Temperature and nutrient conditions modify the effects of phenological shifts in predator-prey communities.

Although there is mounting evidence indicating that the relative timing of predator and prey phenologies determines the outcome of trophic interactions, we still lack a comprehensive understanding of how the environmental context (e.g., abiotic conditions) influences this relationship. Environmental conditions not only frequently drive shifts in phenologies, but they can also affect the very same processes that mediate the effects of phenological shifts on species interactions. Therefore, identifying how environmental conditions shape the effects of phenological shifts is key to predicting community dynamics across a heterogeneous landscape and how they will change with ongoing climate change in the future. Here I tested how environmental conditions shape the effects of phenological shifts by experimentally manipulating temperature, nutrient availability, and relative phenologies in two predator-prey freshwater systems (mole salamander-bronze frog vs. dragonfly larvae-leopard frog). This allowed me to (1) isolate the effects of phenological shifts and different environmental conditions; (2) determine how they interact; and (3) evaluate how consistent these patterns are across different species and environments. I found that delaying prey arrival dramatically increased predation rates, but these effects were contingent on environmental conditions and the predator system. Although nutrient addition and warming both significantly enhanced the effect of arrival time, their effect was qualitatively different across systems: Nutrient addition enhanced the positive effect of early arrival in the dragonfly-leopard frog system, whereas warming enhanced the negative effect of arriving late in the salamander-bronze frog system. Predator responses varied qualitatively across predator-prey systems. Only in the system with a strong gape limitation were predators (salamanders) significantly affected by prey arrival time and this effect varied with environmental context. Correlations between predator and prey demographic rates suggest that this was driven by shifts in initial predator-prey size ratios and a positive feedback between size-specific predation rates and predator growth rates. These results highlight the importance of accounting for temporal and spatial correlations of local environmental conditions and gape limitation when predicting the effects of phenological shifts and climate change on predator-prey systems.

Celebrating 50+ years of research on the reproductive biology and endocrinology of the green frog: An overview.

This issue is dedicated to the late Professor Giovanni Chieffi, and this article is an overview of the research on Comparative Endocrinology of reproduction using Rana esculenta (alias Pelophylax esculentus) as a model system. Starting from the early 1970s till today, a large quantity of work have been conducted both in the fields of experimental endocrinology and in the definition of the diffuse neuroendocrine system, with a major focus on the increasing role of regulatory peptides. The various aspects investigated concerned the histological descriptions of principal endocrine glands of the hypothalamic-pituitary-gonadal (HPG) axis, the localization and distribution in the HPG of several different substances (i.e. neurosteroids, hypothalamic peptide hormones, pituitary gonadotropins, gonadal sex steroids, and other molecules), the determination of sex hormone concentrations in both serum and tissues, the hormone manipulations, as well as the gene and protein expression of steroidogenic enzymes and their respective receptors. All together these researches, often conducted considering different periods of the annual reproductive cycle of the green frog, allowed to understand the mechanism of cascade control/regulation of the HPG axis of R. esculenta, characterizing the role of different hormones in the two sexes, and testing the hypotheses about the function of single hormones in different target organs. It becomes evident from the review that, in their simplest form, several features of this species are specular as compared to those of other vertebrate species and that reproduction in this frog species is either under endogenous multi-hormonal control or by a wide array of different factors. Our excursus of this research, spanning almost five decades, shows that R. esculenta has been intensively and successfully used as an animal model in reproductive endocrinology as well as several field studies such as those involving environmental concerns that focus on the effects of endocrine disruptors and other environmental contaminants.

Influence of exposure to pesticide mixtures on the metabolomic profile in post-metamorphic green frogs (Lithobates clamitans).

Pesticide use in agricultural areas requires the application of numerous chemicals to control target organisms, leaving non-target organisms at risk. The present study evaluates the hepatic metabolomic profile of one group of non-target organisms, amphibians, after exposure to a single pesticide and pesticide mixtures. Five common-use pesticide active ingredients were used in this study, three herbicides (atrazine, metolachlor and 2,4-d), one insecticide (malathion) and one fungicide (propiconazole). Juvenile green frogs (Lithobates clamitans) were reared for 60-90days post-metamorphosis then exposed to a single pesticide or a combination of pesticides at the labeled application rate on soil. Amphibian livers were excised for metabolomic analysis and pesticides were quantified for whole body homogenates. Based on the current study, metabolomic profiling of livers support both individual and interactive effects where pesticide exposures altered biochemical processes, potentially indicating a different response between active ingredients in pesticide mixtures, among these non-target species. Amphibian metabolomic response is likely dependent on the pesticides present in each mixture and their ability to perturb biochemical networks, thereby confounding efforts with risk assessment.

