The genomics of mimicry: Gene expression throughout development provides insights into convergent and divergent phenotypes in a Müllerian mimicry system.

A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which species share a conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which mimicry is produced in poison frogs. We assembled a 6.02-Gbp genome with a contig N50 of 310 Kbp, a scaffold N50 of 390 Kbp and 85% of expected tetrapod genes. We leveraged this genome to conduct gene expression analyses throughout development of four colour morphs of Ranitomeya imitator and two colour morphs from both R. fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes differentially expressed throughout development, many of them related to melanophores/melanin, iridophore development and guanine synthesis. We also identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species, and identify several plausible candidates for colouration in vertebrates (e.g. cd36, ep-cadherin and perlwapin). Finally, we hypothesise that keratin genes (e.g. krt8) are important for producing different structural colours within these frogs.

Invading new climates at what cost? Ontogenetic differences in the thermal dependence of metabolic rate in an invasive amphibian.

Global warming can either promote or constrain the invasive potential of alien species. In ectotherm invaders that exhibit a complex life cycle, success is inherently dependent on the capacity of each developmental stage to cope with environmental change. This is particularly relevant for invasive anurans, which disperse on land while requiring water for reproduction. However, it remains unknown how the different life stages respond in terms of energy expenditure under different climate change scenarios. We here quantified the oxygen uptake of frogs at rest (a proxy of the standard metabolic rate) in the aquatic phase (at the tadpole and climax, i.e. during metamorphosis, stages) and in the terrestrial phase (metamorphosed stage) at three environmental temperatures. To do so, we used marsh frogs (Pelophylax ridibundus), an amphibian with the largest invasive range within the palearctic realm and for which their adaptation to global warming might be key to their invasion success. Beyond an increase of metabolic rate with temperature, our data show variation in thermal adaptation across life stages and a higher metabolic cost during metamorphosis. These results suggest that the cost to shift habitat and face changes in temperature may be a constraint on the invasive potential of species with a complex life cycle which may be particularly vulnerable during metamorphosis.

A Phylogenetically Distinct Group of Glandirana rugosa Found in Kyushu, Japan.

The Japanese wrinkled frog Glandirana rugosa is separated into five genetically different groups. One group in western Japan is further divided into three subgroups, found in Kyushu, Shikoku, and western Honshu. We collected G. rugosa frogs at 39 sites in Kyushu and determined nucleotide sequences of the mitochondrial 12S and 16S rRNA genes for phylogenetic analysis. Unexpectedly, we found a group of frogs in southeastern Kyushu that did not cluster with any of the pre-existing five groups of G. rugosa on the phylogenetic trees. The frogs in the new group and G. rugosa in Kyushu were externally similar, but there were a few significant differences in morphological features between the two populations. In addition, we observed significant differences in the frogs' calls . Thus, the group of the frogs in southeastern Kyushu may represent a new candidate species in the genus Glandirana. We discuss the possibility of a new species.

Experimental Evidence That Metamorphosis Alleviates Genomic Conflict.

Whenever genetically correlated traits experience antagonistic selection, an adaptive response in one trait can lead to a maladaptive response in the correlated trait. This is a form of genome-level conflict that can have important evolutionary consequences by impeding organisms from reaching their adaptive optima. Antagonistic selection should be pervasive in organisms with complex life histories because larval and adult life stages specialize in dramatically different environments. Since individuals express larval and adult morphologies from a single genome, genomic conflict across ontogenetic stages should also be prevalent. Using wood frogs as a study system, we measured natural selection on larval and postmetamorphic life stages and estimated genetic correlations among traits. Alternative life stages experienced a mix of both antagonistic and congruent viability selection. The integration between traits changed over the course of metamorphosis, reducing genetic correlations that cause conflict. Our results provide novel experimental evidence that metamorphosis can alleviate genomic conflict by partitioning life-history stages into modules that can more readily respond to selection. These results highlight the adaptive potential of metamorphosis as a means to avoid ecological specialization trade-offs. Moreover, they provide insights into the prevalence and evolutionary maintenance of complex life cycles.

Sperm is epigenetically programmed to regulate gene transcription in embryos.

For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.

Genetic basis of amphibian larval development along a latitudinal gradient: Gene diversity, selection and links with phenotypic variation in transcription factor C/EBP-1.

