acts as an endocrine disruptor. Such an intricate relationship is unprecedented and reinforces the importance of studying the serious consequences that multiple environmental stressors can cause to aquatic populations.

Skin microbiome disturbance linked to drought-associated amphibian disease.

The onset of global climate change has led to abnormal rainfall patterns, disrupting associations between wildlife and their symbiotic microorganisms. We monitored a population of pumpkin toadlets and their skin bacteria in the Brazilian Atlantic Forest during a drought. Given the recognized ability of some amphibian skin bacteria to inhibit the widespread fungal pathogen Batrachochytrium dendrobatidis (Bd), we investigated links between skin microbiome health, susceptibility to Bd and host mortality during a die-off event. We found that rainfall deficit was an indirect predictor of Bd loads through microbiome disruption, while its direct effect on Bd was weak. The microbiome was characterized by fewer putative Bd-inhibitory bacteria following the drought, which points to a one-month lagged effect of drought on the microbiome that may have increased toadlet susceptibility to Bd. Our study underscores the capacity of rainfall variability to disturb complex host-microbiome interactions and alter wildlife disease dynamics.

The cardiorespiratory system of miniature frogs.

Anurans of the genus Brachycephalus are among the smallest vertebrates in the world, due to an extreme process of miniaturization. As an example of this process, Brachycephalus species show loss of fingers, loss of the eardrum and middle ear, bone fusions, and the presence of paravertebral plates and parotic plaque. However, no studies addressing the consequences of miniaturization on internal organs, such as the lungs and heart, are currently available. Thus, this study aimed to investigate if overall small body size has affected the cardiorespiratory system. We investigated, via dissections, individuals of four Brachycephaloidea species: Brachycephalus rotenbergae, B. pitanga, Eleutherodactylus johnstonei, and Ischnocnema parva. We observed that B. rotenbergae and B. pitanga present a reduction of the atrial septum and absence of the carotid body. On the other hand, despite being a member of the sister genus to Brachycephalus (both genera belong to the Brachycephalidae), individuals of Ischnocnema present a heart with a complete septum and carotid body; this is also observed in E. johnstonei (Eleutherodactylidae). We observed that B. rotenbergae and B. pitanga have thin skin with a one to two cell thick germ layer, and their lungs likely exhibit lower blood supply when compared to individuals of the E. johnstonei and I. parva species. Based on the observed structures, we suggest that in species of Brachycephalus, respiration is performed mainly through the skin, and their lungs may have a reduced respiratory function.

Hidden shifts in allometry scaling between sound production and perception in anurans.

Background: Animal communication consists of signal production and perception, which are crucial for social interactions. The main form used by anurans is auditory communication, in most cases produced as advertisement calls. Furthermore, sound perception happens mainly through an external tympanic membrane, and plays an important role in social behavior. In this study, we evaluated the influence of body and tympanic membrane sizes on call frequency across the phylogeny of anurans. Methods: We use data on snout-vent length, tympanic membrane diameter, and dominant frequency of the advertisement call from the literature and from natural history museum collections. We mapped these traits across the anuran phylogeny and tested different models of diversification. Our final dataset includes data on body size, tympanic membrane size, and call dominant frequency of 735 anuran species. Results: The best explanatory model includes body and tympanum size with no interaction term. Although our results show that call frequency is strongly constrained by body and tympanum size, we identify five evolutionary shifts in allometry from that ancestral constraint. We relate these evolutionary shifts to the background noise experienced by populations. Body size is important for myriad ecological interactions and tympanum size is strongly associated with female call frequency preferences. Thus, allometric escape in frog calls might arise through environmental selection such as breeding in fast flowing or soundscape competition, as well as sexual selection linked to tympanum size.

A dataset for benchmarking Neotropical anuran calls identification in passive acoustic monitoring.

