Responses Of Tadpoles Research Articles

Immunological and physiological responses to predation risk and sublethal concentrations of chlorothalonil and β-endosulfan in Lithobates taylori (Anura: Ranidae) tadpoles

Pesticide exposure and its interaction with other natural stressors can play a role in amphibian population declines because disruptions in stress hormone regulatory mechanisms may inhibit immune responses during metamorphosis. Here, we determined the interactive effects of predation risk and sublethal concentration of two pesticides on immunological and physiological responses in tadpoles of the tropical frog Lithobates taylori. Using mesocosms, we used chronic exposure to three levels of chlorothalonil and β-endosulfan in the presence or absence of Odonate larvae. Our results show that β-endosulfan in high concentrations reduced the weight of the tadpoles and increased the neutrophil count and corticosterone (CORT) levels. Larval development was accelerated by high concentrations of chlorothalonil. Also, this pesticide in low and high concentrations increases the absolute values of lymphocytes. Tadpoles exposed to chlorothalonil increased the numbers of monocytes (in low concentrations), and lymphocytes (in high and low concentrations). The interactions of the low concentrations of both pesticides with and without the predator's presence also increased the number of lymphocytes. A combination of pesticides increases the number of lymphocytes in the blood due to synergistic cytotoxicity. This study proves that β- endosulfan elevates circulating CORT and thus generates physiological stress in tadpoles. Given that both pesticides are widely used within the distribution of L. taylori in Costa Rica, it is likely that tadpoles’ development and immune function are altered by pesticide use. In combination with stressors such as emerging diseases and altered precipitation regimes, widespread agrochemical uses likely caused this species enigmatic decline in recent decades.

Behavioral responses of benthic and nektonic tadpoles to the presence of a benthic predator

Behavioral responses of benthic and nektonic tadpoles to the presence of a benthic predator

Integrated biomarker-based ecological risks assessment of tadpole responses to tris(2-chloroethyl) phosphate, tris(1-chloro-2-propyl) phosphate, and their combined environmental exposure

Tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP) are common chlorinated organophosphorus flame retardants (OPFRs) used in industry. They have been frequently detected together in aquatic environments and associated with various hazardous effects. However, the ecological risks of prolonged exposure to these OPFRs at environmentally relevant concentrations in non-model aquatic organisms remain unexplored. This study investigated the effects of long-term exposure (up to 25 days) to TCEP and TCPP on metamorphosis, hepatic antioxidants, and endocrine function in Polypedates megacephalus tadpoles. Exposure concentrations were set at 3, 30, and 90 μg/L for each substance, conducted independently and in equal-concentration combinations, with a control group included for comparison. The integrated biomarker response (IBR) method developed an optimal linear model for predicting the overall ecological risks of TCEP and TCPP to tadpoles in potential distribution areas of Polypedates species. Results showed that: (1) Exposure to environmentally relevant concentrations of TCEP and TCPP elicited variable adverse effects on tadpole metamorphosis time, hepatic antioxidant enzyme activity and related gene expression, and endocrine-related gene expression, with their combined exposure exacerbating these effects. (2) The IBR value of TCEP was consistently greater than that of TCPP at each concentration, with an additive effect observed under their combined exposure. (3) The ecological risk of tadpoles exposed to the combined presence of TCEP and TCPP was highest in China's Taihu Lake and Vietnam's Hanoi than in other distribution locations. In summary, prolonged exposure to environmentally relevant concentrations of TCEP and TCPP presents potential ecological risks to amphibian tadpoles, offering insights for the development of policies and strategies to control TCEP and TCPP pollution in aquatic ecosystems. Furthermore, the methodology employed in establishing the IBR prediction model provides a methodological framework for assessing the overall ecological risks of multiple OPFRs.

