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Many organisms have evolved to produce different phenotypes in response to environmental variation. Dendropsophus ebraccatus tadpoles develop opposing shifts in morphology and coloration when they are exposed to invertebrate vs vertebrate predators. Each of these alternate phenotypes are adaptive, conferring a survival advantage against the predator with which tadpoles were reared but imposing a survival cost with the mismatched predator. Here, we measured the phenotypic response of tadpoles to graded cues and mixed cues of both fish and dragonfly nymphs. Prey species like D . ebraccatus commonly co-occur with both of these types of predators, amongst many others as well. In our first experiment, tadpoles increased investment in defensive phenotypes in response to increasing concentrations of predator cues. Whereas morphology only differed in the strongest predation cue, tail spot coloration differed even at the lowest cue concentration. In our second experiment, tadpoles reared with cues from both predators developed an intermediate yet skewed phenotype that was most similar to the fish-induced phenotype. Previous studies have shown that fish are more lethal than dragonfly larvae; thus tadpoles responded most strongly to the more dangerous predator, even though the number of prey consumed by each predator was the same. This may be due to D . ebraccatus having evolved a stronger response to fish or because fish produce more kairomones than do dragonflies for a given amount of food. We demonstrate that not only do tadpoles assess predation risk via the concentration of predation cues in the water, they produce a stronger response to a more lethal predator even when the strength of cues is presumed to be identical.

Background Animals in captivity are inherently separated from their natural environments, which both exposes them to new heterospecific organisms as well as reduces contact with naturally occurring predators, prey or microbiota. The microbes that live on and in animals are increasingly recognized as having important impacts on animal health, development and behavior. We raised post-metamorphic treefrogs in 1) naturalistic containers in groups, 2) regularly sterilized containers in groups, or 3) regularly sterilized containers but solitary. Froglets were raised for over eight months; in addition to monitoring growth and development, we collected fecal samples on three occasions, gut samples on two occasions, and skin swab samples once. We compared the diversity of microbial communities across sample types and over time. Results Froglets raised in group housing, either naturalistic or regularly cleaned, had the fastest growth and sexual differentiation, but naturalistic housing also improved survival. Alpha diversity of bacteria on the skin or in the gut did not vary with rearing conditions, whereas diversity in the gut increased over time. Alpha diversity of feces did vary with rearing treatment and changed over time. Bacterial community composition (beta diversity) varied most strongly with sample type, but also with rearing conditions and over time. In addition, bacterial communities of feces were highly correlated with those of guts, indicating that feces can serve as an accurate and non-invasive biomarker of the gut microbiome. Lastly, transferring frogs from regularly sterilized environments to naturalistic vivaria improved bacterial community diversity. Conclusions Our study suggests that naturalistic housing improves the overall health and development of captive amphibians and that these improvements may occur by facilitating a more stable and diverse microbiome.

Environmentally cued plasticity in hatching timing is widespread in animals. As with later life-history switch points, plasticity in hatching timing may have carryover effects that affect subsequent interactions with predators and competitors. Moreover, the strength of such effects of hatching plasticity may be context dependent. We used red-eyed treefrogs, Agalychnis callidryas , to test for lasting effects of hatching timing (four or six days post-oviposition) under factorial combinations of resource levels (high or low) and predation risk (none, caged, or lethal Pantala flavescens dragonfly naiads). Tadpoles were raised in 400-L mesocosms in Gamboa, Panama, from hatching until all animals had metamorphosed or died, allowing assessment of effects across a nearly six-month period of metamorphosis. Hatching early reduced survival to metamorphosis, increased larval growth, and had context-dependent effects on metamorph phenotypes. Early during the period of metamorph emergence, early-hatched animals were larger than late-hatched ones, but this effect attenuated over time. Early-hatched animals also left the water with relatively longer tails. Lethal predators dramatically reduced survival to metamorphosis, with most mortality occurring early in the larval period. Predator effects on the timing of metamorphosis and metamorph size and tail length depended upon resources. For example, lethal predators reduced larval periods, and this effect was stronger with low resources. Predators affected metamorph size early in the period of metamorphosis, whereas resource levels were a stronger determinant of phenotype for animals that metamorphosed later. Effects of hatching timing were detectable on top of strong effects of larval predators and resources, across two subsequent life stages, and some were as strong as or stronger than effects of resources. Plasticity in hatching timing is ecologically important and currently underappreciated. Effects on metamorph numbers and phenotypes may impact subsequent interactions with predators, competitors, and mates, with potentially cascading effects on recruitment and fitness.

