Global change exposes wildlife to a variety of environmental stressors and is affecting biodiversity worldwide, with amphibian population declines being at the forefront of the global biodiversity crisis. The use of non-invasive methods to determine the physiological state in response to environmental stressors is therefore an important advance in the field of conservation physiology. The glucocorticoid hormone corticosterone (CORT) is one useful biomarker to assess physiological stress in amphibians, and sampling water-borne (WB) CORT is a novel, non-invasive collection technique. Here, we tested whether WB CORT can serve as a valid proxy of organismal levels of CORT in larvae of the common frog (Rana temporaria). We evaluated the association between tissue and WB CORT levels sampled from the same individuals across ontogenetic stages, ranging from newly-hatched larvae to froglets at ten days after metamorphosis. We also investigated how both tissue and WB CORT change throughout ontogeny. We found that WB CORT is a valid method in pro-metamorphic larvae as values for both methods were highly correlated. In contrast, there was no correlation between tissue and WB CORT in newly-hatched, pre-metamorphic larvae, metamorphs, or post-metamorphic froglets probably due to ontogenetic changes in respiratory and skin morphology and physiology affecting the transdermal CORT release. Both collection methods consistently revealed a non-linear pattern of ontogenetic change in CORT with a peak at metamorphic climax. Thus, our results indicate that WB CORT sampling is a promising, non-invasive conservation tool for studies on late-stage amphibian larvae. However, we suggest considering that different contexts might affect the reliability of WB CORT and consequently urge future studies to validate this method whenever it is used in new approaches. We conclude proposing some recommendations and perspectives on the use of WB CORT that will aid in broadening its application as a non-invasive tool in amphibian conservation physiology. 

Tadpoles are important components of many aquatic food webs as they influence populations at other trophic levels and, ultimately, energy transfer between aquatic and terrestrial ecosystems. What tadpoles consume is a key factor to their ecological functions. Neotropical anuran assemblages include many species with a larval stage, however, the diets of neotropical tadpoles are relatively poorly known. We performed a systematic review on the available literature on in situ studies of neotropical tadpole diets and found out that most studies are descriptive and temporally restricted, failing to consider seasonal variations. Studies frequently also fail to consider possible ontogenetic dietary variations or tadpoles' capacity to select food based on what is available to them in their habitats. We contributed to this topic by investigating seasonal variations and comparing the diets of two developmental stages of Scinax curicica tadpoles. Tadpole diet varied between dry and rainy seasons, but not between the studied developmental stages. These results, together with published studies, suggest that multiple ecological/physiological factors are likely to influence tadpole food intake and should not be overlooked. Studies on neotropical tadpole feeding ecology, digestion capacity, physiological effects of different diets and microbiome versus diet interactions are highly desirable but largely unexplored. Such studies are likely to add valuable information to understand tadpole roles in aquatic food webs, their growth and development performance resulting from food acquisition and assimilation, and consequently their effects on other species in aquatic and terrestrial ecosystems.

Microplastics (MP) are an abundant, long-lasting, and widespread type of environmental pollution that is of increasing concern as it might pose a serious threat to ecosystems and species. However, these threats are still largely unknown for amphibians. Here, we used the African clawed frog (Xenopus laevis) as a model species to investigate whether polyethylene MP ingestion affects amphibian growth and development and leads to metabolic changes across two consecutive life stages (larvae and juveniles). Furthermore, we examined whether MP effects were more pronounced at higher rearing temperatures. Larval growth, development, and body condition were recorded, and standard metabolic rate (SMR) and levels of stress hormone (corticosterone, CORT) were measured. We determined variation in size, morphology, and hepatosomatic index in juveniles to identify any potential consequences of MP ingestion across metamorphosis. In both life stages, MP accumulation in the body was assessed. MP ingestion was found to result in sublethal effects on larval growth, development, and metabolism, to lead to allometric carry-over effects on juvenile morphology, and to accumulate in the specimens at both life stages. In larvae, SMR and developmental rate increased in response to MP ingestion; there additionally was a significant interaction of MP ingestion and temperature on development. CORT levels were higher in larvae that ingested MP, except at higher temperature. In juveniles, body was wider, and extremities were longer in animals exposed to MP during the larval stage; a high rearing temperature in combination with MP ingestion counteracted this effect. Our results provide first insights into the effects of MP on amphibians throughout metamorphosis and demonstrate that juvenile amphibians may act as a pathway for MP from freshwater to terrestrial environments. To allow for generalizations across amphibian species, future experiments need to consider the field prevalence and abundance of different MP in amphibians at various life stages.

