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.

Traffic Noise Impacts Glucocorticoid Response, Activity, and Growth in Two Species of Tadpoles.

There is a large body of evidence linking increased noise to negative health effects for animals. Anthropogenic noise induces behavioral and physiological reactions across a range of taxa and increased traffic noise affects glucocorticoid (GC) hormones associated with the stress response in amphibians. GCs help to maintain homeostasis while balancing energetic trade-offs between reproduction, growth, and activity. Stressors during early development can impact fitness at later life stages. We measured growth, activity, and GCs in response to high levels of traffic noise in two tadpole species that differ in life history: Acris crepitans and Rana berlandieri. We predicted that earlier exposures to traffic noise will slow down the development and alter the behavior and GC concentrations differently than later exposures. Subjects were initially either exposed to natural levels of traffic noise for 8 days (early exposure) or a white noise control (later exposure), then the treatment was switched. Activity was measured via focal sampling and tadpoles were categorized as active if movement was detected. Tadpoles exposed to white noise initially maintained mass and activity throughout the experiment and early exposure to traffic noise had a greater impact on mass, activity, and GCs. Tadpoles exposed to traffic noise initially lost mass, with A. crepitans regaining mass but not R. berlandieri. When exposed earlier to traffic noise, R. berlandieri increased movement when shifted to the white noise treatment while A. crepitans did not significantly change activity. Acris creptians had higher corticosterone release rates compared to R. berlandieri, and in both species, release rates were higher for tadpoles exposed to noise earlier. The longer-lived R. berlandieri allocated more of their energetic resources into activity, while the shorter-lived A. crepitans allocated energy toward growth. Rana berlandieri and A. crepitans utilized different coping strategies to contend with early exposure to traffic noise, potentially due to differences in life histories. Our findings suggest that these tadpoles employ different coping mechanisms to modulate stress responses in noise-polluted environments, and these mechanisms could influence their fitness later in life. Further study is needed to understand the impact in more sensitive tadpole species.

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.

Influence of octylphenol on gene expression of gonadotropins and their receptors, testicular structure and mating behavior of male Rana chensinensis.

In the present study, responses of the Chinese brown frog (Rana chensinensis) to exposure to different doses and duration of Octyphenol (OP) which degraded from alkylphenol ethoxylates (APEs) were characterized during the adult periods. The effects of OP on growth, development and reproduction and the expression of genes in gonad were investigated. The expression levels of fshß, lhß, fshr and lhr had significant differences as the exposure time increased. The pathological and morphological changes were also observed in the OP treatments. Furthermore, the number of TUNEL positive cells and the TUNEL index was elevated after exposed to OP. Besides that, OP treatment could influence its mating behavior and reduce the fertilization rates. Taken together, these results indicated that OP disrupt sex steroid signaling, normal development of spermatogenesis, courtship behavior of male frogs and decline fertilization rate in R. chensinensis.

Fatter or stronger: Resource allocation strategy and the underlying metabolic mechanisms in amphibian tadpoles.

The allocation of resources between storage and somatic growth is an essential physiological phenomenon in animals. Allocation mechanisms have broad theoretical and applied implications. The real-time resource allocation patterns in animals remain to be elucidated, and there is limited understanding of the metabolic mechanisms. We investigated the resource allocation strategy of Rana omeimontis tadpoles. Their ontogenetic fat accumulation began when body weight increased to 30-50 mg, at which time storage had a high priority in resource allocation. Beyond this weight range, somatic growth accelerated but storage investment was maintained, resulting in a positive correlation between body fat index and body weight at the population level. This pattern could be explained by assuming a positive relationship between storage abundance and growth investment, and this was supported by the prioritized increment of body fat to body weight when tadpoles were provided with increased food. At the metabolic level, hepatic fat accumulation was accompanied by upregulated utilization of fat storage, and the tadpoles presented lipid-based energy metabolism. Activating the mobilization of hepatic fat storage promoted somatic growth. In short, the liver is like a reservoir with valves that regulate energy flow for downstream developmental processes. These results provide novel mechanistic insights into resource allocation.

Temperature-mediated trade-off between development and performance in larval wood frogs (Rana sylvatica).

Countergradient variation has been detected in diverse taxa. In a common manifestation, individuals from colder environments develop faster than conspecifics from warmer environments when placed in a common garden. Where such a pattern exists, it implies a trade-off: Individuals from warmer environments have intrinsic rates of development lower than those demonstrated by other individuals of the same species. We explored a trade-off between development rate and locomotor performance in the wood frog (Rana sylvatica), an amphibian for which countergradient variation has been well documented. We reared wood frogs from 10 populations under two temperature regimes, bracketing the temperatures observed in local natural ponds. Individuals reared under warmer conditions developed more rapidly but exhibited burst speeds 20% lower than individuals reared under colder conditions. The slope of the reaction norm was consistent across the 10 populations and thus, we found no evidence of countergradient variation in performance. Burst speed assays of wild-caught tadpoles from the same populations showed a similar but nonsignificant trend, with greater variability among ponds. Overall, our findings support the existence of a development-performance trade-off that may be of broad importance and which may help explain the widespread occurrence of countergradient variation.