Ectotherm development rates often show adaptive divergence along climatic gradients, but the genetic basis for this variation is rarely studied. Here, we investigated the genetic basis for phenotypic variation in larval development in the moor frog Rana arvalis from five regions along a latitudinal gradient from Germany to northern Sweden. We focused on the C/EBP-1 gene, a transcription factor associated with larval development time. Allele frequencies at C/EBP-1 varied strongly among geographical regions. Overall, the distribution of alleles along the gradient was in concordance with the dual post-glacial colonization routes into Scandinavia, with a large number of alleles exclusively present along the southern colonization route. Only three of 38 alleles were shared between the routes. Analysis of contemporary selection on C/EBP-1 showed divergent selection among the regions, probably reflecting adaptation to the local environmental conditions, although this was especially strong between southern and northern regions coinciding also with lineages from different colonization routes. Overall, the C/EBP-1 gene has historically been under purifying selection, but two specific amino acid positions showed significant signals of positive selection. These positions showed divergence between southern and northern regions, and we suggest that they are functionally involved in the climatic adaptation of larval development. Using phenotypic data from a common garden experiment, we found evidence for specific C/EBP-1 alleles being correlated with larval development time, suggesting a functional role in adaptation of larval development to large-scale climatic variation.

Effects of dietary antioxidants and environmental stressors on immune function and condition in Lithobates (Rana) sylvaticus.

The negative effects of abiotic and biotic stressors on animal condition, physiology, behaviour, and fitness have become increasingly recognized, making it critical to understand how these may be mitigated, such as through diet. When consumed, dietary antioxidants (secondary metabolites produced by plants) provide protection from oxidative damage that can result from exposure to stressors. We examined the effects of dietary antioxidants (ß-carotene and vitamin E) on the ability of Lithobates sylvaticus (wood frog) tadpoles to overcome the detrimental effects imposed by two common environmental stressors - exposure to the herbicide atrazine or infection by a pathogenic helminth parasite (Echinostoma trivolvis). Tadpoles which were fed high concentrations of either vitamin E or ß-carotene had a greater immune response compared to those given trace diets of either compound as measured by circulating white blood cells and a phytohemaglutination assay (PHA). Similarly, tadpoles in the herbicide and parasitism treatments that were given trace antioxidant diets had significant weight loss, whereas those consuming high levels of vitamin E or ß-carotene did not. Our results suggest that dietary antioxidants, specifically ß-carotene and vitamin E, have a positive effect on tadpole immune systems and overall condition that likely allows them to better cope with natural stressors, with potential implications for their foraging behaviour.

Bioaccumulation of methylmercury in wood frogs and spotted salamanders in Vermont vernal pools.

Mercury (Hg) has accumulated in forested landscapes in the Northeastern U.S., and hotspots with enhanced deposition have been identified throughout the region. Due to a variety of favorable landscape characteristics, including relatively high dissolved organic carbon (DOC), fluctuating water levels, and low pH and dissolved oxygen, vernal pools provide ideal conditions for the conversion of Hg to its more toxic and bioavailable form, methylmercury (MeHg). Yet little is known about the concentrations, speciation, and bioavailability of Hg in vernal pools, or its bioaccumulation in vernal pool fauna and potential export into terrestrial systems. We investigated the role of forest cover type on the bioaccumulation of MeHg in wood frog (Lithobates sylvatica) and spotted salamander (Ambystoma maculatum) eggs, larvae, and adults, and investigated relationships among MeHg and water chemistry (pH, DOC). Water samples from pools located in coniferous stands had greater concentrations of THg and MeHg compared to deciduous pool water, and showed significant positive correlation to DOC (r = 0.683, P < 0.001) and correlated negatively with pH (r = -0.613, P < 0.001). Methylmercury levels in amphibian embryos were similar between the two species (L. sylvatica mean = 5.4 ng/g dw; A. maculatum mean = 3.5 ng/g dw). Concentrations of MeHg increased substantially in larvae, and were significantly greater in A. maculatum (mean = 237.6 ng/g ± 18.5 SE) than L. sylvatica larvae (62.5 ng/g ± 5.7 SE). Forest cover type did not explain variation in MeHg concentration among amphibian embryos or larvae. Methylmercury levels in adult tissue samples were significantly greater in A. maculatum (mean = 79.9 ng/g ± 8.9 SE) compared to L. sylvatica (mean = 47.7 ng/g ± 9.7 SE). This research demonstrates that vernal pools are important hotspots where amphibians bioaccumulate MeHg, which may then be transferred to terrestrial ecosystems. The abundance of amphibian larvae suggests they could be important bioindicators for monitoring MeHg loading and bioavailability.