Among a multitude of stressors to which wildlife is exposed, environmental pollution is a pervasive one that poses a serious threat. The permeable skin of amphibians is likely to increase direct contact of the body with pollutants, making them a group worth studying to access environmental quality. Consequently, finding reliable and complementary biomarkers that will present detectable and predictable changes in response to pollutants is essential to identify pollution sublethal effects on amphibians and to investigate whether these are in part responsible for population declines. The glucocorticoid hormone corticosterone (CORT), involved in many metabolic functions, is often used to measure the physiological stress response to environmental stressors in amphibians. In this study, we evaluated whether water-borne CORT can serve as a non-invasive biomarker for nitrate pollution stress in the European common frog (Rana temporaria) by comparing the effect of nitrate exposure on hormone release rates and on other physiological downstream biomarkers, i.e., ultimate physiological effects of the stressor. Specifically, we investigated the effect of different nitrate concentrations (0, 10, 50, and 100 mg/L) on water-borne CORT release rates, age, size, and body condition. Exposure to nitrate pollution significantly increased age at metamorphosis and water-borne CORT release rates, and led to reduced mass and body condition, but only at higher nitrate concentrations (i.e., 50 and 100 mg/L). Considering this similar sensitivity to other acknowledged biomarkers, water-borne CORT was a reliable biomarker of physiological stress in R. temporaria exposed to nitrate pollution stress in a controlled single-stressor laboratory approach. Thus, water-borne CORT is a promising method to be included in more holistic approaches. We recommend that such approaches keep testing multiple biomarker combinations, as species are exposed to several stressors likely to interact and produce varied outcomes in different biomarkers in their natural habitats.

Throughout the year, wild animals are exposed to a variety of challenges such as changing environmental conditions and reproductive activity. These challenges may affect their stress hormone levels for varying durations and in varying intensities and impacts. Measurements of the glucocorticoid hormone cortisol in the hair of mammals are considered a good biomarker for measuring physiological stress and are increasingly used to evaluate stress hormone levels of wild animals. Here, we examined the influence of season, reproductive activity, sex, as well as body condition on hair cortisol concentrations (HCC) in Lepilemur edwardsi, a small Malagasy primate species. L. edwardsi lives in the seasonal dry forests of western Madagascar, which are characterized by a strongly changing resource availability throughout the year. We hypothesized that these seasonal changes of resource availability and additionally the reproductive cycle of this species would influence HCC of L. edwardsi. Results revealed that hair cortisol concentration of females did not change seasonally or with the reproductive cycle. However, we found a significant increase of hair cortisol levels in males from the early wet season during the early dry season (mating season). This increase is presumably due to changed behavior during the mating season, as sportive lemurs travel more and show aggressive behavior during this time of the year. This behavior is energy‐costly and stressful, and presumably leads to elevated HCC. As elevated cortisol levels may impair immune function, L. edwardsi males might also be more susceptible to parasites and diseases, which is unfavorable in particular during a period of low resource availability (dry season).