Assessing the impact of antiviral drugs commonly utilized during the COVID-19 pandemic on the embryonic development of Xenopus laevis

The antiviral drugs favipiravir and oseltamivir are widely used to treat viral infections, including coronavirus 2019 (COVID-19), and their levels are expected to increase in the aquatic environment. In this study, the potential toxic and teratogenic effects of these drugs were evaluated using the frog embryo teratogenesis assay Xenopus (FETAX). In addition, glutathione S-transferase (GST), glutathione reductase (GR), catalase, carboxylesterase (CaE), and acetylcholinesterase (AChE) enzyme activities and malondialdehyde levels were measured as biochemical markers in embryos and tadpoles for comparative assessment of the sublethal effects of the test compounds. Prior to embryo exposure, drug concentrations in the exposure medium were measured with high-performance liquid chromatography. The 96-h median lethal concentration (LC50) was 137.9 and 32.3 mg/L for favipiravir and oseltamivir, respectively. The teratogenic index for favipiravir was 4.67. Both favipiravir and oseltamivir inhibited GR, CaE, and AChE activities in embryos, while favipiravir increased the GST and CaE activities in tadpoles. In conclusion, favipiravir, for which teratogenicity data are available in mammalian test organisms and human teratogenicity is controversial, inhibited Xenopus laevis embryo development and was teratogenic. In addition, sublethal concentrations of both drugs altered the biochemical responses in embryos and tadpoles, with differences between the developmental stages.

Functional traits drive tadpole responses to land use in coastal areas

Abstract Land use changes are considered a significant cause of amphibian declines worldwide. However, little is known regarding which land uses have more pervasive impacts and which help to supplement the biodiversity conserved in protected areas. In addition, amphibian species respond to a broad array of geographical constraints, which may blur responses to disturbance, depending on the species' natural history. Here, we explored whether functional diversity patterns of amphibian assemblages are linked to human land use while considering spatial autocorrelation and phylogenetic relatedness among species. We surveyed tadpoles along 500 km in coastal wetlands in southern Brazil and quantified four traits related to tadpole habitat use and predator avoidance. Spatial components influenced functional richness to a greater extent than land use. Functional evenness was higher in ponds surrounded by more exotic reforestation, and to a lesser extent an effect of crop area. Functional divergence was also associated with reforestation areas. Our results showed that some land uses (reforestation with exotic species and temporary crops) have more pervasive impacts on tadpole communities. Such changes in amphibian communities occurred even at low levels of landscape change. Our findings highlight that conversion to human land use requires complementary approaches (i.e., tadpole and adult responses) to provide early warning signals about habitat modification effects.

Limited contribution of photoenzymatic DNA repair in mitigating carry-over effects from larval UVB exposure: Implications for frog recruitment

Solar ultraviolet-B (UVB) radiation has increased due to stratospheric ozone depletion, climate and ecosystem changes and is a driver of amphibian population declines. Photoenzymatic repair (PER) is a critical mechanism for limiting UVB lethality in amphibian larvae. However, the link between PER and the UVB-induced effects remains understudied through long-term investigations in vivo. Here, we assessed how larval PER determines the lethal and sublethal effects induced by environmentally relevant acute UVB exposure until the juvenile phase in the Neotropical frog Odontophrynus americanus. We conducted laboratory-based controlled experiments in which tadpoles were or were not exposed to UVB and subsequently were exposed to light (for PER activation) or dark treatments. Results showed that the rates of mortality and apoptosis observed in post-UVB dark treatment are effectively limited in post-UVB light treatment, indicating PER (and not dark repair, i.e. nucleotide excision repair) is critical to limit the immediate genotoxic impact of UVB-induced pyrimidine dimers. Nonetheless, even tadpoles that survived UVB exposure using PER showed sublethal complications that extended to the juvenile phase. Tadpole responses included alterations in morphology, chromosomal instability, increased skin susceptibility to fungal proliferation, as well as increased generation of reactive oxygen species. The short-term effects were carried over to later stages of life because metamorphosis time increased and juveniles were smaller. No body abnormalities were visualized in tadpoles, metamorphs, and juveniles, suggesting that O. americanus is UVB-resistant concerning these responses. This study reveals that even frog species equipped with an effective PER are not immune to carry-over effects from early UVB exposure, which are of great ecological relevance as late metamorphosis and smaller juveniles may impact individual performance and adult recruitment to breeding. Future ecological risk assessments and conservation and management efforts for amphibian species should exercise caution when linking PER effectiveness to UVB resistance.