Global change is predicted to induce shifts in anuran acoustic behavior, which can be studied through passive acoustic monitoring (PAM). Understanding changes in calling behavior requires automatic identification of anuran species, which is challenging due to the particular characteristics of neotropical soundscapes. In this paper, we introduce a large-scale multi-species dataset of anuran amphibians calls recorded by PAM, that comprises 27 hours of expert annotations for 42 different species from two Brazilian biomes. We provide open access to the dataset, including the raw recordings, experimental setup code, and a benchmark with a baseline model of the fine-grained categorization problem. Additionally, we highlight the challenges of the dataset to encourage machine learning researchers to solve the problem of anuran call identification towards conservation policy. All our experiments and resources have been made available at https://soundclim.github.io/anuraweb/ .

Coinfection with chytrid genotypes drives divergent infection dynamics reflecting regional distribution patterns.

By altering the abundance, diversity, and distribution of species-and their pathogens-globalization may inadvertently select for more virulent pathogens. In Brazil's Atlantic Forest, a hotspot of amphibian biodiversity, the global amphibian trade has facilitated the co-occurrence of previously isolated enzootic and panzootic lineages of the pathogenic amphibian-chytrid (Batrachochytrium dendrobatidis, 'Bd') and generated new virulent recombinant genotypes ('hybrids'). Epidemiological data indicate that amphibian declines are most severe in hybrid zones, suggesting that coinfections are causing more severe infections or selecting for higher virulence. We investigated how coinfections involving these genotypes shapes virulence and transmission. Overall, coinfection favored the more virulent and competitively superior panzootic genotype, despite dampening its transmission potential and overall virulence. However, for the least virulent and least competitive genotype, coinfection increased both overall virulence and transmission. Thus, by integrating experimental and epidemiological data, our results provide mechanistic insight into how globalization can select for, and propel, the emergence of introduced hypervirulent lineages, such as the globally distributed panzootic lineage of Bd.

Low microbiome diversity in threatened amphibians from two biodiversity hotspots.

Microbial diversity positively influences community resilience of the host microbiome. However, extinction risk factors such as habitat specialization, narrow environmental tolerances, and exposure to anthropogenic disturbance may homogenize host-associated microbial communities critical for stress responses including disease defense. In a dataset containing 43 threatened and 90 non-threatened amphibian species across two biodiversity hotspots (Brazil's Atlantic Forest and Madagascar), we found that threatened host species carried lower skin bacterial diversity, after accounting for key environmental and host factors. The consistency of our findings across continents suggests the broad scale at which low bacteriome diversity may compromise pathogen defenses in species already burdened with the threat of extinction.

Chytrid fungus in amphibians from the lowland Brazilian Amazon.

Infectious diseases are one of the main threats to biodiversity. The fungus Batrachochytrium dendrobatidis (Bd) is associated with several amphibian losses around the globe, and environmental conditions may dictate the success of pathogen spread. The Brazilian Amazon has been considered climatically unsuitable for chytrid fungus, but additional information on Bd dynamics in this ecoregion is still lacking. We sampled 462 amphibians (449 anurans, 4 caudatans and 9 caecilians), representing 57 species from the Brazilian Amazon, and quantified Bd infections using qPCR. We tested whether abiotic variables predicted the risk of Bd infections, and tested for relationships between biotic variables and Bd. Finally, we experimentally tested the effects of Bd strains CLFT 156 and CLFT 102 (from the southern and northern Atlantic Forest, respectively) on Atelopus manauensis. We detected higher Bd prevalence than those previously reported for the Brazilian Amazon, and positive individuals in all 3 orders of amphibians sampled. Both biotic and abiotic predictors were related to prevalence, and no variable explained infection load. Moreover, we detected higher Bd prevalence in forested than open areas, while the host's reproductive biology was not a factor. We detected higher mortality in the experimental group infected with CLFT 156, probably because this strain was isolated from a region characterized by discrepant climatic conditions (latitudinally more distant) when compared with the host's sampling site in Amazon. The lowland Brazilian Amazon is still underexplored and future studies targeting all amphibian orders are essential to better understand Bd infection dynamics in this region.

