Reptiles and amphibians (herptiles) are some of the most endangered and threatened species on the planet and numerous conservation strategies are being implemented with the goal of ensuring species recovery. Little is known, however, about the gut microbiome of wild herptiles and how it relates to the health of these populations. Here, we report results from the gut microbiome characterization of both a broad survey of herptiles, and the correlation between the fungus Basidiobolus, and the bacterial community supported by a deeper, more intensive sampling of Plethodon glutinosus, known as slimy salamanders. We demonstrate that bacterial communities sampled from frogs, lizards, and salamanders are structured by the host taxonomy and that Basidiobolus is a common and natural component of these wild gut microbiomes. Intensive sampling of multiple hosts across the ecoregions of Tennessee revealed that geography and host:geography interactions are strong predictors of distinct Basidiobolus operational taxonomic units present within a given host. Co-occurrence analyses of Basidiobolus and bacterial community diversity support a correlation and interaction between Basidiobolus and bacteria, suggesting that Basidiobolus may play a role in structuring the bacterial community. We further the hypothesis that this interaction is advanced by unique specialized metabolism originating from horizontal gene transfer from bacteria to Basidiobolus and demonstrate that Basidiobolus is capable of producing a diversity of specialized metabolites including small cyclic peptides.IMPORTANCEThis work significantly advances our understanding of biodiversity and microbial interactions in herptile microbiomes, the role that fungi play as a structural and functional members of herptile gut microbiomes, and the chemical functions that structure microbiome phenotypes. We also provide an important observational system of how the gut microbiome represents a unique environment that selects for novel metabolic functions through horizontal gene transfer between fungi and bacteria. Such studies are needed to better understand the complexity of gut microbiomes in nature and will inform conservation strategies for threatened species of herpetofauna.

As threats to amphibian health increase, there is a growing need for diagnostic tools to assess and monitor their health status. Plasma protein electrophoresis has proven to be useful in other nonmammalian species. It enables quantification of protein fractions in plasma that may be altered in various disease processes, and is therefore useful in narrowing down differential diagnoses and detecting inflammation, in combination with other modalities such as biochemical and hematologic testing. The amphibian electrophoretogram must be defined before baseline reference intervals are obtained across species. Agarose gel electrophoresis was performed on plasma samples collected from presumed clinically normal individuals of one anuran and six urodelans: Osteopilus septentrionalis (n=2), Gyrinophilus porphyriticus (n=1), Notophthalmus viridescens (n=1), Eurycea guttolineata (n=2), Amphiuma tridactylum (n=2), Cryptobranchus alleganiensis (n=5), and Siren lacertina (n=6). The electrophoretograms varied in number of fractions between each species; however, the number of fractions was consistent within a species. An albumin migrating fraction was consistently observed in all species. A prealbumin migrating fraction was identified in species that primarily use organs other than skin for respiration. This study provides preliminary examples of a normal plasma protein electrophoretogram for seven amphibian species. Further studies quantifying reference intervals and identification of protein fractions will help establish protein electrophoresis as a useful tool in amphibian health investigations.

Deepening droughts and unprecedented wildfires are at the leading edge of climate change. Such events pose an emerging threat to species maladapted to these perturbations, with the potential for steeper declines than may be inferred from the gradual erosion of their climatic niche. This study focused on two species of amphibians-Philoria kundagungan and Philoria richmondensis (Limnodynastidae)-from the Gondwanan rainforests of eastern Australia that were extensively affected by the \"Black Summer\" megafires of 2019/2020 and the severe drought associated with them. We sought to assess the impact of these perturbations by quantifying the extent of habitat affected by fire, assessing patterns of occurrence and abundance of calling males post-fire, and comparing post-fire occurrence and abundance with that observed pre-fire. Some 30% of potentially suitable habitat for P. kundagungan was fire affected, and 12% for P. richmondensis. Field surveys revealed persistence in some burnt rainforest; however, both species were detected at a higher proportion of unburnt sites. There was a clear negative effect of fire on the probability of site occupancy, abundance and the probability of persistence for P. kundagungan. For P. richmondensis, effects of fire were less evident due to the limited penetration of fire into core habitat; however, occupancy rates and abundance of calling males were depressed during the severe drought that prevailed just prior to the fires, with the reappearance of calling males linked to the degree of rehydration of breeding habitat post-fire. Our results highlight the possibility that severe negative impacts of climate change for montane rainforest endemics may be felt much sooner than commonly anticipated under a scenario of gradual (decadal-scale) changes in mean climatic conditions. Instead, the increased rate of severe stochastic events places these narrow range species at a heightened risk of extinction in the near-term.