Effects of low doses Trichlorfon exposure on Rana chensinensis tadpoles.

Trichlorfon is an organophosphate insecticide widely used in aquaculture and agriculture. Little is known about the effects of long-term of low doses trichlorfon exposure on amphibians. In this study, we investigated the effects of low doses trichlorfon on Rana chensinensis tadpoles after exposure to 0.01, 0.1, and 1.0 mg/L trichlorfon for 2 and 4 weeks. Survival, growth, development and mortality were monitored regularly over the course of exposure. The results showed that trichlorfon led to a decrease in tadpole survival. Reductions in growth and disruptions to the development of tadpoles were observed in trichlorfon treatments. Morphological abnormalities of affected tadpoles included axial flexures, skeletal malformations and lateral kinks. Trichlorfon increased the frequency of micronucleus (MN) formation in circulating erythrocytes of tadpoles exposed for 2 weeks to 0.1 and 1.0 mg/L trichlorfon. At all concentrations, an enhanced frequency of MN formation was observed in tadpoles exposed for 4 weeks. Exposure to trichlorfon induced other nuclear abnormalities such as lobed and notched nuclei only in tadpoles exposed to 1.0 mg/L trichlorfon for 4 weeks. In addition, exposure to trichlorfon within the 0.01-1.0 mg/L range increased the genetic damage index in hepatic tissues in all treatments. Apoptosis-associated DNA fragmentation in hepatic tissues occurred in a weak ladder-like pattern. This study presents evidence of low doses trichlorfon effects on amphibians, highlighting the properties of this organophosphate insecticide that jeopardize nontarget species exposed to trichlorfon.

Interacting effects of predation risk and resource level on escape speed of amphibian larvae along a latitudinal gradient.

Fast-growing genotypes living in time-constrained environments are often more prone to predation, suggesting that growth-predation risk trade-offs are important factors maintaining variation in growth along climatic gradients. However, the mechanisms underlying how fast growth increases predation-mediated mortality are not well understood. Here, we investigated if slow-growing, low-latitude individuals have faster escape swimming speed than fast-growing high-latitude individuals using common frog (Rana temporaria) tadpoles from eight populations collected along a 1500 km latitudinal gradient. We measured escape speed in terms of burst and endurance speeds in tadpoles raised in the laboratory at two food levels and in the presence and absence of a predator (Aeshna dragonfly larvae). We did not find any latitudinal trend in escape speed performance. In low food treatments, burst speed was higher in tadpoles reared with predators but did not differ between high-food treatments. Endurance speed, on the contrary, was lower in high-food tadpoles reared with predators and did not differ between treatments at low food levels. Tadpoles reared with predators showed inducible morphology (increased relative body size and tail depth), which had positive effects on speed endurance at low but not at high food levels. Burst speed was positively affected by tail length and tail muscle size in the absence of predators. Our results suggest that escape speed does not trade-off with fast growth along the latitudinal gradient in R. temporaria tadpoles. Instead, escape speed is a plastic trait and strongly influenced by the interaction between resource level and predation risk.

Critical disease windows shaped by stress exposure alter allocation trade-offs between development and immunity.