Perception of con- and heterospecific injury cues in tadpoles of dendrobatid, ranid and bufonid frogs (Anura)

Predator-prey interactions are vital for organismal survival. They shape anti-predator mechanisms and often depend on sensory abilities. Tadpoles use chemical cues, such as injury cues (alarm cues), to assess predation risks and modify their life-history, morphology, and behaviours accordingly. However, the prevalence of chemically mediated anti-predator responses in species with distinct ecological niches (e.g. within phytotelmata) remains unknown, hindering our understanding of the ecological significance and evolution of alarm substances. Therefore, our study aimed to investigate chemically mediated anti-predator responses in tadpoles of two Neotropical poison dart frogs, Ranitomeya sirensis and Epipedobates anthonyi (and compare their responses to two Palearctic model organisms, Rana temporaria and Bufo bufo, which are known to utilise alarm substances). Through behavioural bioassays, we exposed predator-naïve tadpoles to extracts of each species (i.e. con- and heterospecific cues), including water as a control (i.e. five treatments per species). We assessed changes in their activity before and after stimulus introduction. Our results show that E. anthonyi did not respond to any of the stimuli, whereas R. sirensis displayed increased activity levels exclusively in response to conspecific cues, but not to heterospecific cues. With this, our findings suggest a specialized recognition system in R. sirensis, potentially directed at conspecific competitors but likely unrelated to anti-predator mechanisms. In contrast, E. anthonyi may be insensitive to injury cues or utilize alternative sensory modalities to respond to acute predation events. This study sheds light on the chemical alarm response system of Neotropical poison dart frog tadpoles, providing foundational understanding of how dendrobatids react to injury cues. It prompts questions about the ecological significance and evolutionary implications of chemical communication in species facing extreme resource limitation during development and underscores the importance of comparative research for understanding chemical communication in diverse aquatic ecosystems.

Behavioural response of Dryophytes plicatus tadpoles to the cues of non-native rainbow trout and a native snake

Abstract Non-native species have numerous and significant, often negative, effects on amphibians, but that threat may be ameliorated if the native species is able to respond behaviourally to the non-native predators. We experimentally compared the behavioural response of tadpoles of the Ridged Tree Frog, Dryophytes plicatus, to cues from non-native Rainbow Trout, Oncorhynchus mykiss, to those to a native predator, the Short-tail Alpine Garter Snake, Thamnophis scaliger. Dryophytes plicatus tadpoles did not alter activity in response to cues from O. mykiss but increased activity in the presence of cues from T. scaliger, although it is possible this increase is related, at least in part, to the order of the presentation of the cues. The activity of D. plicatus tadpoles was higher in the absence of the vegetation than in its presence when exposed to predator cues, both T. scaliger and O. mykiss, but not in the control. In conclusion, our results show D. plicatus tadpoles alter their overall activity when exposed to cues from a native predator, T. scaliger, but not the non-native O. mykiss. These results may explain, in part, why D. plicatus can co-occur with T. scaliger but not O. mykiss.

Widespread learned predator recognition to an alien predator across populations in an amphibian species

Alien predators are a major cause of decline and extinction of species worldwide, since native organisms are rarely equipped with specific antipredatory strategies to cope with them. However, phenotypic plasticity and learned predator recognition may help prey populations to survive novel predators. Here we examine geographical variation in the learning ability of larval spadefoot toads (Pelobates cultripes) to recognize invasive predatory crayfish (Procambarus clarkii). We compare the learning-mediated behavioural responses of tadpoles from six populations across two regions in Spain (central and southern), with different histories of exposure to the presence of the invasive species. Two of the populations showed innate recognition of chemical cues from the invasive crayfish, whereas three of them learned to recognize such cues as a threat after conditioning with conspecific alarm cues. Learning abilities did not differ among southern populations, but they did among central populations. We assessed patterns of genetic variation within and among these two regions through microsatellite markers and found low genetic divergence among the southern populations but greater differentiation among the central ones. We hypothesize that similar responses to the invasive crayfish in southern populations may have arisen from a combination of extended historical exposure to this introduced predator (~ 50 y) and higher levels of gene flow, as they inhabit a highly interconnected pond network. In contrast, populations from central Spain show lower connectivity, have been exposed to the invasive crayfish for a shorter period of time, and are more divergent in their plastic responses.