Tadpole development is influenced by environmental cues and hypoxia can favor the emergence of the neural networks driving air breathing. Exposing isolated brainstems from pre-metamorphic tadpoles to acute hypoxia (∼0% O2; 15 min) leads to a progressive increase in fictive air breaths (∼3 fold) in the hours that follow stimulation. Here, we first determined whether this effect persists over longer periods (<18 h); we then evaluated maturity of the motor output by comparing the breathing pattern of hypoxia-exposed brainstems to that of preparations from adult bullfrogs under basal conditions. Because progressive withdrawal of GABAB-mediated inhibition contributes to the developmental increase in fictive lung ventilation, we then hypothesised that hypoxia reduces respiratory sensitivity to baclofen (selective GABAB-agonist). Experiments were performed on isolated brainstem preparations from pre-metamorphic tadpoles (TK stages IV to XIV); respiratory-related neural activity was recorded from cranial nerves V/VII and X before and 18 h after exposure to hypoxia (0% O2 + 2% CO2; 25 min). Time-control experiments (no hypoxia) were performed. Exposing pre-metamorphic tadpoles to hypoxia did not affect gill burst frequency, but augmented the frequency of fictive lung bursts and the incidence of episodic breathing levels intermediate between pre-metamorphic and adult preparations. Addition of baclofen to the aCSF (0,2 μM - 20 min) reduced lung burst frequency, but the response of hypoxia-exposed brainstems was greater than controls. We conclude that acute hypoxia facilitates development and maturation of the motor command driving air breathing. We propose that a greater number of active rhythmogenic neurons expressing GABAb receptors contributes to this effect.

BACKGROUND: Functional diversity is important to maintain ecosystem functioning. Species with different ecomorphological traits may display distinct functional roles in ecosystems. Accordingly, functionally extreme species are more important as they can exhibit specific strategies. However, little is known about the distribution patterns of functionally extreme species at a local scale and whether the prior extinction of extreme species can cause significant effects on functional diversity. In addition, no empirical studies have been conducted on the microhabitat determinants of extreme species to maintain the functional diversity.
RESULTS: This study collected 1470 tadpoles belonging to 6 families and 20 anuran species. These species were subsequently divided into 65 functional entities based on their developmental stages to incorporate intraspecific traits variability. As a result, we detected seven extreme functional entities, accounting for 10.7% of the total number of entities. Moreover, the prior extinction of extreme entities can lead to a significant decrease in functional diversity compared with the random extinction of entities. Microhabitat variables such as conductivity, water depth, and current velocity determined the distribution of extreme entities.
CONCLUSIONS: Although the functionally extreme entities only represented a small proportion of the total number of tadpoles, they played irreplaceable roles in maintaining functional diversity. Their extinction may induce high functional vulnerability in tadpole communities. Therefore, anuran species with extreme tadpole traits need to be projected for amphibian conservation.

We conduct a phylogeographic and population genetic study of the Asiatic toad (Bufo gargarizans) to understand its evolutionary history, and the influence of geology and climate. A total of 292 individuals from 94 locations were genotyped for two mitochondrial loci (cytb, ND2) and five nuclear introns (Sox9-2, Rho-3, CCNB2-3, UCH-2, and DBI-2), and we performed a suite of phylogenetic, population genetic, and divergence dating analyses. The phylogenetic trees constructed using mitochondrial loci inferred B. gargarizans being divided into two major groups: China mainland and Northeast Asia (Northeast China, Russia, and Korean Peninsula). As with previous studies of this species, we recover population genetic structure not tied to geographic region. Additionally, we discover a new genetic clade restricted to Northeast Asia that points towards the Korean Peninsula being a glacial refugium during the Pleistocene. The weak phylogeographic pattern of B. gargarizans is likely the result of multiple biological, anthropogenic, and historical factors - robust dispersal abilities as a consequence of physiological adaptations, human translocation, geologic activity, and glacial cycles of the Pleistocene. We highlight the complex geologic and climatic history of Northeast Asia and encourage further research to understand its impact on the biodiversity in the region.

