Development of a multiplex real-time PCR surveillance assay for monitoring the health status of Ecuadorian amphibians at risk of extinction

Chytrid fungi and viruses within the genus Ranavirus have been associated with mass mortality events and declines in amphibian populations worldwide. The fungus Batrachochytrium dendrobatidis (Bd) was reported in Ecuador; however, other chytrid fungi like Batrachochytrium salamandrivorans (Bsal) or ranaviruses have not been described in the country so far. To prevent the introduction of pathogens into amphibian populations under conservation programs and to implement a successful disease surveillance program, the development of a sensitive and specific diagnostic assay was required. We describe here the optimization of one TaqMan probe-based multiplex quantitative polymerase chain reaction (qPCR) assay that enables the simultaneous detection of Bsal and ranavirus, and one monoplex TaqMan qPCR assay for the detection of Bd. Standard curves, with a high linear correlation (r2 > 0.995), were generated using a synthetic genome template (gBlocks®) containing the target sequences from all three pathogens. Different samples from skin, liver, kidney, spleen, and lung from six different amphibian species were tested, and both qPCR assays showed highly reproducible and reliable results. To our knowledge, this method is the first multiplex qPCR system developed in Ecuador for identifying amphibian pathogens and represents a valuable tool for the early detection of these pathogens and for infection and co-infection monitoring in future epidemiological surveillance of amphibian species at risk of extinction

Elevational and local climate variability predicts thermal breadth of mountain tropical tadpoles

The climate variability hypothesis posits that increased environmental thermal variation should select for thermal generalists, while stable environments should favor thermal specialists. This hypothesis has been tested on large spatial scales, such as latitude and elevation, but less so on smaller scales reflective of the experienced microclimate. Here, we estimated thermal tolerance limits of 75 species of amphibian tadpoles from an aseasonal tropical mountain range of the Ecuadorian Andes, distributed along a 3500 m elevational range, to test the climatic variability hypothesis at a large (elevation) and a small (microhabitat) scale. We show how species from less variable thermal habitats, such as lowlands and those restricted to streams, exhibit narrower thermal tolerance breadths than highland and pond-dwelling species respectively. Interestingly, while broader thermal tolerance breadths at large scales are driven by higher cold tolerance variation (heat-invariant hypothesis), at local scales they are driven by higher heat tolerance variation. This contrasting pattern may result from divergent selection on both thermal limits to face environmental thermal extremes at different scales. Specifically, within the same elevational window, exposure to extreme maximum temperatures could be avoided through habitat shifts from temporary ponds to permanent ponds or streams, while minimum peak temperatures remained invariable between habitats but steadily decreased with elevation. Therefore an understanding of the effects of habitat conversion is crucial for future research on resilience to climate change

Post‐epizootic microbiome associations across communities of neotropical amphibians

Microbiome–pathogen interactions are increasingly recognized as an important element of host immunity. While these host-level interactions will have consequences for community disease dynamics, the factors which influence host microbiomes at larger scales are poorly understood. We here describe landscape-scale pathogen–microbiome associations within the context of post-epizootic amphibian chytridiomycosis, a disease caused by the panzootic chytrid fungus Batrachochytrium dendrobatidis. We undertook a survey of Neotropical amphibians across altitudinal gradients in Ecuador ~30 years following the observed amphibian declines and collected skin swab-samples which were metabarcoded using both fungal (ITS-2) and bacterial (r16S) amplicons. The data revealed marked variation in patterns of both B. dendrobatidis infection and microbiome structure that are associated with host life history. Stream breeding amphibians were most likely to be infected with B. dendrobatidis. This increased probability of infection was further associated with increased abundance and diversity of non-Batrachochytrium chytrid fungi in the skin and environmental microbiome. We also show that increased alpha diversity and the relative abundance of fungi are lower in the skin microbiome of adult stream amphibians compared to adult pond-breeding amphibians, an association not seen for bacteria. Finally, stream tadpoles exhibit lower proportions of predicted protective microbial taxa than pond tadpoles, suggesting reduced biotic resistance. Our analyses show that host breeding ecology strongly shapes pathogen–microbiome associations at a landscape scale, a trait that may influence resilience in the face of emerging infectious diseases.info:eu-repo/semantics/publishedVersio

Microbiological and cytological characterization of coelomic fluid from three captive endangered amphibian Gastrotheca species with edema syndrome: preliminary analysis

Objective: Edema syndrome is highly prevalent but under researched in captive frogs around the world. The objective of the present study was to characterize at a basic microbiological and cytological level of the bacteria of the edema fluid of 20 individuals of the genus Gastrotheca to determine the presence of possible anaerobic and aerobic bacteria. Results: Fourteen types of bacteria were identified in the edema fluid, 12 of them at the species level (Pasteurella haemolytica, Hafnia alvei, Enterobacter agglomerans, Aeromonas hydrophila, Pseudomonas fluorescens, Burkholderia pseudomallei, Salmonella arizonae, Enterobacter gergoviae, Enterobacter sakazakii, Yersinia enterocolitica, Klebsiella oxytoca, and Klebsiella ozaenae) and two at the genus level (Enterococcus spp. and Streptococcus spp.). The most frequently identified cells were lymphocytes (37.7% in females and 46.4% in males), erythrocytes (23.5% in females and 17.5% in males) and neutrophils (4.2% in females and 2.8% in males). Finally, no relationship was found between the data obtained and the sex of the individuals studied

Critical Thermal Limits Do Not Vary between Wild- caught and Captive-bred Tadpoles of Agalychnis spurrelli (Anura: Hylidae)