Cryptic lineages and standing genetic variation across independent cane toad introductions.

Widespread introduced species can be leveraged to investigate the genetic, ecological and adaptive processes underlying rapid evolution and range expansion, particularly the contributions of genetic diversity to adaptation. Rhinella marina, the cane toad, has been a focus of invasion biology for decades in Australia. However, their introduction history in North America is less clear. Here, we investigated the roles of introduction history and genetic diversity in establishment success of cane toads across their introduced range. We used reduced representation sequencing (ddRAD) to obtain 34,000 SNPs from 247 toads in native (French Guiana, Guyana, Ecuador, Panama, Texas) and introduced (Bermuda, southern Florida, northern Florida, Hawai'i, Puerto Rico) populations. Unlike all other cane toad introductions, we found that Florida populations were more closely related to native Central American lineages (R. horribilis), than to native Southern American lineages (R. marina). Furthermore, we found high levels of diversity and population structure in the native range, corroborating suggestions that R. marina is a species complex. We also found that introduced populations exhibit only slightly lower genetic diversity than native populations. Together with demographic analyses, this indicates founding populations of toads in Florida were larger than previously reported. Lastly, within R. marina, only one of 245 putatively adaptive SNPs showed fixed differences between native and introduced ranges, suggesting that putative selection in these introduced populations is based upon existing genetic variation. Our findings highlight the importance of genetic sequencing in understanding biological introductions and hint at the role of standing genetic variation in range expansion.

Torrent frogs emit acoustic signals of a narrower spectral range in habitats with longer-lasting biotic background noise.

Animal acoustic communication can be challenging under intense noise. Amphibians that inhabit noisy environments offer a great opportunity to study potential mechanisms that maximise signal transmission. Based on the hypothesis that frogs can adjust their calls to circumvent communication problems, we tested the effect of the duration of biotic sounds (intermittent acoustic signals from two syntopic species) on calling properties of a torrent frog species. We recorded 54 focal males at four localities with varied levels of synchronous noise. Duration of noise for each recording was obtained by summing the duration of calls emitted by two closely related species. Using linear mixed-effect models with air temperature and body size as co-factors, we found that focal males emitted calls at a narrower frequency bandwidth when they were exposed to longer durations of heterospecific signals. We suggest that males reduce the frequency bandwidth of their calls to minimise the masking effect of the background noise and to amplify auditory induction for the receiver individuals. This is the first study reporting that biotic noise can trigger changes in frequency bandwidth in frogs, shedding light on the role of the environment in shaping animal behaviour.

Genetic structure of American bullfrog populations in Brazil.

Non-native species are a major problem affecting numerous biomes around the globe. Information on their population genetics is crucial for understanding their invasion history and dynamics. We evaluated the population structure of the non-native American bullfrog, Aquarana catesbeiana, in Brazil on the basis of 324 samples collected from feral and captive groups at 38 sites in seven of the nine states where feral populations occur. We genotyped all samples using previously developed, highly polymorphic microsatellite loci and performed a discriminant analysis of principal components together with Jost's D index to quantify pairwise differentiation between populations. We then amplified 1,047 base pairs of the mitochondrial cytochrome b (cytb) gene from the most divergent samples from each genetic population and calculated their pairwise differences. Both the microsatellite and cytb data indicated that bullfrogs comprise two populations. Population grouping 1 is widespread and possesses two cytb haplotypes. Population grouping 2 is restricted to only one state and possesses only one of the haplotypes from Population grouping 1. We show that there were two imports of bullfrogs to Brazil and that there is low genetic exchange between population groupings. Also, we find that there is no genetic divergence among feral and captive populations suggesting continuous releases. The limited genetic variability present in the country is associated to the small number of introductions and founders. Feral bullfrogs are highly associated to leaks from farms, and control measures should focus on preventing escapes using other resources than genetics, as feral and captive populations do not differ.

Presence of invasive American bullfrogs may reduce infectious disease in a native frog species.