Behavioral responses of laboratory-reared and wild-caught Polypedates maculatus (Anura: Rhacophoridae) tadpoles to dietary cues from the carnivorous tadpoles of Hoplobatrachus tigerinus (Anura: Dicroglossidae)


 
 
 
 The behavioral responses of laboratory-reared and wild-caught tadpoles of Polypedates maculatus to predatory tadpoles of Hoplobatrachus tigerinus were studied in the laboratory. The predator’s diet-derived metabolites released in excreta of predator after consumption of P. maculatus tadpoles were used to simulate predation threat. Both laboratory-reared and wild-caught tadpoles of P. maculatus showed antipredator behavioral responses i.e., reduced swimming movements and overall time spent in swimming and had a higher burst speed in response to water borne dietary cues of predators. Further, the antipredator responses of wild-caught tadpoles were significantly higher than those exhibited by laboratory-reared tadpoles. The studythus shows that antipredator behavior in these tadpoles is innate. Further, an enhanced antipredator behavior of wild-caught tadpoles may suggest their prior experience with predators in natural waters.
 
 
 

Behavioral responses of tadpoles of Clinotarsus curtipes (Anura: Ranidae) to odor cues of dragonfly larvae


 
 
 
 In aquatic environments, many prey animals, including anuran larvae, predominantly use chemical cues to assess predation risk. In such systems, a variety of chemical cues (e.g., kairomones, alarm, dietary) affect the behavioral responses of the prey tadpoles. Many anuran tadpoles are able to discriminate different chemical cues and exhibit differential antipredator behavioral responses according to the perceived risk. The behavioral responses of tadpoles of Clinotarsus curtipes to predatory larvae of the dragonfly Pantala flavescens were studied in the laboratory. The predator’s kairomones (water conditioned by a starved predator) or its diet-derived metabolites released in excreta of a predator after consumption of conspecific (C. curtipes) or heterogeneric (Indosylvirana temporalis) prey tadpoles were used to simulate predation threat. The tadpoles of C. curtipes had no behavioral response to predator kairomones. However, the tadpoles showed antipredator behavioral responses i.e., reduced swimming movements and overall time spent swimming, and had a higher burst speed in response to water-borne cues released from the excreta of predators fed both conspecific and heterogeneric prey. The antipredator behavioral responses of tadpoles were most intense in response to cues of predators fed on conspecific prey. The findings of the present study show that tadpoles of C. curtipes are capable of assessing levels of predation threat and modulating the intensity of their defense behavior in accordance with the perceived threat.
 
 
 

Adaptive plasticity in anuran metamorphosis: response of tadpoles of Polypedates maculatus (Anura: Rhacophoridae) to pond drying


 
 
 
 The influence of desiccation on metamorphic traits (larval duration and size at emergence) was studied in Polypedates maculatus under laboratory conditions. Gosner Stage 23 tadpoles were exposed to decreasing water levels (gradual or rapid) until the beginning of metamorphic climax (Stage 42). A control group was reared in constant water levels. Tadpoles reared in decreasing water levels reached the metamorphic climax earlier and metamorphosed at a smaller size than those reared in constant water levels. Further, tadpoles experiencing rapid depletion of water reached the metamorphic climax earlier and metamorphosed at a smaller size than those experiencing gradual depletion of water levels. Tadpoles of P. maculatus showed adaptive plasticity in metamorphosis to pond drying. Survival of tadpoles in treatments and the control was 100%. The study revealed that tadpoles of P. maculatus have plastic development in response to water levels; the trade-off between growth and development favors development, which results in early metamorphosis at a small size.
 
 
 

One of these things is not like the other: Mixed predator cues result in lopsided phenotypic responses in a Neotropical tadpole.

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.