Climate change is expected to increase mean temperatures and the frequency of extreme weather events, that can lead to earlier/extended breeding seasons in temperate taxa. As a consequence, many organisms that show climate-induced phenological shifts might be exposed to environmental conditions they are not well adapted to while breeding, and their ability to cope with stressful conditions might be influenced. Here, we investigated how parental breeding time shapes the sensitivity to nitrate exposure at three consecutive life stages (embryonic, larval, juvenile) in the European common frog (Rana temporaria). We compared hatching success and life-stage specific survival, growth, standard metabolic rate, body condition, and acute thermal sensitivity of offspring from an earlier-breeding parental cohort (early cohort) vs. a later-breeding parental cohort (late cohort) exposed to a range of environmentally relevant concentrations of nitrate (0–100 mg/L). We also investigated whether nitrate exposure experienced during the embryonic and larval stages affects physiological performance in later life stages (after metamorphosis). Our study reveals that parental breeding time affects the sensitivity to nitrate pollution at three consecutive life stages in Rana temporaria. Breeding later in spring reduced hatching size and survival at high nitrate exposure, but also induced compensatory growth of the offspring. In both early and late cohorts, exposure to nitrate pollution reduced developmental rate and led to larger, but older larvae at the onset of metamorphosis with a greater sensitivity to warmer environmental temperatures. Standard metabolic rate, on the contrary, was neither affected by parental breeding time nor by exposure to nitrate. Exposure to nitrate pollution during embryonic and larval development led to carry-over in juvenile froglets as their sensitivity to temperature was higher. In a world highly impacted by humans, it is thus essential to give stressors a more holistic approach in order to better predict their consequences on species subjected to them.

The composition of cutaneous (skin-associated) bacterial communities of amphibians has been intensively studied in light of the potential of some of these commensal bacterial taxa to mitigate infection with the chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). However, surprisingly, the absolute densities in which these bacteria occur on the skin are only poorly known. We here combine quantification of bacterial 16SrDNA copies from skin swabs by quantitative PCR with counts from scanning electron microscopy (SEM) images to assess and compare bacterial abundances on the skin of various central European amphibians. We focus on the fire salamander (Salamandra salamandra) which is severely threatened by the spread of Bsal. Based on counts from SEM pictures of selected skin parts, local densities of ~43,000 bacteria per mm² were ascertained in frogs, although the data are insufficient to understand whether these values apply to the whole body surface. Bacterial densities are doubtless much lower in fire salamanders in which we observed almost no bacteria in SEM examination. From qPCR data, we find: (i) statistically relevant differences in bacterial abundances among species, with the lowest abundances in terrestrial salamanders and the highest abundances in toads; (ii) higher bacterial loads in captive compared to wild fire salamanders, and much higher loads in Bsal-infected captive specimens, in agreement with the hypothesis that Bsal-infection leads to blooming of opportunistic bacteria that may cause secondary infection; (iii) an only weak decrease of bacterial load after repeated swabbing, in agreement with the hypothesis that skin swabs capture only a part of the bacteria of the swabbed skin surface. We discuss the multiple sources of uncertainty in absolute estimates of abundances of cutaneous bacteria and suggest further research to clarify and reduce

these uncertainties.

Temperature is one of the most prominent environmental factors influencing amphibian physiology and also an important life history trait for amphibian pathogens, such as the chytrid fungus Batrachochytrium dendrobatidis (Bd). This pathogen has been reported from wild amphibian communities on Madagascar, but it remains poorly known if field body temperatures (TB) of wild Malagasy amphibians are in a range suitable for infection by this fungus. Here, we evaluated field TB of 845 rainforest frogs from 38 species in two hyperdiverse amphibian communities in Madagascar during the warm-wet and cool-dry seasons, the first such comprehensive data set from the island. TB of Malagasy rainforest frogs differed between habitat types and thus appears to correlate with ecological niche. In particular, frogs measured in pond habitats had a higher TB across seasons than those sampled from streams, riparian zones, and terrestrial (including arboreal) habitats. Overall, TB of the 38 frog species ranged between 9.3 and 33.5 across habitats and diel cycle. This temperature range matches the optimal temperature range for Bd growth (17–23°C). We hypothesize that susceptibility to Bd might differ between frogs occupying different habitats. Pond-dwelling frogs may have more opportunities to clear Bd infection by choosing warm operative temperatures in sun-heated pond water. Our results highlight the need for more studies evaluating the seasonal fluctuations of field body temperatures in the studied amphibian species to contribute to a more reliable disease risk assessment.