Nonlinear processing of a multicomponent communication signal by combination-sensitive neurons in the anuran inferior colliculus.

Diverse animals communicate using multicomponent signals. How a receiver's central nervous system integrates multiple signal components remains largely unknown. We investigated how female green treefrogs (Hyla cinerea) integrate the multiple spectral components present in male advertisement calls. Typical calls have a bimodal spectrum consisting of formant-like low-frequency (~0.9 kHz) and high-frequency (~2.7 kHz) components that are transduced by different sensory organs in the inner ear. In behavioral experiments, only bimodal calls reliably elicited phonotaxis in no-choice tests, and they were selectively chosen over unimodal calls in two-alternative choice tests. Single neurons in the inferior colliculus of awake, passively listening subjects were classified as combination-insensitive units (27.9%) or combination-sensitive units (72.1%) based on patterns of relative responses to the same bimodal and unimodal calls. Combination-insensitive units responded similarly to the bimodal call and one or both unimodal calls. In contrast, combination-sensitive units exhibited both linear responses (i.e., linear summation) and, more commonly, nonlinear responses (e.g., facilitation, compressive summation, or suppression) to the spectral combination in the bimodal call. These results are consistent with the hypothesis that nonlinearities play potentially critical roles in spectral integration and in the neural processing of multicomponent communication signals.

Thermal Acclimatization in Overwintering Tadpoles of the Green Frog, Lithobates clamitans (Latreille, 1801).

Seasonal acclimatization permits organisms to maintain function in the face of environmental change. Tadpoles of the green frog (Lithobates clamitans) overwinter as tadpoles in much of their range. Because they are active in winter, we hypothesized that green frog tadpoles would display acclimatization of metabolic and locomotor function. We collected tadpoles in Sewanee, Tennessee (35.2°N) in winter and summer. Tadpoles collected during each season were tested at both winter (8°C) and summer (26°C) temperatures. Winter tadpoles were able to maintain swimming performance at both temperatures, whereas swimming performance decreased at cold temperatures in summer tadpoles. There was no evidence for seasonal acclimatization of whole-animal metabolic rate. Although whole-animal metabolic acclimatization was not observed, the activities of cytochrome c oxidase, citrate synthase, and lactate dehydrogenase measured in skeletal muscle homogenates showed higher activity in winter-acclimatized tadpoles indicating compensation for temperature. Further, the composition of muscle membranes of winter tadpoles had less saturated and more monounsaturated fatty acids and a higher ω-3 balance, unsaturation index, and peroxidation index than summer tadpoles. These data indicate that reversible phenotypic plasticity of thermal physiology occurs in larval green frog tadpoles. They appear to compensate for colder temperatures to maintain burst-swimming velocity and the ability to escape predators without the cost of maintaining a constant, higher standard metabolic rate in the winter.

Spatial arrangement of prey affects the shape of ratio-dependent functional response in strongly antagonistic predators.

Predators play a key role in shaping natural ecosystems, and understanding the factors that influence a predator's kill rate is central to predicting predator-prey dynamics. While prey density has a well-established effect on predation, it is increasingly apparent that predator density also can critically influence predator kill rates. The effects of both prey and predator density on the functional response will, however, be determined in part by their distribution on the landscape. To examine this complex relationship we experimentally manipulated prey density, predator density, and prey distribution using a tadpole (prey)-dragonfly nymph (predator) system. Predation was strongly ratio-dependent irrespective of prey distribution, but the shape of the functional response changed from hyperbolic to sigmoidal when prey were clumped in space. This sigmoidal functional response reflected a relatively strong negative effect of predator interference on kill rates at low prey: predator ratios when prey were clumped. Prey aggregation also appeared to promote stabilizing density-dependent intraguild predation in our system. We conclude that systems with highly antagonistic predators and patchily distributed prey are more likely to experience stable dynamics, and that our understanding of the functional response will be improved by research that examines directly the mechanisms generating interference.