Amphibians breeding in aquatic environments may encounter a myriad of threats during their life cycle. One species known to prey on native amphibians in aquatic habitats is the invasive North American bullfrog Lithobates catesbeianus, which, besides being a voracious predator and competitor, often acts as a pathogen carrier and disease superspreader because it tolerates high infection loads of the frog-killing fungus Batrachochytrium dendrobatidis (Bd). Here, we hypothesized that the presence of the bullfrogs in microcosms should either (1) decrease Bd disease severity in native frog species by discouraging them from using the aquatic environment, or (2) increase the mortality of the native species. We tested these 2 mutually exclusive hypotheses by co-housing the snouted treefrog Scinax x-signatus (native to our study area) with L. catesbeianus in the laboratory, exposing them to Bd, and using qPCR analysis to quantify the resulting Bd infection loads in the native frogs. Our experiment had the following replicated treatments: (1) native-only treatment (3 individuals of S. x-signatus), (2) native-predominant treatment (2 S. x-signatus + 1 L. catesbeianus), and (3) exotic-predominant treatment (1 S. x-signatus + 2 L. catesbeianus). We found that Bd infection loads in the native S. x-signatus were highest in the native-only treatment, and lowest in the exotic-predominant treatment, indicating that bullfrogs may discourage native frogs from occupying the aquatic habitat, thus reducing encounter rates between native frogs and the waterborne pathogen. This effect could be driven by the bullfrogs' predatory behavior and their high philopatry to aquatic habitats. Our results highlight that predation risk adds to the complexity of host-species interactions in Bd epidemiology.

Non-detection of mycoviruses in amphibian chytrid fungus (Batrachochytrium dendrobatidis) from Australia.

Mycoviruses may influence the pathogenicity of disease-causing fungi. Although mycoviruses have been found in some chytrid fungi, limited testing has not detected them in Batrachochytrium dendrobatidis (Bd), the cause of the devastating amphibian disease, chytridiomycosis. Here we conducted a survey for mycovirus presence in 38 Bd isolates from Australia (n = 31), Brazil (n = 5) and South Korea (n = 2) with a combination of modern high-throughput sequencing and conventional dsRNA cellulose chromatography. Mycoviruses were not detected in any isolates. This result was unexpected, given the long evolutionary history of Bd, as well as the high prevalence of mycoviruses in related fungal species. Given our widespread sampling in Australia and the limited number of Bd introductions, we suggest that mycoviruses are uncommon or absent from Australian Bd. Testing more isolates from regions where Bd originated, as well as regions with high diversity or low fungal virulence may identify mycoviruses that could aid in disease control.

Initial Phylotranscriptomic Confirmation of Homoplastic Evolution of the Conspicuous Coloration and Bufoniform Morphology of Pumpkin-Toadlets in the Genus Brachycephalus.

The genus Brachycephalus is a fascinating group of miniaturized anurans from the Brazilian Atlantic Forest, comprising the conspicuous, brightly colored pumpkin-toadlets and the cryptic flea-toads. Pumpkin-toadlets are known to contain tetrodotoxins and therefore, their bright colors may perform an aposematic function. Previous studies based on a limited number of mitochondrial and nuclear-encoded markers supported the existence of two clades containing species of pumpkin-toadlet phenotype, but deep nodes remained largely unresolved or conflicting between data sets. We use new RNAseq data of 17 individuals from nine Brachycephalus species to infer their evolutionary relationships from a phylogenomic perspective. Analyses of almost 5300 nuclear-encoded ortholog protein-coding genes and full mitochondrial genomes confirmed the existence of two separate pumpkin-toadlet clades, suggesting the convergent evolution (or multiple reversals) of the bufoniform morphology, conspicuous coloration, and probably toxicity. In addition, the study of the mitochondrial gene order revealed that three species (B. hermogenesi, B. pitanga, and B. rotenbergae) display translocations of different tRNAs (NCY and CYA) from the WANCY tRNA cluster to a position between the genes ATP6 and COIII, showing a new mitochondrial gene order arrangement for vertebrates. The newly clarified phylogeny suggests that Brachycephalus has the potential to become a promising model taxon to understand the evolution of coloration, body plan and toxicity. Given that toxicity information is available for only few species of Brachycephalus, without data for any flea-toad species, we also emphasize the need for a wider screening of toxicity across species, together with more in-depth functional and ecological study of their phenotypes.