Captive-bred organisms are widely used in ecology, evolution and conservation research, especially in scenarios where natural populations are scarce or at risk of extinction. Yet, it is still unclear whether captivity may alter thermal tolerances, crucial traits to predict species resilience to global warming. Here, we study whether captive-bred tadpoles of the gliding treefrog (Agalychnis spurrelli) show different thermal tolerances than wild-caught individuals. Our results show that there are no differences between critical thermal limits (CTmax and CTmin) of captive-bred and wild-caught tadpoles exposed to three-day acclimatization at 20 °C. Therefore, we suggest that the use of captive-bred amphibians is valid and may be appropriate in experimental comparisons to thermal physiological studies of wild populations

Extreme colour variation in the larvae of the executioner clownfrog, Dendropsophus carnifex (Anura: Hylidae), living in nearby ponds of different light exposure and duration

Here, we describe the extreme plasticity in colour variation in the tadpole of the executioner clownfrog, Dendropsophus carnifex (Anura: Hylidae), found across nearby ponds in Mindo (Ecuador). Tadpole coloration was compared between individuals from four distinct ponds revealing two explicit colour schemes, a pale phenotype which is commonly described for this species and a bright phenotype which, to our knowledge, has not been described before. In addition, the bright phenotype revealed reversibility to the pale phenotype in laboratory conditions. We discuss the functionality and driving factors for these contrasting colour phenotypes including the potential role of distinct ecological conditions

Stepping inside the niche: microclimate data are critical for accurate assessment of species' vulnerability to climate change.

To asses a species´vulnerability to climate change, we commonly use mapped environmental data that are coarsely resolved in time and space. Coarsely resolved temperature data are typically inaccurate at predicting temperatures in microhabitats used by an organism and may also exhibit spatial bias in topographically complex areas. One consequence of these inaccuracies is that coarsely resolved layers may predict thermal regimes at a site that exceed species´known thermal limits. In this study, we use statistical downscaling to account for environmental factors and develop high-resolution estimates of daily maximum temperatures for a 36 000 km2 study area over a 38-year period. We then demonstrate that this statistical downscaling provides temperature estimates that consistently place focal species within their fundamental thermal niche, whereas coarsely resolved layers do not. Our results highlight the need for incorporation of fine-scale weather data into species´vulnerability analyses and demosntrate that a statistical downscaling approach can yield biologically relevant estimates of thermal regimes

Predators like it hot: thermal mismatch in a predator–prey system across an elevational tropical gradient

Climate change may have dramatic consequences for communities through both direct effects of peak temperatures upon individual species and through interspecific mismatches in thermal sensitivities of interacting organisms which mediate changes in interspecific interactions (i.e. predation). Despite this, there is a paucity of information on the patterns of spatial physiological sensitivity of interacting species (at both landscape and local scales) which could ultimately influence geographical variation in the effects of climate change on community processes. In order to assess where these impacts may occur, we first need to evaluate the spatial heterogeneity in the degree of mismatch in thermal tolerances between interacting organisms. We quantify the magnitude of interspecific mismatch in maximum (CTmax) and minimum (CTmin) thermal tolerances among a predator–prey system of dragonfly and anuran larvae in tropical montane (242–3,631 m) and habitat (ponds and streams) gradients. To compare thermal mismatches between predator and prey, we coined the parameters maximum and minimum predatory tolerance margins (PTMmax and PTMmin), or difference in CTmax and CTmin of interacting organisms sampled across elevational and habitat gradients. Our analyses revealed that: (a) predators exhibit higher heat tolerances than prey (~4°C), a trend which remained stable across habitats and elevations. In contrast, we found no differences in minimum thermal tolerances between these groups. (b) Maximum and minimum thermal tolerances of both predators and prey decreased with elevation, but only maximum thermal tolerance varied across habitats, with pond species exhibiting higher heat tolerance than stream species. (c) Pond-dwelling organisms from low elevations (0–1,500 m a.s.l.) may be more susceptible to direct effects of warming than their highland counterparts because their maximum thermal tolerances are only slightly higher than their exposed maximum environmental temperatures. The greater relative thermal tolerance of dragonfly naiad predators may further increase the vulnerability of lowland tadpoles to warming due to potentially enhanced indirect effects of higher predation rates by more heat-tolerant dragonfly predators. However, further experimental work is required to establish the individual and population-level consequences of this thermal tolerance mismatch upon biotic interactions such as predator–prey

Development of a multiplex real-time PCR surveillance assay for monitoring the health status of Ecuadorian amphibians at risk of extinction

Chytrid fungi and viruses within the genus Ranavirus have been associated with mass mortality events and declines in amphibian populations worldwide. The fungus Batrachochytrium dendrobatidis (Bd) was reported in Ecuador; however, other chytrid fungi like Batrachochytrium salamandrivorans (Bsal) or ranaviruses have not been described in the country so far. To prevent the introduction of pathogens into amphibian populations under conservation programs and to implement a successful disease surveillance program, the development of a sensitive and specific diagnostic assay was required. We describe here the optimization of one TaqMan probe-based multiplex quantitative polymerase chain reaction (qPCR) assay that enables the simultaneous detection of Bsal and ranavirus, and one monoplex TaqMan qPCR assay for the detection of Bd. Standard curves, with a high linear correlation (r2 > 0.995), were generated using a synthetic genome template (gBlocks®) containing the target sequences from all three pathogens. Different samples from skin, liver, kidney, spleen, and lung from six different amphibian species were tested, and both qPCR assays showed highly reproducible and reliable results. To our knowledge, this method is the first multiplex qPCR system developed in Ecuador for identifying amphibian pathogens and represents a valuable tool for the early detection of these pathogens and for infection and co-infection monitoring in future epidemiological surveillance of amphibian species at risk of extinction