Disease dynamics of red-spotted newts and their anuran prey in a montane pond community.

Long-term monitoring of amphibians is needed to clarify population-level effects of ranaviruses (Rv) and the fungal pathogen Batrachochytrium dendrobatidis (Bd). We investigated disease dynamics of co-occurring amphibian species and potential demographic consequences of Rv and Bd infections at a montane site in the Southern Appalachians, Georgia, USA. Our 3-yr study was unique in combining disease surveillance with intensive population monitoring at a site where both pathogens are present. We detected sub-clinical Bd infections in larval and adult red-spotted newts Notophthalmus viridescens viridescens, but found no effect of Bd on body condition of adult newts. Bd infections also occurred in larvae of 5 anuran species that bred in our fishless study pond, and we detected co-infections with Bd and Rv in adult newts and larval green frogs Lithobates clamitans. However, all mortality and clinical signs in adult newts and larval anurans were most consistent with ranaviral disease, including a die-off of larval wood frogs Lithobates sylvaticus in small fish ponds located near our main study pond. During 2 yr of drift fence monitoring, we documented high juvenile production in newts, green frogs and American bullfrogs L. catesbeianus, but saw no evidence of juvenile recruitment in wood frogs. Larvae of this susceptible species may have suffered high mortality in the presence of both Rv and predators. Our findings were generally consistent with results of Rv-exposure experiments and support the purported role of red-spotted newts, green frogs, and American bullfrogs as common reservoirs for Bd and/or Rv in permanent and semi-permanent wetlands.

Effect of temporal and spectral noise features on gap detection behavior by calling green treefrogs.

Communication plays a central role in the behavioral ecology of many animals, yet the background noise generated by large breeding aggregations may impair effective communication. A common behavioral strategy to ameliorate noise interference is gap detection, where signalers display primarily during lulls in the background noise. When attempting gap detection, signalers have to deal with the fact that the spacing and duration of silent gaps is often unpredictable, and that noise varies in its spectral composition and may thus vary in the degree in which it impacts communication. I conducted playback experiments to examine how male treefrogs deal with the problem that refraining from calling while waiting for a gap to appear limits a male's ability to attract females, yet producing calls during noise also interferes with effective sexual communication. I found that the temporal structure of noise (i.e., duration of noise and silent gap segments) had a stronger effect on male calling behavior than the spectral composition. Males placed calls predominantly during silent gaps and avoided call production during short, but not long, noise segments. This suggests that male treefrogs use a calling strategy that maximizes the production of calls without interference, yet allows for calling to persist if lulls in the background noise are infrequent.

The response of amphibian larvae to exposure to a glyphosate-based herbicide (Roundup WeatherMax) and nutrient enrichment in an ecosystem experiment.

Herbicides and fertilizers are widely used throughout the world and pose a threat to aquatic ecosystems. Using a replicated, whole ecosystem experiment in which 24 small wetlands were split in half with an impermeable barrier we tested whether exposure to a glyphosate-based herbicide, Roundup WeatherMax™, alone or in combination with nutrient enrichment has an effect on the survival, growth or development of amphibians. The herbicide was applied at one of two concentrations (low=210 µg a.e./L, high=2880 µg a.e./L) alone and in combination with nutrient enrichment to one side of wetlands and the other was left as an untreated control. Each treatment was replicated with six wetlands, and the experiment was repeated over two years. In the high glyphosate and nutrient enrichment treatment the survival of wood frog (Lithobates sylvaticus) larvae was lower in enclosures placed in situ on the treated sides than the control sides of wetlands. However, these results were not replicated in the second year of study and they were not observed in free swimming wood frog larvae in the wetlands. In all treatments, wood frog larvae on the treated sides of wetlands were slightly larger (<10%) than those on the control side, but no effect on development was observed. The most dramatic finding was that the abundance of green frog larvae (Lithobates clamitans) was higher on the treated sides than the control sides of wetlands in the herbicide and nutrient treatments during the second year of the study. The results observed in this field study indicate that caution is necessary when extrapolating results from artificial systems to predict effects in natural systems. In this experiment, the lack of toxicity to amphibian larvae was probably due to the fact the pH of the wetlands was relatively low and the presence of sediments and organic surfaces which would have mitigated the exposure duration.