Seasonal variation of Batrachochytrium dendrobatidis in a threatened anuran species from Uruguay.

Chytridiomycosis, an emergent infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), is considered one of the drivers of the current amphibian biodiversity loss. To inform endangered species conservation efforts, it is essential to improve our knowledge about the abiotic and biotic factors that influence Bd infection dynamics in the wild. Here, we analyzed variation of Bd infection in the redbelly toad Melanophryniscus montevidensis, a threatened bufonid from Uruguay. We tested the influence of temperature, precipitation, season, and host population size on Bd prevalence and intensity. Additionally, considering the sub-lethal effects of Bd, we tested if these variables, potentially through their effect on Bd, also explain the variation in host body condition. We determined a high Bd prevalence of 41% (100/241), and that population size influenced both Bd prevalence and infection intensity. We identified an effect of precipitation and season on Bd infection intensity and an effect of season on toad body condition. In addition, we found a negative effect of infection intensity on body condition; moreover, while some toads cleared the infection, their body condition did not improve, suggesting a long-term cost. This is the first report on host population size as an important factor in Bd infection dynamics in a threatened anuran species, and seasonal demographic changes appear to play an important role in the dynamics. Finally, we highlight the need for monitoring Bd in this and other endangered amphibian populations, especially those within the genus Melanophryniscus, which includes several Endangered and Data Deficient species in South America.

Forest cover influences chytrid infections in populations of Boana curupi, a threatened treefrog of south Brazil.

Complex interactions among hosts, pathogens, and the environment affect the vulnerability of amphibians to the emergence of infectious diseases such as chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Boana curupi is a forest-dwelling amphibian endemic to the southern Atlantic Forest of South America, a severely fragmented region. Here, we evaluated whether abiotic factors (including air and water temperature, relative air humidity, and landscape) are correlated with chytrid infection intensity and prevalence in B. curupi. We found individuals infected with Bd in all populations sampled. Prevalence ranged from 25-86%, and the infection burden ranged from 1 to over 130000 zoospore genomic equivalents (g.e.) (mean ± SD: 4913 ± 18081 g.e.). The infection load differed among populations and was influenced by forest cover at scales of 100, 500, and 1000 m, with the highest infection rates recorded in areas with a higher proportion of forest cover. Our results suggest that the fungus is widely distributed in the populations of B. curupi in southern Brazil. Population and disease monitoring are necessary to better understand the relationships between host, pathogen, and environment, especially when, as in the case of B. curupi, threatened species are involved.

Amphibian chytrid infections along the highest elevational gradient of the Brazilian Atlantic Forest.

Environmental variation along elevational gradients shapes conditions for pathogen development, which influences disease outcomes. Chytridiomycosis is a non-vectored disease caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd) and is responsible for massive declines of amphibian populations all over the world. Several biotic and abiotic factors are known to influence Bd infection dynamics in amphibians, including temperature and host species richness. Here, we quantified Bd prevalence and load along an elevational gradient in the Caparaó National Park (CNP), Brazil, and tested for associations of Bd infections with elevation, temperature, and species richness. We hypothesized that Bd infections would increase as local species richness decreased with elevation. We detected Bd along the entire elevational gradient and found a negative association between infection load and elevation. We did not detect significant associations between infection prevalence and elevation. Our findings are consistent with other wide elevational gradient studies, but are contrary to 2 other studies performed in the Atlantic Forest. We did not find the minimum elevational range that should be sampled to detect the influence of elevation on Bd variation. Our study represents the widest elevational gradient that has been sampled in Brazil and contributes to a better understanding of Bd distribution and dynamics in natural systems.

