Batrachochytrium dendrobatidis (Bd) is a pathogenic chytrid fungus that is particularly lethal for amphibians. Bd can extirpate amphibian populations within a few weeks and remain in water in the absence of amphibian hosts. Most efforts to determine Bd presence and quantity in the field have focused on sampling hosts, but these data do not give us a direct reflection of the amount of Bd in the water, which are useful for parameterizing disease models, and are not effective when hosts are absent or difficult to sample. Current methods for screening Bd presence and quantity in water are time, resource, and money intensive. Here, we developed a streamlined method for detecting Bd in water with low turbidity (e.g., water samples from laboratory experiments and relatively clear pond water from a natural lentic system). We centrifuged water samples with known amounts of Bd to form a pellet and extracted the DNA from that pellet. This method was highly effective and the resulting concentrations across all tested treatments presented a highly linear relationship with the expected values. While the experimentally derived values were lower than the inoculation doses, the values were highly correlated and a conversion factor allows us to extrapolate the actual Bd concentration. This centrifuge-based method is effective, repeatable, and would greatly expand the domain of tractable questions to be explored in the field of Bd ecology. Importantly, this method increases equity in the field, because it is time- and cost-efficient and requires few resources.

The environment of an organism exerts selective pressures that affect mobility, feeding, reproduction as well as predator-prey and conspecific interactions. Land use changes induced by human activities modify these selective pressures and may result in the adaptation of organisms. Amphibians are ectotherms that typically show a biphasic life cycle with an aquatic and terrestrial phase, which makes them particularly sensitive to environmental change. We studied the impact of habitat modifications on palmate newt populations in the Ile de France region across four types of habitats: urban, mixed, agricultural, and natural with at least two replicates for each habitat type. We measured the morphology of newts using callipers, quantified maximal running and swimming speed and acceleration using high-speed video recordings, and quantified the swelling of the hind limb linked to an inflammatory reaction. Our results show that in urban habitats, newts are larger and heavier and have a better body condition. Females, moreover, have a larger head in natural habitats, possibly due to diet specialisation of females during the breeding season. In mixed and agricultural habitats, newts have longer limbs and show a tendency to run faster, possibly associated with the selective pressures on movement in mixed habitats. Differences in inflammatory responses were observed between sexes but not habitat types. Overall, our results show differences in morphology and trends for differences in performance in newts living in different habitats suggesting that animals are adapting to human-induced changes in their environment.

Among terrestrial ectotherms, hibernation is a common response to extreme cold temperatures and is associated with reduced physiological rates, including immunity. When winter wanes and temperatures increase, so too do vital rates of both ectothermic hosts and their parasites. Due to metabolic scaling, if parasite activity springs back faster than host immune functions then cold seasons and transitions between cold and warm seasons may represent periods of vulnerability for ectothermic hosts. Understanding host regulation of physiological rates at seasonal junctions is a first step toward identifying thermal mismatches between hosts and parasites. Here we show that immune gene expression is responsive to transitions into and out of the cold season in a winter-adapted amphibian, the wood frog (Lithobates sylvaticus), and that frogs experienced parasitism by at least two nematode species throughout the entirety of the cold season. In both splenic and skin tissues, we observed a decrease in immune gene expression going from fall to winter, observed no changes between winter and emergence from hibernation, and observed increases in immune gene expression after hibernation ended. At all timepoints, differentially expressed genes from spleens were more highly enriched for immune system processes than those from ventral skin, especially with respect to terms related to adaptive immune processes. Infection with nematode lungworms was also associated with upregulation of immune processes in the spleen. We suggest that rather than being a period of stagnation, during which physiological processes and infection potential cease, the cold season is immunologically dynamic, requiring coordinated regulation of many biological processes, and that the reemergence period may be an important time during which hosts invest in preparatory immunity.