Ubiquitous environmental stressors are often thought to alter animal susceptibility to pathogens and contribute to disease emergence. However, duration of exposure to a stressor is likely critical, because while chronic stress is often immunosuppressive, acute stress can temporarily enhance immune function. Furthermore, host susceptibility to stress and disease often varies with ontogeny; increasing during critical developmental windows. How the duration and timing of exposure to stressors interact to shape critical windows and influence disease processes is not well tested. We used ranavirus and larval amphibians as a model system to investigate how physiological stress and pathogenic infection shape development and disease dynamics in vertebrates. Based on a resource allocation model, we designed experiments to test how exposure to stressors may induce resource trade-offs that shape critical windows and disease processes because the neuroendocrine stress axis coordinates developmental remodelling, immune function and energy allocation in larval amphibians. We used wood frog larvae (Lithobates sylvaticus) to investigate how chronic and acute exposure to corticosterone, the dominant amphibian glucocorticoid hormone, mediates development and immune function via splenocyte immunohistochemistry analysis in association with ranavirus infection. Corticosterone treatments affected immune function, as both chronic and acute exposure suppressed splenocyte proliferation, although viral replication rate increased only in the chronic corticosterone treatment. Time to metamorphosis and survival depended on both corticosterone treatment and infection status. In the control and chronic corticosterone treatments, ranavirus infection decreased survival and delayed metamorphosis, although chronic corticosterone exposure accelerated rate of metamorphosis in uninfected larvae. Acute corticosterone exposure accelerated metamorphosis increased survival in infected larvae. Interactions between stress exposure (via glucocorticoid actions) and infection impose resource trade-offs that shape optimal allocation between development and somatic function. As a result, critical disease windows are likely shaped by stress exposure because any conditions that induce changes in differentiation rates will alter the duration and susceptibility of organisms to stressors or disease.

Comparison of triadimefon and its metabolite on acute toxicity and chronic effects during the early development of Rana nigromaculata tadpoles.

Pesticides are one of major causes for amphibian population declines and the behavior of pesticide metabolite products to amphibians has become a rising concern. In this study, the acute toxicity and the chronic effects of triadimefon and triadimenol (the metabolite of triadimefon) on Rana. nigromaculata were investigated. In the acute assay, significant differences were observed in antioxidant enzyme activities and malondialdehyde levels between the triadimefon and triadimenol. The 96 h-acute toxicity of triadimefon (25.97 mg/L) and triadimenol (34.55 mg/L) to tadpoles was low. In 28d-chronic exposure, we studied the relative expression of tadpoles genes related to thyroid hormone-dependent metamorphic development, histological examination of liver and some biological index, including wet weight, snout-to-vent length (SVL) and development stages. The results revealed that the effects of triadimefon and triadimenol on tadpole development are driven by a disruption of the hormonal pathways involved in metamorphosis. Interestingly, triadimefon was more harmful on R. nigromaculata than triadimenol at high dose, whereas the reverse result was observed at low doses. According to the relative expression of thyroid hormone-dependent genes, we also found that the two compounds may have different mechanisms of toxic action on R. nigromaculata. Our study developed a pragmatic approach for use in the risk assessment of pesticide and its metabolite，and increased the information and understanding of the impacts of fungicides and other potential endocrine disrupting environmental contaminants on amphibians.

Influence of DEHP on thyroid, sex steroid-related genes and gonadal differentiation in Rana chensinensis tadpoles.

In the present study, responses of the Chinese brown frog (Rana chensinensis) to exposure to di-2-ethylhexyl phthalate (DEHP), a common plasticizer, during the larval period were characterized. The effects of DEHP on metamorphosis rate, thyroid hormone, thyroid histology and the expression of genes involved in the steroid hormone synthesis in gonad were investigated. Metamorphosis rate and 50 percent of the tadpoles to reach Gosner stage 42 (T0.5 ) were significantly slower in all DEHP groups. The thyroid glands of the tadpoles exposed to DEHP clearly exhibited colloid depletion. In addition, decreased concentrations of T4 and T3 were observed in the tadpoles exposed to DEHP. Moreover, the highest DEHP exposure (10 µmol/L DEHP) showed increased ratio of females significantly. Also, up-regulation significantly of transcripts of cytochrome P450 aromatase (CYP19) gene was detected in male tadpoles exposed to DEHP. The present results indicate that this increase in estrogens could lead to female-biased sex ratio in DEHP exposure group. Taken together, the present study indicates that DEHP disrupt thyroid hormone and sex steroid signaling in R. chensinensis tadpoles. Our present observations support evidence of a crosstalk between TH and sex steroids in gonad differentiation.

Exogenous stress hormones alter energetic and nutrient costs of development and metamorphosis.