Embryonic exposure to native and alien predator cues tunes tadpole defensive behaviour

When exposed to predation risk, some amphibian species show innate responses, while others recognize their predators by learning. To explore the role played by each mechanism in the assessment of predation risk, we investigated the effects of embryonic and larval exposure to predator chemical cues on tadpole defensive responses, including behavioural, morphological and life history traits. In the first experiment, agile frog (Rana dalmatina) embryos were exposed to the odour of either native (Aeshna cyanea larvae) or alien (Procambarus clarkii) predators each day from egg collection to hatchling (14 days). Body measures (mass, developmental stage, body length, tail length and tail depth) were recorded at hatching and a behavioural test was conducted to explore tadpole responses to predator cues and the potential interaction with their previous embryonic experience. In general, embryonic conditioning did not affect life history traits, except for a slight reduction in tail depth:length ratio for tadpoles exposed to odonate odours. Controls (embryos treated with water) after hatchling reduced their activity when exposed to gammarid-fed odonate cues, suggesting that responses were at least partially innate. Tadpoles exposed to odonate cues as embryos showed a strong defensive response when exposed to dragonfly kairomones. Tadpoles exposed to gammarid-fed crayfish as embryos showed clear behavioural responses towards the same cue (irrespectively of predator diet). Overall, our results suggest that embryonic exposure may tune the defensive responses of the larval stage and early exposure to naïve stimuli may promote their cautionary associations with predation risk.

Influence of temperature on the biomarker responses of bullfrog tadpoles (Lithobates catesbeianus) to 2-hydroxyatrazine exposure

The influence of temperature (25 and 32 °C) on the biomarker responses of bullfrog tadpoles (Lithobates catesbeianus) to different concentrations of the atrazine metabolite 2-hydroxyatrazine (2-HA, 0, 10, 50 and 200 ng.L−1, 16 days), was evaluated. Temperature affected the activities of superoxide dismutase, glutathione S-transferase and acetylcholinesterase. The activities of catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase and carboxylesterase presented no alterations. Frequencies of micronuclei and nuclear abnormalities were also not altered. 2-HA decreased SOD activity at 25 °C and caused histopathological changes in the liver and the kidney at both temperatures, with the kidney being more affected by the combination of higher temperature and 2-HA exposure, presenting glomerular shrinkage and an increase in Bowman's space. Our results indicate that at environmentally relevant concentrations, 2-HA can cause changes in biomarker responses as well as in the morphology of liver and kidney in L. catesbeianus tadpoles. Temperature has an important influence on biomarker response and histopathological alterations.

Copper-mediated shifts in transcriptomic responses of intestines in Bufo gargarizans tadpoles to lead stress.

The differential transcriptomic responses of intestines in Bufo gargarizans tadpoles to Pb alone or in the presence of Cu were evaluated. Tadpoles were exposed to 30μg/L Pb individually and in combination with Cu at 16 or 64μg/L from Gosner stage (Gs) 26 to Gs 38. After de novo assembly, 105,107 unigenes were generated. Compared to the control group, 7387, 6937, and 11139 differentially expressed genes (DEGs) were identified in the treatment of Pb + Cu0, Pb + Cu16, and Pb + Cu64, respectively. In addition, functional annotation and enrichment analysis of DEGs revealed substantial transcriptional reprogramming of diverse molecular and biological pathways were induced in all heavy metal treatments. The relative expression levels of genes associated with intestinal epithelial barrier and bile acids (BAs) metabolism, such as mucin2, claudin5, ZO-1, Asbt, and Ost-β, were validated by qPCR. This study demonstrated that Pb exposure induced transcriptional responses in tadpoles, and the responses could be modulated by Cu.