Characterization of gene expression endpoints during postembryonic development of the northern green frog (Rana clamitans melanota).

Postembryonic development of a larval tadpole into a juvenile frog involves the coordinated action of thyroid hormone (TH) across a diversity of tissues. Changes in the frog transcriptome represent a highly sensitive endpoint in the detection of developmental progression, and for the identification of environmental chemical contaminants that possess endocrine disruptive properties. Unfortunately, in contrast with their vital role as sentinels of environmental change, few gene expression tools currently exist for the majority of native North American frog species. We have isolated seven expressed gene sequences from the Northern green frog (Rana clamitans melanota) that encode proteins associated with TH-mediated postembryonic development and global stress response, and established a quantitative real-time polymerase chain reaction (qPCR) assay. We also obtained three additional species-specific gene sequences that functioned in the normalization of the expression data. Alterations in mRNA abundance profiles were identified in up to eight tissues during R. clamitans postembryonic development, and following exogenous administration of TH to premetamorphic tadpoles. Our results characterize tissue distribution and sensitivity to TH of select mRNA of a common North American frog species and support the potential use of this qPCR assay in identification of the presence of chemical agents in aquatic environments that modulate TH action.

Adaptive forgetting in Iberian green frog tadpoles (Pelophylax perezi): learned irrelevance and latent inhibition may avoid predator misidentification.

Predator recognition often requires learning by prey individuals. Iberian green frog tadpoles (Pelophylax perezi) have the ability to learn to recognize new potential predators when their chemical cues are found paired with conspecific alarm cues. However, a random pairing of alarm cues and chemical stimuli of a nonpredator might later induce costly antipredator responses to nondangerous species. Here, we studied the potential existence in this frog species of two phenomena (learned irrelevance and latent inhibition) that could help tadpoles to avoid these nonadaptive responses to chemical cues of nonpredator species. Our results showed that, when tadpoles experienced a random pattern of presence of alarm cues alone or predator cues alone over the 4 days before or after the simultaneous detection of these two cues paired, no learned association was formed. These results showed the existence of an effect of learned irrelevance on learning in Iberian green frog tadpoles. Also, tadpoles clearly inhibited the formation of a learning association between predator and alarm cues after a 4-day period during which they had been exposed to the predator cues alone. This result showed the existence of an effect of latent inhibition on learning about cues related to increased predation risk. Thus, both learned irrelevance and latent inhibition, rather than being considered to be failed predator recognition, can rather be seen as adaptive ways for dealing with conflicting information and as strategies to avoid learning irrelevant information and costly antipredatory responses to nonpredatory stimuli.

Synergistic effects of predators and trematode parasites on larval green frog (Rana clamitans) survival.

Parasites and predators can have complex, nonadditive effects on a shared group of victims, which can have important consequences for population dynamics. In particular, parasites can alter host traits that influence predation risk, and predators can have nonconsumptive effects on prey traits which influence susceptibility (i.e., infection intensity and tolerance) to parasites. Here, we examined the combined effects of trematode parasites (Digenea: Echinostomatidae) and odonate (Anax) predators on the survival of larval green frogs (Rana clamitans). First, in a large-scale mesocosm experiment, we manipulated the presence or absence of parasites in combination with the presence of no predator, caged predators, or free predators, and measured survival, traits, and infection. Parasites, caged predators, and free predators decreased survival, and we found a strong negative synergistic effect of parasites in combination with free predators on survival. Importantly, we then examined the potential mechanisms that explain the observed synergistic effect of parasites and predators in a series of follow-up experiments. Results of the follow-up experiments suggest that increased predation susceptibility due to elevated activity levels in the presence of free-swimming parasite infective stages (i.e., an avoidance response) is the most likely mechanism responsible for the observed synergism. These results suggest a potential trade-off in susceptibility to parasites and predators, which can drive nonadditive effects that may have important consequences for natural enemy interactions in natural populations and amphibian conservation.