Outbreaks of emerging infectious diseases are influenced by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Deterioration in the population of the micro-endemic Tanzanian Kihansi spray toad (Nectophrynoides asperginis) occurred after the construction of a hydropower dam, implicating habitat modification in this species decline. Population recovery followed habitat augmentation; however, a subsequent outbreak of chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) led to the spray toad\'s extinction in the wild. We show using spatiotemporal surveillance and mitogenome assembly of Bd from archived toad mortalities that the outbreak was caused by invasion of the BdCAPE lineage and not the panzootic lineage BdGPL. Molecular dating reveals an emergence of BdCAPE across southern Africa overlapping with the timing of the spray toad\'s extinction. That our post-outbreak surveillance of co-occurring amphibian species in the Udzungwa Mountains shows widespread infection by BdCAPE yet no signs of ill-health or decline suggests these other species can tolerate Bd when environments are stable. We conclude that, despite transient success in mitigating the impact caused by dams\' construction, invasion by BdCAPE caused the ultimate die-off that led to the extinction of the Kihansi spray toad.

The amount of genetic diversity within a population can affect ecological processes at population, community, and ecosystem levels. However, the magnitude, consistency, and scope of these effects are largely unknown. To investigate these issues, we conducted two experiments manipulating the amount of genetic diversity and environmental factors in larval amphibians. The first experiment manipulated wood frog genetic diversity, the presence or absence of caged predators, and competition from leopard frogs to test whether these factors affected survival, growth, and morphology of wood frogs and leopard frogs. The second experiment manipulated wood frog genetic diversity, the presence or absence of uncaged predators, and resource abundance to test whether these factors affected wood frog traits (survival, morphology, growth, development, and behavior) and other components of the ecological community (zooplankton abundance, phytoplankton, periphyton, and bacterial community structure). Genetic diversity did not affect wood frog survival, growth, and development in either experiment. However, genetic diversity did affect the mean morphology of wood frog tadpoles in the first experiment and the abundance and distribution of zooplankton in the second experiment. It did not affect phytoplankton abundance, periphyton abundance, or bacterial community structure. While effect sizes (Cohen\'s d) of genetic diversity were approximately half those of environment treatments, the greatest effect sizes were for interaction effects between genetic diversity and environment. Our results indicate that genetic diversity can have a large effect on ecological processes, but the direction of those effects is highly dependent upon environmental conditions, and not easily predicted from simple measures of traits.

Many animals exchange chemicals during courtship and mating. In some amphibians, sexual chemical communication is mediated by pheromones produced in male breeding glands that are transferred to the female\'s nostrils during mating. This has been mostly studied in salamanders, despite frogs having similar glands and courtship behaviours suggestive of chemical communication. In Neotropical poison frogs (Dendrobatidae and Aromobatidae), males of many species develop breeding glands in their fingers, causing certain fingers to visibly swell. Many also engage in cephalic amplexus, whereby the male\'s swollen fingers are placed in close contact with the female\'s nares during courtship. Here, we investigate the possible roles of swollen fingers in pheromone production using whole-transcriptome sequencing (RNAseq). We examined differential gene expression in the swollen versus non-swollen fingers and toes of two dendrobatid species, Leucostethus brachistriatus and Epipedobates anthonyi, both of which have specialised mucous glands in finger IV, the latter of which has cephalic amplexus. The overwhelming pattern of gene expression in both species was strong upregulation of sodefrin precursor-like factors (SPFs) in swollen fingers, a well-known pheromone system in salamanders. The differentially expressed SPF transcripts in each species were very high (>40), suggesting a high abundance of putative protein pheromones in both species. Overall, the high expression of SPFs in the swollen fingers in both species, combined with cephalic amplexus, supports the hypothesis that these traits, widespread across members of the subfamilies Colostethinae and Hyloxalinae (ca. 141 species), are involved in chemical signalling during courtship.
Muchos animales intercambian sustancias químicas durante el cortejo y el apareamiento. En algunos anfibios, la comunicación química sexual está mediada por feromonas producidas en las glándulas reproductoras de los machos que se transfieren a las hembras durante el apareamiento. Esto se ha estudiado sobre todo en salamandras, a pesar de que las ranas tienen glándulas similares y comportamientos de cortejo que sugieren una comunicación química. En las ranas venenosas neotropicales (Dendrobatidae y Aromobatidae), los machos de muchas especies desarrollan glándulas en los dedos, lo que hace que algunos dedos se vean hinchados. Asimismo, varias especies presentan amplexo cefálico, comportamiento de cortejo en el cual los dedos hinchados entran en estrecho contacto con las narinas y boca de la hembra. En este estudio investigamos las posibles funciones de los dedos hinchados en la producción de feromonas mediante la secuenciación del transcriptoma completo (RNAseq). Examinamos la expresión génica diferencial en los dedos hinchados y no hinchados de dos especies de dendrobátidos, Leucostethus brachistriatus y Epipedobates anthonyi, ambos con glándulas mucosas especializadas en el dedo IV, y esta última especie, con amplexo cefálico. El patrón abrumador de expresión génica en ambas especies fue la alta expression de Sodefrin Precursor‐Like Factor (SPF) en los dedos hinchados, un sistema de feromonas ampliamente conocido en las salamandras. El número de transcritos SPF expresados diferencialmente en cada especie fue muy elevado (>40), lo que sugiere una gran abundancia de feromonas proteicas putativas en ambas especies. En general, la elevada expresión de SPF en los dedos hinchados en ambas especies, combinada con el amplexo cefálico, apoya la hipótesis de que estos rasgos, muy extendidos entre los miembros de las subfamilias Colostethinae e Hyloxalinae (aprox 141 especies), están implicados en la señalización química durante el cortejo.

The plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads.

Microplastics (MPs) usually appear in the aquatic environment as complex pollutants with other environmental pollutants, such as levofloxacin (LVFX). After 45-day exposure to LVFX and MPs with different particle sizes at environmental levels, we measured the weight, snout-to-vent length (SVL), and development stages of Rana nigromaculata. Furthermore, we analyzed proteins and genes related to immune system and thyroid axis regulation, intestinal histological, and bioaccumulation of LVFX and MPs in the intestine and brain to further explore the toxic mechanism of co-exposure. We found MPs exacerbated the effect of LVFX on growth and development, and the order of inhibitory effects is as follows: LVFX-MP3>LVFX-MP1>LVFX-MP2. 0.1 and 1 μm MP could penetrate the blood-brain barrier, interact with LVFX in the brain, and affect growth and development by regulating thyroid axis. Besides, LVFX with MPs caused severer interference on thyroid axis compared with LVFX alone. However, 10 μm MP was prone to accumulating in the intestine, causing severe histopathological changes, interfering with the intestinal immune system and influencing growth and development through immune enzyme activity. Thus, we concluded that MPs could regulate the thyroid axis by interfering with the intestinal immune system.

Chytridiomycosis is a devastating disease and is a key cause of amphibian population declines around the world. Despite active research on this amphibian disease system for over 2 decades, we still do not have treatment methods that are safe and that can be broadly used across species. Here, we show evidence that voriconazole is a successful method of treatment for 1 species of amphibian in captivity and that this treatment could offer benefits over other treatment options like heat or itraconazole, which are not able to be used for all species and life stages. We conducted 2 treatments of chytridiomycosis using voriconazole. The treatment was effective and resulted in 100% pathogen clearance, and mortality ceased. Additionally, treating frogs with voriconazole requires less handling than treatment methods like itraconazole and requires no specialized equipment, like heat treatment. We highlight that clinical treatment trials should be conducted to identify an optimum dosage and treatment time and that trials should test whether this treatment is safe and effective for tadpoles and other species.

Environmental conditions like temperature and photoperiod can strongly shape organisms\' growth and development. For many ectotherms with complex life cycles, global change will cause their offspring to experience warmer conditions and earlier-season photoperiods, two variables that can induce conflicting responses. We experimentally manipulated photoperiod and temperature during gray treefrog (Hyla versicolor) larval development to examine effects at metamorphosis and during short (10-day) and long (56-day) periods post-metamorphosis. Both early- and late-season photoperiods (April and August) decreased age and size at metamorphosis relative to the average-season (June) photoperiod, while warmer temperatures decreased age but increased size at metamorphosis. Warmer larval temperatures reduced short-term juvenile growth but had no long-term effect. Conversely, photoperiod had no short-term carryover effect, but juveniles from early- and late-season larval photoperiods had lower long-term growth rates than juveniles from the average-season photoperiod. Similar responses to early- and late-season photoperiods may be due to reduced total daylight compared with average-season photoperiods. However, juveniles from late-season photoperiods selected cooler temperatures than early-season juveniles, suggesting that not all effects of photoperiod were due to total light exposure. Our results indicate that despite both temperature and photoperiod affecting metamorphosis, the long-term effects of photoperiod may be much stronger than those of temperature.