Temperature-induced developmental plasticity could allow amphibian larvae to complete metamorphosis successfully despite new thermal challenges and increased desiccation risk due to climate change. Here we investigated how the capacity for temperature-induced developmental plasticity varies with latitude and whether population-specific biogeographic background accounts for the different degree of plastic responses to temperature. We carried out a combined analysis based on the data from 150 studies (93 articles) performed on 64 amphibian species. We collected empirical data for age and size at metamorphosis in amphibian larvae acclimated to different temperatures during development and found that all larvae from all populations in these studies revealed a change in metamorphic traits with a given change in temperature and thus, were able to exhibit temperature-induced developmental plasticity. Age at metamorphosis was more affected by temperature than size at metamorphosis. Age and size at the onset of metamorphosis were generally lowest at warmest temperatures during development. Furthermore, populations from tropical latitudes were less sensitive to a change in developmental temperature compared to populations from higher latitudes. Accordingly, we suggest tropical populations to be the most vulnerable to increasing temperatures during metamorphosis. Our analyses reveal biases with respect to taxonomy, biogeographic distribution of species, and study design. Data from tropical populations are underrepresented and thus, the capacity for developmental plasticity of the most threatened species probably remains poorly understood. Future studies should focus on under-represented regions, most threatened species, and include a broader range of temperatures during development in order to make robust projections on future sensitivity of populations to climate change.

Global changes in temperature, predator introductions, and pollution might challenge animals by altering food conditions. A fast-growing source of environmental pollution are microplastics. If ingested with the natural food source, microplastics act as artificial fibers that reduce food quality by decreasing nutrient and energy density with possible ramifications for growth and development. Animals might cope with altered food conditions with digestive plasticity. We examined experimentally whether larvae of the African clawed frog (Xenopus laevis) exhibit digestive morphology plasticity (i.e., gut length, mass, and diameter) in response to microplastics ingestion. As natural systems contain non-digestible particles similar in size and shape to microplastics, we included cellulose as a natural fiber control group. Gut length and mass increased in response to microplastics and cellulose ingestion indicating that both types of fibers induced digestive plasticity. Body mass and body condition were similar across experimental groups, indicating that larvae fully compensated for low nutrient and energy density by developing longer intestines. The ability of a species to respond plastically to environmental variation, as X. laevis responded, indicates that this species might have the potential to cope with new conditions during global change, although it is uncertain whether this potential may be reduced in a multi-stressor environment.

Phenotypic plasticity may allow ectotherms with complex life histories such as amphibians to cope with climate-driven changes in their environment. Plasticity in thermal tolerance (i.e., shifts of thermal limits via acclimation to higher temperatures) has been proposed as a mechanism to cope with warming and extreme thermal events. However, thermal tolerance and, hence, acclimation capacity, is known to vary with life stage. Using the common frog (Rana temporaria) as a model species, we measured the capacity to adjust lower (CT_min) and upper (CT_max) critical thermal limits at different acclimation temperatures. We calculated the acclimation response ratio as a metric to assess the stage-specific acclimation capacity at each of seven consecutive ontogenetic stages and tested whether acclimation capacity was influenced by body mass and/or age. We further examined how acclimation temperature, body mass, age, and ontogenetic stage influenced CT_min and CT_max. In the temperate population of R. temporaria that we studied, thermal tolerance and acclimation capacity were affected by ontogenetic stage. However, acclimation capacity at both thermal limits was well below 100% at all life stages tested. The lowest and highest acclimation capacity in thermal limits was observed in young and late larvae, respectively. The relatively low acclimation capacity of young larvae highlights a clear risk of amphibian populations to ongoing climate change. Ignoring stage-specific differences in thermal physiology may drastically underestimate the climate vulnerability of species which will hamper successful conservation actions.

Despite an enormous boost of digital teaching due to Covid-19-related restrictions, the amount of open-access online teaching resources in zoology is relatively scarce beyond rather generalized high-school-level materials. For our own teaching, Katharina Ruthsatz, Mark D. Scherz, and Miguel Vences have developed a season of 14 guided video courses in animal anatomy and morphology for a general zoology lab for undergraduates at Braunschweig University of Technology (in German language). We are also making these videos available in English via a dedicated YouTube channel called Anatomy Insights. The videos give a brief overview of the taxonomy, biology, and ecology of the respective taxa and provide a step-by-step/real-time manual for dissection. While there is no substitute for practical anatomical experience, and dissection videos should supplement and must not replace hands-on dissection in anatomy courses at university level, such globally available resources serve to democratize access to education opportunities in animal morphology and taxonomy, while reducing the associated environmental and ethical footprint.