Many animals respond to predation risk by altering their morphology, behavior, or life-history. We know a great deal about the cues prey respond to and the changes to prey that can be induced by predation risk, but less is known about how plastic responses to predators may be affected by separate plastic responses occurring earlier in life, particularly during the embryonic period. Embryos of a broad array of taxa can respond to egg- or larval-stage risks by altering hatching timing, which may alter the way organisms respond to future predators. Using the red-eyed treefrog (Agalychnis callidryas), a model for understanding the effects of plasticity across life-stages, we assessed how the combined effects of induced variation in the timing of embryo hatching and variation in the larval predator community impacted tadpole morphology, pigmentation and swimming performance. We found that A. callidryas tadpoles developed deeper tail muscles and fins and darker pigmentation in response to fish predators, either when alone or in diverse community with other predators. Tadpoles altered morphology much less so to dragonfly naiads or water bugs. Interestingly, morphological responses to predators were also affected by induced differences in hatching age, with early and late-hatched tadpoles exhibiting different allometric relationships between tail height and body length in different predator environments. Beyond induced morphological changes, fish predators often damaged tadpoles' tails without killing them (i.e., sublethal predation), but these tadpoles swam equally quickly to those with fully intact tails. This was due to the fact that tadpoles with more damaged tails increased tail beats to achieve equal swimming speed. This study demonstrates that plastic phenotypic responses to predation risk can be influenced by a complex combination of responses to both the embryo and larval environments, but also that prey performance can be highly resilient to sublethal predation.

Vector control strategies are among the most effective measures to combat mosquito-borne diseases, such as malaria. These strategies work by altering the mosquito age structure through increased mortality of the older female mosquitoes that transmit pathogens. However, methods to monitor changes to mosquito age structure are currently inadequate for programmatic implementation. Female mosquitoes generally mate a single time soon after emergence and draw down spermatozoa reserves with each oviposition cycle. Here, we demonstrate that measuring spermatozoa quantity in female Anopheles mosquitoes is an effective approach to assess mosquito age. Using multiplexed qPCR targeted at male spermatozoa, we show that Y-linked genes in female mosquitoes are exclusively found in the spermatheca, the organ that houses spermatozoa, and the quantity of these gene sequences significantly declines with age. The method can accurately identify mosquitoes more than 10 days old and thus old enough to potentially transmit pathogens harbored in the salivary glands during blood feeding. Furthermore, mosquito populations that differ by 10% in daily survivorship have a high likelihood of being distinguished using modest sample sizes, making this approach scalable for assessing the efficacy of vector intervention control programs.

To effectively balance investment in predator defenses versus other traits, organisms must accurately assess predation risk. Chemical cues caused by predation events are indicators of risk for prey in a wide variety of systems, but the relationship between how prey perceive risk in relation to the amount of prey consumed by predators is poorly understood. While per capita predation rate is often used as the metric of relative risk, studies aimed at quantifying predator-induced defenses commonly control biomass of prey consumed as the metric of risk. However, biomass consumed can change by altering either the number or size of prey consumed. In this study we determine whether phenotypic plasticity to predator chemical cues depends upon prey biomass consumed, prey number consumed, or both. We examine the growth response of red-eyed treefrog tadpoles (Agalychnis callidryas) to cues from a larval dragonfly (Anax amazili). Biomass consumed was manipulated by either increasing the number of prey while holding individual prey size constant, or by holding the number of prey constant and varying individual prey size. We address two questions. (i) Do prey reduce growth rate in response to chemical cues in a dose dependent manner? (ii) Does the magnitude of the response depend on whether prey consumption increases via number or size of prey? We find that the phenotypic response of prey is an asymptotic function of prey biomass consumed. However, the asymptotic response is higher when more prey are consumed. Our findings have important implications for evaluating past studies and how future experiments should be designed. A stronger response to predation cues generated by more individual prey deaths is consistent with models that predict prey sensitivity to per capita risk, providing a more direct link between empirical and theoretical studies which are often focused on changes in population sizes not individual biomass.