Co-infecting pathogen lineages have additive effects on host bacterial communities.

Amphibian skin bacteria may confer protection against the fungus Batrachochytrium dendrobatidis (Bd), but responses of skin bacteria to different Bd lineages are poorly understood. The global panzootic lineage (Bd-GPL) has caused amphibian declines and extinctions globally. However, other lineages are enzootic (Bd-Asia-2/Brazil). Increased contact rates between Bd-GPL and enzootic lineages via globalization pose unknown consequences for host-microbiome-pathogen dynamics. We conducted a laboratory experiment and used 16S rRNA amplicon-sequencing to assess: (i) whether two lineages (Bd-Asia-2/Brazil and Bd-GPL) and their recombinant, in single and mixed infections, differentially affect amphibian skin bacteria; (ii) and the changes associated with the transition to laboratory conditions. We determined no clear differences in bacterial diversity among Bd treatments, despite differences in infection intensity. However, we observed an additive effect of mixed infections on bacterial alpha diversity and a potentially antagonistic interaction between Bd genotypes. Additionally, observed changes in community composition suggest a higher ability of Bd-GPL to alter skin bacteria. Lastly, we observed a drastic reduction in bacterial diversity and a change in community structure in laboratory conditions. We provide evidence for complex interactions between Bd genotypes and amphibian skin bacteria during coinfections, and expand on the implications of experimental conditions in ecological studies.

Skin microbiome correlates with bioclimate and Batrachochytrium dendrobatidis infection intensity in Brazil's Atlantic Forest treefrogs.

In Brazil's Atlantic Forest (AF) biodiversity conservation is of key importance since the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the rapid loss of amphibian populations here and worldwide. The impact of Bd on amphibians is determined by the host's immune system, of which the skin microbiome is a critical component. The richness and diversity of such cutaneous bacterial communities are known to be shaped by abiotic factors which thus may indirectly modulate host susceptibility to Bd. This study aimed to contribute to understanding the environment-host-pathogen interaction determining skin bacterial communities in 819 treefrogs (Anura: Hylidae and Phyllomedusidae) from 71 species sampled across the AF. We investigated whether abiotic factors influence the bacterial community richness and structure on the amphibian skin. We further tested for an association between skin bacterial community structure and Bd co-occurrence. Our data revealed that temperature, precipitation, and elevation consistently correlate with richness and diversity of the skin microbiome and also predict Bd infection status. Surprisingly, our data suggest a weak but significant positive correlation of Bd infection intensity and bacterial richness. We highlight the prospect of future experimental studies on the impact of changing environmental conditions associated with global change on environment-host-pathogen interactions in the AF.

Seasonal prevalence of the amphibian chytrid in a tropical pond-dwelling tadpole species.

Infection by the chytrid fungus Batrachochytrium dendrobatidis (Bd) is a major threat to amphibians and has caused catastrophic global declines of amphibian populations. Some studies have detected a seasonal pattern of infection associated with the local climate, and although most of them have focused on investigating the seasonality of Bd in relation to its impacts on amphibians, fewer have aimed to understand the chytrid persistence in the amphibian assemblage over seasons by investigating reservoir hosts. Since tadpoles are generally tolerant to Bd infection, they often play a relevant role in local disease dynamics. Thus, we hypothesized that tadpoles of Boana faber, a species that can be found in permanent ponds throughout the seasons, would function as Bd reservoirs. We therefore investigated Bd infection prevalence in tadpoles of this species over 2 yr in a nature reserve. As expected, we detected a seasonal variation of Bd infection, with a higher prevalence of Bd during the coldest months (winter) when compared to the warmer months (summer). Interestingly, our seasonal-trend decomposition analysis showed that Bd prevalence is increasing annually in the area, which could represent either a natural fluctuation of this pathogen, or an imminent threat to that anuran assemblage. With this study, we highlight the tadpole of B. faber as a potential reservoir for Bd, and we suggest that monitoring Bd in such hosts could be a powerful tool for identifying priority areas for amphibian conservation.