UNASSIGNED: To measure medium borne bystander effects, to study the influence of radioadaptive response (RAR) on bystander response, and to discover reliable radioresponsive biomarkers in radio-adapting frogs from Duke Swamp contaminated with an above-background radiation level and in naïve frogs from Twin Lake as the background control site.
UNASSIGNED: Frogs were captured at Duke Swamp and Twin Lake and brought to the lab at the Canadian Nuclear Laboratories facility. Half of the frogs from each site were irradiated with 4 Gy while the other half of the frogs were left with no further radiation treatment. Frog bladders were removed and placed in sterile culture media. Upon arrival at McMaster University, the bladders were processed for tissue cultures. After 48 h, the culture media conditioned by the bladder explants were harvested for clonogenic reporter survival assay and calcium flux measurements for assessing bystander effects. HPV-G cells were used as bystander reporter cells in all radiation-induced bystander effect (RIBE) assays. The frog bladder cultures were incubated for another 10-12 days followed by immunochemical staining for bcl-2 and c-myc expressions to analyze cellular anti-apoptotic (pro-survival) and pro-apoptotic (pro-death) responses, respectively.
UNASSIGNED: Only culture media conditioned by bladders from 4-Gy-irradiated naïve frogs from Twin Lake induced bystander effects (reduction of HPV-G reporter cells\' clonogenic survival and presence of strong calcium flux activities). The 4 Gy irradiation dose increased pro-apoptotic c-myc expression in naïve frogs\' bladder explants. Culture media conditioned by bladders from radio-adapting frogs from Duke Swamp enhanced HPV-G\'s clonogenic survival and a 4 Gy irradiation challenge did not change the enhanced clonogenic survival nature nor induce calcium flux. In bladder explants from both control and 4-Gy-irradiated radio-adapting frogs, anti-apoptotic bcl-2 expression for pro-survival responses was ubiquitous while c-myc expression for pro-death responses was limited to a small fraction of cells.
UNASSIGNED: The clonogenic RIBE reporter assay using HPV-G and calcium flux measurements are useful diagnostic tools for RIBE assessment of field biological samples, specifically those from frogs. RAR induced by environmentally relevant low-dose radiation induces protective bystander response. Bcl-2 and c-myc are reliable biomarkers for evaluating low dose radiation responses in wild populations of amphibians. Overall, this pilot study emphasizes the importance of looking at non-targeted effects (NTEs) in natural populations of non-human biota that could be vulnerable to chronic low-dose radiation exposures.

Monitoring programs serve to detect trends in the distribution and abundance of species. To do so, monitoring programs often use static state variables. Dynamic state variables that describe population dynamics might be more valuable because they allow for a mechanistic understanding of the processes that lead to population trends. We fit multistate occupancy models to data from a country-wide multispecies amphibian occupancy monitoring program and estimated occupancy and breeding probabilities. If breeding probabilities are determinants of occupancy dynamics, then they may serve in monitoring programs as state variables that describe dynamic processes. The results showed that breeding probabilities were low and that a large proportion of the populations had to be considered to be non-breeding populations (i.e., populations where adults are present but no breeding occurs). For some species, the majority of populations were non-breeding populations. We found that non-breeding populations have lower persistence probabilities than populations where breeding occurs. Breeding probabilities may thus explain trends in occupancy but they might also explain other ecological phenomena, such as the success of invasive species, which had high breeding probabilities. Signs of breeding, i.e., the presence of eggs and larvae, were often hard to detect. Importantly, non-breeding populations also had low detection probabilities, perhaps because they had lower abundances. We suggest that monitoring programs should invest more in the detection of life history stages indicative of breeding, and also into the detection of non-breeding populations. We conclude that breeding probability should be used as a state variable in monitoring programs because it can lead to deeper insights into the processes driving occupancy dynamics.

Using rotors to expose animals to different levels of hypergravity is an efficient means of understanding how altered gravity affects physiological functions, interactions between physiological systems and animal development. Furthermore, rotors can be used to prepare space experiments, e.g., conducting hypergravity experiments to demonstrate the feasibility of a study before its implementation and to complement inflight experiments by comparing the effects of micro- and hypergravity. In this paper, we present a new platform called the Gravitational Experimental Platform for Animal Models (GEPAM), which has been part of European Space Agency (ESA)\'s portfolio of ground-based facilities since 2020, to study the effects of altered gravity on aquatic animal models (amphibian embryos/tadpoles) and mice. This platform comprises rotors for hypergravity exposure (three aquatic rotors and one rodent rotor) and models to simulate microgravity (cages for mouse hindlimb unloading and a random positioning machine (RPM)). Four species of amphibians can be used at present. All murine strains can be used and are maintained in a specific pathogen-free area. This platform is surrounded by numerous facilities for sample preparation and analysis using state-of-the-art techniques. Finally, we illustrate how GEPAM can contribute to the understanding of molecular and cellular mechanisms and the identification of countermeasures.

Amphibians are now recognized as the most endangered group. One of this decline causes is the degradation of their habitat through direct contamination of water, soil leaching, or runoff from surrounding contaminated soils and environments. In the North of France, the extensive industrial activities resulted in massive soil contamination by metal compounds. Mineral amendments were added to soils to decrease trace metal mobility. Because of the large areas to be treated, the use of inexpensive industrial by-products was favored. Two types of fly ashes were both tested in an experimental site with the plantation of trees in 2000. Aim of the present work was to investigate the effects of extracts from metal-contaminated soils treated or not for 10 years with fly ashes on Xenopus laevis oocyte using cell biology approaches. Indeed, our previous studies have shown that the Xenopus oocyte is a relevant model to study the metal ion toxicity. Survival and maturation of oocyte exposed to the soil extracts were evaluated by phenotypic approaches and electrophysiological recordings. An extract derived from a metal-contaminated soil treated for 10 years with sulfo-calcic ashes induced the largest effects. Membrane integrity appeared affected and ion fluxes in exposed oocytes were changed. Thus, it appeared that extracted elements from certain mineral amendments used to prevent the mobility of metals in the case of highly metal-contaminated soils could have a negative impact on X. laevis oocytes.