In response to environmental stressors, organisms often demonstrate flexible responses in morphology, life history or behaviour. However, it is currently unclear if such plastic responses are coordinated or operate independently of one another. In vertebrates, this may partly result from studies examining population- or species-level mean responses, as opposed to finer grained analyses of individuals or families. We measured predator-specific morphological and coloration plasticity in 42 families of tadpoles of the treefrog Dendropsophus ebraccatus and behavioural plasticity from 18 of these families, allowing us to examine the correlation between three predator-induced plastic responses. For all three plastic responses, tadpoles showed strong opposing responses to each of two predators, providing the appearance of covariation in plasticity. However, the examination of individual families revealed a strong correlation between morphological and coloration plasticity, but no correlations between either morphology or colour and behavioural plasticity. Thus, our analysis shows that some aspects of the plastic phenotype develop together while others function independently. This highlights the importance of examining individual- and family-level variation for understanding the adaptive significance of developmental plasticity, which is crucial for a holistic appreciation of phenotypic plasticity and its importance in ecology and evolution.

Laying eggs out of water was crucial to the transition to land and has evolved repeatedly in multiple animal phyla. However, testing hypotheses about this transition has been difficult because extant species only breed in one environment. The pantless treefrog, Dendropsophus ebraccatus, makes such tests possible because they lay both aquatic and arboreal eggs. Here, we test the oviposition site choices of D. ebraccatus under conflicting risks of arboreal egg desiccation and aquatic egg predation, thereby estimating the relative importance of each selective agent on reproduction. We also measured discrimination between habitats with and without predators and development of naturally laid aquatic and arboreal eggs. Aquatic embryos in nature developed faster than arboreal embryos, implying no cost to aquatic egg laying. In choice tests, D. ebraccatus avoided habitats with fish, showing that they can detect aquatic egg predators. Most importantly, D. ebraccatus laid most eggs in the water when faced with only desiccation risk, but switched to laying eggs arboreally when desiccation risk and aquatic predators were both present. This provides the first experimental evidence to our knowledge that aquatic predation risk influences non-aquatic oviposition and strongly supports the hypothesis that it was a driver of the evolution of terrestrial reproduction.

Like many animals, tadpoles often produce different, predator-specific phenotypes when exposed to risk of predation. It is generally assumed that such plasticity enhances survival in the presence of the predator and is costly elsewhere, but evidence remains surprisingly scarce. We measured (1) the survival trade-off of opposing phenotypes developed by Dendropsophus ebraccatus tadpoles when exposed to different predators and (2) which specific aspects of morphology drive any potential survival benefit or cost. Tadpoles developed predator-specific phenotypes after being reared with caged fish or dragonfly predators for two weeks. In 24 h predation trials with either a fish or a dragonfly, survival was highest in the groups with their matched predator, and lowest among with those the mismatched predator, with predator-naive controls being relatively intermediate. Then, using a large group of phenotypically variable predator-naive tadpoles, we found that increased survival rates are directly related to the morphological changes that are induced by each predator. This demonstrates that induced phenotypes are indeed adaptive and the product of natural selection. Furthermore, our data provide clear evidence of an environmental cost for phenotypic plasticity in a heterogeneous environment. Such costs are fundamental for understanding the evolution and maintenance of inducible phenotypes.

Nonaquatic reproduction has evolved repeatedly, but the factors that select for laying eggs on land are not well understood. The treefrogDendropsophus ebraccatushas plasticity in its reproductive mode, laying eggs that successfully develop in or out of water. This permits the first experimental comparison of the selective agents that shape adult oviposition behavior and embryo developmental capacity. I quantified the sources and strengths of arboreal and aquatic egg mortality and how mortality varies with weather patterns, and I assessed 39 years of daily rainfall patterns to infer historic levels of egg mortality and effects of climate change on the selective balance between aquatic and nonaquatic egg deposition. Aquatic predators and desiccation were the strongest selective agents in water and air, respectively. Egg mortality varied with weather such that aquatic oviposition was advantageous when rainfall was low but laying eggs out of water increased survival when rainfall was high. Additionally, I found that since 1972 there have been significant changes in the rainfall patterns in central Panama, and this has altered the selective landscape acting on egg-laying behavior. This work provides insight into the evolution and maintenance of adaptive phenotypic plasticity as well as historic and current selection on reproduction.