Metabolic response of the Siberian wood frog Rana amurensis to extreme hypoxia.

The Siberian wood frog Rana amurensis is a recently discovered example of extreme hypoxia tolerance that is able to survive several months without oxygen. We studied metabolomic profiles of heart and liver of R. amurensis exposed to 17 days of extreme hypoxia. Without oxygen, the studied tissues experience considerable stress with a drastic decrease of ATP, phosphocreatine, and NAD+ concentrations, and concomitant increase of AMP, creatine, and NADH. Heart and liver switch to different pathways of glycolysis with differential accumulation of lactate, alanine, succinate, as well as 2,3-butanediol (previously not reported for vertebrates as an end product of glycolysis) and depletion of aspartate. We also observed statistically significant changes in concentrations of certain osmolytes and choline-related compounds. Low succinate/fumarate ratio and high glutathione levels indicate adaptations to reoxygenation stress. Our data suggest that maintenance of the ATP/ADP pool is not required for survival of R. amurensis, in contrast to anoxia-tolerant turtles.

A threshold dosage of estrogen for male-to-female sex reversal in the Glandirana rugosa frog.

Steroid hormones play very important roles in gonadal differentiation in many vertebrate species. Previously, we have determined a threshold dosage of testosterone (T) to induce female-to-male sex reversal in Glandirana rugosa frogs. Genetic females formed a mixture of testis and ovary, the so-called ovotestis, when tadpoles of G. rugosa were reared in water containing the dosage of T, which enabled us to detect primary changes in the histology of the masculinizing gonads. In this study, we determined a threshold dosage of estradiol-17ß (E2) to cause male-to-female sex reversal in this frog. We observed first signs of histological changes in the ovotestes, when tadpoles were reared in water containing the dosage of E2. Ovotestes were significantly larger than wild-type testes in size. By E2 treatment, male germ cells degenerated in the feminizing testis leading to their final disappearance. In parallel, oocytes appeared in the medulla of the ovotestis and later in the cortex as well. Quantitative polymerase chain reaction analysis revealed that the expression of sex-related genes involved in testis formation was significantly decreased in the ovotestis. In addition, immuno-positive signals of CYP17 that is involved in testis differentiation in this frog disappeared in the medulla first and then in the cortex. These results suggested that oocytes expanded in the feminizing gonad (ovary) contemporaneously with male germ cell disappearance. Primary changes in the histology of the gonads during male-to-female sex reversal occurred in the medulla and later in the cortex. This direction was opposite to that observed during female-to-male sex reversal in the G. rugosa frog.

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.

Host age alters amphibian susceptibility to Batrachochytrium dendrobatidis, an emerging infectious fungal pathogen.

Parasites and pathogens are often aggregated in a minority of susceptible hosts within a population, with a majority of individuals harboring low infection intensities. However, determining the relative importance of host traits to explain this heterogeneity is a challenge. One ecologically important pathogen is Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis and has been associated with many amphibian population declines worldwide. For many hosts, post-metamorphic stages are generally more susceptible than the larval stage. Yet, examination of the effects of Bd infection at different ages within a life stage, has received little attention. This study investigated the hypothesis that recently-post-metamorphic frogs were more sensitive to chytridiomycosis than older frogs, and that sensitivity to Bd infection decreased as frogs aged. We examined this relationship with Pacific treefrogs (Pseudacris regilla) and red legged frogs (Rana aurora). Age had a strong effect on susceptibility to infection, infection intensity, and survival-but not in the directions we had predicted. In both host species, an increase in age was associated with frogs becoming more susceptible to Bd infection, harboring larger infection intensities, and greater risk of mortality. This suggests that the timing of Bd exposure may influence amphibian population dynamics.

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.

Assessment of Sublethal Effects of Neonicotinoid Insecticides on the Life-History Traits of 2 Frog Species.

Neonicotinoid insecticides are used extensively in agriculture and, as a consequence, are now detectable in nearby aquatic environments. Few studies have evaluated the effects of neonicotinoids on amphibians in these aquatic environments. In the present study, we examined the effects of 2 commercial formulations of neonicotinoids (active ingredients clothianidin and thiamethoxam) on survival and life-history traits of wood frogs (Lithobates sylvaticus) and northern leopard frogs (Lithobates pipiens). We used artificial pond mesocosms to assess the effects of these neonicotinoids, at nominal concentrations of 2.5 and 250 µg/L, on amphibian larval development through metamorphosis. We found no differences between controls and neonicotinoid exposure for any of the endpoints assessed for either wood frogs or leopard frogs. The present study suggests that concentrations meeting or exceeding observed levels of clothianidin and thiamethoxam in surface waters will not directly affect metamorphosis in 2 amphibians. Environ Toxicol Chem 2019;38:1967-1977. © 2019 SETAC.

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.

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.

The effect of intensified illuminance and artificial light at night on fitness and susceptibility to abiotic and biotic stressors.