Variation in environmental conditions during larval life stages can shape development during critical windows and have lasting effects on the adult organism. Changes in larval developmental rates in response to environmental conditions, for example, can trade off with growth to determine body size and condition at metamorphosis, which can affect adult survival and fecundity. However, it is unclear how use of energy and nutrients shape trade-offs across life-stage transitions because no studies have quantified these costs of larval development and metamorphosis. We used an experimental approach to manipulate physiological stress in larval amphibians, along with respirometry and 13C-breath testing to quantify the energetic and nutritional costs of development and metamorphosis. Central to larval developmental responses to environmental conditions is the hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis, which regulates development, as well as energy homeostasis and stress responses across many taxa. Given these pleiotropic effects of HPA/I activity, manipulation of the HPA/I axis may provide insight into costs of metamorphosis. We measured the energetic and nutritional costs across the entire larval period and metamorphosis in a larval amphibian exposed to exogenous glucocorticoid (GC) hormones - the primary hormone secreted by the HPA/I axis. We measured metabolic rates and dry mass across larval ontogeny, and quantified lipid stores and nutrient oxidation via 13C-breath testing during metamorphosis, under control and GC-exposed conditions. Changes in dry mass match metamorphic states previously reported in the literature, but dynamics of metabolism were influenced by the transition from aquatic to terrestrial respiration. GC-treated larvae had lower dry mass, decreased fat stores and higher oxygen consumption during stages where controls were conserving energy. GC-treated larvae also oxidized greater amounts of 13C-labelled protein stores. These results provide evidence for a proximate cause of the physiological trade-off between larval growth and development, and provide insight into the energetic and nutrient costs that shape fitness trade-offs across life stages.

Intraspecific priority effects modify compensatory responses to changes in hatching phenology in an amphibian.

In seasonal environments, modifications in the phenology of life-history events can alter the strength of time constraints experienced by organisms. Offspring can compensate for a change in timing of hatching by modifying their growth and development trajectories. However, intra- and interspecific interactions may affect these compensatory responses, in particular if differences in phenology between cohorts lead to significant priority effects (i.e. the competitive advantage that early-hatching individuals have over late-hatching ones). Here, we conducted a factorial experiment to determine whether intraspecific priority effects can alter compensatory phenotypic responses to hatching delay in a synchronic breeder by rearing moor frog (Rana arvalis) tadpoles in different combinations of phenological delay and food abundance. Tadpoles compensated for the hatching delay by speeding up their development, but only when reared in groups of individuals with identical hatching phenology. In mixed phenology groups, strong competitive effects by non-delayed tadpoles prevented the compensatory responses and delayed larvae metamorphosed later than in single phenology treatments. Non-delayed individuals gained advantage from developing with delayed larvae by increasing their developmental and growth rates as compared to single phenology groups. Food shortage prolonged larval period and reduced mass at metamorphosis in all treatments, but it did not prevent compensatory developmental responses in larvae reared in single phenology groups. This study demonstrates that strong intraspecific priority effects can constrain the compensatory growth and developmental responses to phenological change, and that priority effects can be an important factor explaining the maintenance of synchronic life histories (i.e. explosive breeding) in seasonal environments.

Effects of Echinostoma trivolvis metacercariae infection during development and metamorphosis of the wood frog (Lithobates sylvaticus).

Many organisms face energetic trade-offs between defense against parasites and other host processes that may determine overall consequences of infection. These trade-offs may be particularly evident during unfavorable environmental conditions or energetically demanding life history stages. Amphibian metamorphosis, an ecologically important developmental period, is associated with drastic morphological and physiological changes and substantial energetic costs. Effects of the trematode parasite Echinostoma trivolvis have been documented during early amphibian development, but effects during later development and metamorphosis are largely unknown. Using a laboratory experiment, we examined the energetic costs of late development and metamorphosis coupled with E. trivolvis infection in wood frogs, Lithobates [=Rana] sylvaticus. Echinostoma infection intensity did not differ between tadpoles examined prior to and after completing metamorphosis, suggesting that metacercariae were retained through metamorphosis. Infection with E. trivolvis contributed to a slower growth rate and longer development period prior to the initiation of metamorphosis. In contrast, E. trivolvis infection did not affect energy expenditure during late development or metamorphosis. Possible explanations for these results include the presence of parasites not interfering with pronephros degradation during metamorphosis or the mesonephros compensating for any parasite damage. Overall, the energetic costs of metamorphosis for wood frogs were comparable to other species with similar life history traits, but differed from a species with a much shorter duration of metamorphic climax. Our findings contribute to understanding the possible role of energetic trade-offs between parasite defense and host processes by considering parasite infection with simultaneous energetic demands during a sensitive period of development.