Anurans exhibit plasticity in the timing of metamorphosis and tadpoles show phenotypic plasticity in age and size at metamorphosis as a response to temperature variation. This developmental plasticity to changing thermal conditions is expected to be a primary factor that dictates the vulnerability of amphibians to increasing ambient temperatures such as are predicted in climate change scenarios. We analyzed the patterns of thermal effects on size and age at metamorphosis to investigate whether the intraspecific “temperature-size rule” is applicable over a broad range of anuran species by carrying out a combined analysis based on the data from 25 studies performed on 18 anuran species. Furthermore, we tested whether the thermal background of respective populations impacts the capacity for a plastic response in metamorphic traits. We could confirm this pattern for across-population comparisons. All included populations developed faster and 75% were smaller at the onset of metamorphosis when developmental temperatures were warmer, but the sensitivity of growth and developmental rate to a given temperature change was different. We found that the thermal background of a population influences the sensitivity of metamorphic traits and thus, the capacity for a plastic response in growth and developmental rate. Warm adapted populations were less sensitive to temperature variation indicating a reduced capacity for developmental plasticity and therefore, those species may be more vulnerable to the impacts of climate change. Future studies should include a broader range of rearing temperatures and temperature fluctuations to determine full knowledge of the capacity for developmental plasticity within a species-specific thermal window.

Chemical, physical, and biological environmental stressors may affect the endocrine system such as the thyroid hormone axis in larval amphibians with consequences for energy partitioning among development, growth, and metabolism. We studied the effects of two thyroid hormone (TH) level affecting compounds, exogenous L-thyroxine (T4) and sodium perchlorate (SP), on various measures of development and body condition in larvae of the African clawed frog (Xenopus laevis). We calculated the scaled mass index, the hepatosomatic index, and the relative tail muscle mass as body condition indices to estimate fitness. Altered TH levels significantly altered the growth, development, survival, and body condition in metamorphic larvae in different directions. While exogenous T4 reduced growth and accelerated development, SP treatment increased growth but slowed down development. Altered TH levels improved body conditions in both treatments and especially in larvae of the SP treatment but to the detriment of lower survival rates in both TH level altering treatments. The hepatosomatic index was negatively affected by exogenous T4, but not by SP treatment indicating a lower lipid reserve in the liver in larvae of T4 treatment. These altered TH levels as caused by several environmental stressors may have influence on individual fitness across life since body condition at the onset of metamorphosis determines metamorphic and juvenile survival. Further research is needed to determine synergetic effects of environmental stressors on TH levels and its effects on physiological traits such as metabolic rate.

Environmental variation induced by natural and anthropogenic processes including climate change may threaten species by causing environmental stress. Anuran larvae experiencing environmental stress may display altered thyroid hormone (TH) status with potential implications for physiological traits. Therefore, any capacity to adapt to environmental changes through plastic responses provides a key to determine species vulnerability to environmental variation. We investigated whether developmental temperature (Tdev), altered TH levels, and the interactive effect of both affect standard metabolic rate (SMR), body condition (BC), survival, and thermal tolerance in larvae of the African clawed frog (Xenopus laevis) reared at five temperatures with experimentally altered TH levels. At metamorphosis, SMR, BC, and survival were significantly affected by Tdev, TH status and their interaction with the latter often intensifying impacts. Larvae developing at warmer temperatures exhibited significantly higher SMRs and BC was reduced at warm Tdev and high TH levels suggesting decreased ability to acclimate to variation in temperature. Accordingly, tadpoles that developed at warm temperatures had higher maximum thermal limits but more narrow thermal tolerance windows. High and low TH levels decreased and increased upper thermal limits, respectively. Thus, when experiencing both warmer temperatures and environmental stress, larvae may be less able to compensate for changes in Tdev. Our results demonstrate that physiological traits in larvae of X. laevis are strongly affected by increased TH levels and warmer temperatures. Altered TH levels and increasing Tdev due to global change may result in a reduced capacity for physiological plasticity. This has far reaching consequences since the energetic requirement at the onset of metamorphosis is known to determine metamorphic success and thus, is indirectly linked to individual fitness in later life stages.