Changing light conditions due to human activities represents an important emerging environmental concern. Although changes to natural light conditions can be independently detrimental, in nature, organisms commonly face multiple stressors. To understand the consequences of altered light conditions, we exposed a model amphibian (wood frog; Lithobates sylvaticus) to a control and two anthropogenic light conditions: intensified daytime illuminance and artificial light at night - ALAN (intensified daytime illuminance + extended photoperiod). We measured (1) metrics of fitness (hatching success as well as survival to, size at, and time to metamorphosis) (2) susceptibility (time to death) to a commonly co-occurring anthropogenic stressor, road salt (NaCl) and (3) susceptibility (infection load) to a common parasite (trematode). We also explored behavioral (swimming activity) and physiological (baseline corticosterone (CORT) release rates) changes induced by these light conditions, which may mediate changes in the other measured parameters. We found that both intensified daytime illuminance and ALAN reduced hatching success. In contrast, for amphibians that successfully hatched, neither treatment affected amphibian survival or time to metamorphosis but individuals exposed to ALAN were larger at metamorphosis. The light treatments also had marginal effects; individuals in ALAN treatments were more susceptible to NaCl and trematodes. Finally, tadpoles exposed to ALAN moved significantly less than tadpoles in the control and intensified daytime illuminance treatments, while light had no effect on CORT release rate. Overall, changes in light conditions, in particular ALAN, significantly impacted an amphibian model in laboratory conditions. This work underscores the importance of considering not only the direct effects of light on fitness metrics but also the indirect effects of light with other abiotic and biotic stressors. Anthropogenic-induced changes to light conditions are expected to continue increasing over time so understanding the diverse consequences of shifting light conditions will be paramount to protecting wildlife populations.

Ontogenetic development of the oral apparatus and oropharyngeal cavity in bullfrog tadpoles (Lithobates catesbeianus, Shaw 1802).

OBJECTIVE: The present study aimed to describe the morphology of oral apparatus and oral cavity of bullfrog tadpoles during their development and metamorphosis. DESIGN: The oral apparatus and oropharyngeal cavity of tadpoles from hatching up to metamorphosis stage was dissected for further analysis. These structures were fixed in Karnovsky solution, afterwards in osmium tetroxide and metalized in palladium gold and electron-micrographed using the scanning electron microscope. RESULTS: The development of oral apparatus started with the formation and keratinization of the jaw sheaths and labial teeth followed by the formation of marginal and sub-marginal papillae. Degeneration of oral apparatus and formation of mouth was observed during metamorphosis. From stage-42 (metamorphic climax) to stage-43, the jaw sheath and labial tooth rows were disappeared progressively while the size and number of labial papillae were decreased. At stage-44, mouth formation started with the development of anterior and posterior labium though the labial papillae were still present. At stage-45 and 46, mouth was already formed, being very similar to the adult and characterized by the progressive increase in size. CONCLUSION: The sequence of events that happen during the development of oral apparatus of Lithobates catesbeianus Shaw, 1802 tadpoles follows the same pattern as occur in other anuran species but metamorphic atrophy of the oral apparatus follows the sequence of morphogenesis.

Experimental evidence for maternal provisioning of alkaloid defenses in a dendrobatid frog.

Dendrobatid frogs sequester alkaloid defenses from dietary arthropods. Here, we provide experimental evidence that mother strawberry poison frogs (Oophaga pumilio) provision alkaloids to tadpoles. Captive-raised females were fed the synthetic alkaloid decahydroquinoline (DHQ), which we subsequently quantified in their skin, eggs, and developing tadpoles. DHQ quantity was positively associated with tadpole mass/development, suggesting high sequestration rates by tadpoles. These data confirm that tadpoles obtain nutrition and alkaloids by feeding exclusively on maternally provisioned eggs.

Amphibian (Rana nigromaculata)exposed to cyproconazole: Changes in growth index, behavioral endpoints, antioxidant biomarkers, thyroid and gonad development.

Pesticides are a major cause of reduction in the global population of amphibians. This study investigates the effect of varying concentrations of cyproconazole (1 and 10 mg/L) on Rana nigromaculata during a chronic 90 days exposure period. High levels of cyproconazole (10 mg/L) induced declined body weight, short snout-vent length, slow metamorphic development and abnormal behavioral endpoints in R. nigromaculata tadpoles. Tadpoles exposed to 10 mg/L did not survive beyond 42 days. Abnormal behaviors were observed more frequently with exposure to the highest concentration of cyproconazole. Compared with controls, the concentrations of dismutase (SOD), catalase (CAT) and glutathione (GSH) were significantly increased in tadpoles exposed to 1 mg/L cyproconazole. However, when the concentration of cyproconazole increased to 10 mg/L, concentrations of SOD, GSH and CAT activity began to decline. In addition, thyroid and gonad development were also affected at the gene and hormone level, with varied effects observed with different exposure levels and days. Exposure to cyproconazole at the lower level of 1 mg/L induced damage to histological structures of the thyroid gland. Stereoselective tissue distribution and bioaccumulation of cyproconazole was observed in tadpoles. The ranked order of bioaccumulation was: enantiomer -4 > 3> 2 > 1, with the level of cyproconazole highest in the gut. These findings reflect the toxicity of cyproconazole to R. nigromaculata and further our understanding of the effects of pesticide exposure on global amphibian population declines.

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.

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.