Effects of α-cypermethrin enantiomers on the growth, biochemical parameters and bioaccumulation in Rana nigromaculata tadpoles of the anuran amphibians.

Populations of many amphibian species are declining worldwide in part because of pesticide contamination. As a surface water contaminant, α-cypermethrin may have severe ecological impacts on amphibians. Here, we examined the acute toxicity of α-cypermethrin enantiomers to dark-spotted frog Rana nigromaculata tadpoles at 24, 48, 72 and 96h, finding that the tadpoles were indeed sensitive to α-cypermethrin. The (S)-(1R, 3R)-enantiomer was approximately 29 times more toxic than the (R)-(1S, 3S)-enantiomer at 96h. A significant delayed growth in R. nigromaculata tadpoles after exposure to 0.5µgL-1 of S-(1R, 3R)-cypermethrin was observed. Additionally, increased superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and malondialdehyde (MDA) levels indicate the presence of oxidative stress in the tadpoles. Further, tadpoles exposed to sublethal concentrations of α-cypermethrin enantiomers exhibited enantioselective growth and oxidative damage. Bioaccumulation experiments showed that the tadpoles could rapidly accumulate α-cypermethrin. The (R)-(1S, 3S)-enantiomer was preferentially accumulated over the (S)-(1R, 3R)-enantiomer, and it was also eliminated more quickly, as evidenced in the subsequent depuration experiments.

Tissue-specific transcriptome characterization for developing tadpoles of the northern leopard frog (Lithobates pipiens).

A potential cause of amphibian population declines are the impacts of environmental degradation on tadpole development. We conducted RNA sequencing on developing northern leopard frog tadpoles and through de novo transcriptome assembly we annotated a large number of open reading frames comparable in number and extent to genes identified in Xenopus. Using our transcriptome, we found transcript level changes between early (Gosner 26-31) and late (Gosner 36-41) stage tadpoles were the greatest in the tail, which is reabsorbed throughout development. There was an up-regulation of immunity genes in both the head and tail of the late tadpoles and a down-regulation of genes associated with the energy pathways of the mitochondria and the production of myosin. Overall, transcript level changes across development were consistent with studies on Xenopus and our findings highlight the broader utility of using RNA-seq to identify genes differentially expressed throughout development and in response to environmental pressures.

Analysis of fatty acid composition revealed differences in the diets of tadpoles of two amphibian species.

The study of the composition of fatty acid markers of tadpoles of cohabiting amphibian species for the first time revealed differences in their diets: the moor frog Rana arvalis prefers bacteria not associated with plant detritus, whereas the diet of the common spadefoot Pelobates fuscus is based on cyanobacteria, green algae, diatoms, and possibly higher plants. Major differences in the fatty acid composition are determined by the difference in the percentage of eicosapentaenoic and myristic acids.

Stoichiometric implications of a biphasic life cycle.

Animals mediate flows of elements and energy in ecosystems through processes such as nutrient sequestration in body tissues, and mineralization through excretion. For taxa with biphasic life cycles, the dramatic shifts in anatomy and physiology that occur during ontogeny are expected to be accompanied by changes in body and excreta stoichiometry, but remain little-explored, especially in vertebrates. Here we tested stoichiometric hypotheses related to the bodies and excreta of the wood frog (Lithobates sylvaticus) across life stages and during larval development. Per-capita rates of nitrogen (N) and phosphorus (P) excretion varied widely during larval ontogeny, followed unimodal patterns, and peaked midway through development (Taylor-Kollros stages XV and XII, respectively). Larval mass did not increase steadily during development but peaked at stage XVII and declined until the termination of the experiment at stage XXII. Mass-specific N and P excretion rates of the larvae decreased exponentially during development. When coupled with population-biomass estimates, population-level excretion rates were greatest at stages VIII-X. Percent carbon (C), N, and C:N of body tissue showed weak trends across major life stages; body P and C:P, however, increased sixfold during development from egg to adult. Our results demonstrate that intraspecific ontogenic changes in nutrient contents of excretion and body tissues can be significant, and that N and P are not always excreted proportionally throughout life cycles. These results highlight the dynamic roles that species play in ecosystems, and how the morphological and physiological changes that accompany ontogeny can influence ecosystem-level processes.