Pump my wetland: potential benefits of using water pumps fitted with large-mesh screens to conserve anurans in regulated floodplain environments

Context Water pumps fitted with filtering screens are effective for delivering water to floodplain environments and excluding large-bodied exotic fish; yet, the benefits to wetland fish and anurans are unknown. Aims To quantify fish and tadpole responses to refilling wetlands with water pumps fitted with large-mesh screens following drawdown v. overland reconnections in the mid-Murrumbidgee region of New South Wales. Methods Frog and fish communities were compared between wetlands under managed inundation and overland reconnections by using PERMANOVA, and non-metric multidimensional scaling was used to evaluate community divergence between watering strategies. Classification and regression trees were used to identify thresholds in explanatory variables and predicted threshold responses in fish and tadpole abundance. Key results Fish and tadpole communities differed in relation to watering strategies. Managed inundation resulted in a higher abundance of tadpole species and one native fish, whereas overland reconnections resulted in a high abundance of exotic fish species and fewer tadpoles. Water depth was a driver of tadpole abundance in two species, including the threatened southern bell frog (Litoria raniformis). Conclusions and implications Using filtered pumps with large-mesh screens to deliver water to wetlands of high conservation value may be an effective strategy for reducing large-bodied exotic fish and enhancing frog and fish populations in regulated floodplain systems.

Comparison of transcriptome responses of the liver, tail fin, and olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles disrupted by thyroid hormones and estrogen

Thyroid hormones (THs) are important developmental regulators in vertebrates, including during the metamorphosis of a tadpole into a frog. Metamorphosis is a post-embryonic developmental period initiated by TH production in the tadpole thyroid gland. The two main bioactive forms of TH are L-thyroxine (T4) and 3,5,3’-triiodothyronine (T3); these hormones have overlapping but distinct mechanisms of action. Premetamorphic tadpoles are highly responsive to TH and can be induced to metamorphose through exogenous TH exposure, making them an important model for both the study of vertebrate TH signaling and endocrine disrupting chemicals (EDCs). It is important to differentiate TH-mediated responses from estrogenic responses in premetamorphic tadpoles when assessing dysregulation by EDCs as crosstalk between the two endocrine systems is well-documented. Herein, we compare the RNA-sequencing-derived transcriptomic profiles of three TH-responsive tissues (liver, olfactory epithelium, and tail fin) in premetamorphic bullfrog (Rana [Lithobates] catesbeiana) tadpoles exposed to T3, T4, and estradiol (E2). These profiles were generated using the latest available genome assembly for the species. The data indicate that there is a clear distinction, and little overlap, between the transcriptomic responses elicited by E2 and the THs. In contrast, within the THs, the T3- and T4-induced transcriptomic profiles generally show considerable overlap; however, the degree of overlap is highly tissue-dependent, illustrating the importance of distinguishing the two THs and the affected signaling pathways within the target tissue type when evaluating hormone active agents. The data herein also show that E2 and TH treatment can uniquely induce significant changes in expression of their respective “classic” bioindicator transcripts vtg (E2) and thra, thrb, and thibz (THs). However, care must be taken in the interpretation of increased vep or esr1 transcripts as a change in transcript levels can be induced by THs rather than solely E2.

Effects of a Neonicotinoid Insecticide and Population Density on Behavior and Development of Wood Frogs (Rana sylvatica).

Amphibians have been facing global declines over the last decades from direct and indirect effects of anthropogenic activities. A contributor to declines is waterway contamination from agricultural runoffs of pesticides such as neonicotinoids. Beyond direct and indirect effects of the pesticide, few studies have investigated the possible interactions between neonicotinoids and natural environmental stressors across larval development, which could alter the strength and direction of observed neonicotinoid effects. The present study used a fully crossed design to investigate how a concentration of imidacloprid (a neonicotinoid; 10 µg/L) measured in surface waters interacted with low and high population densities (0.33 and 1 tadpole/L, respectively), an important environmental stressor, to influence behavior and development across metamorphosis in wood frogs (Rana sylvatica), known to breed in agricultural landscapes. Behaviors were measured in the absence and presence of predation cues using open-field tests at three distinct developmental stages, up to the metamorph stage. We found that imidacloprid did not interact with population density or independently affect behaviors in the absence of predation cues. However, individuals raised at high density compared with low density were more active at an early developmental stage but less active at metamorphic climax. Furthermore, both density and imidacloprid independently decreased the natural freezing response of tadpoles to predation cues. Finally, we found that distance traveled in the open-field test was weakly repeatable between aquatic stages but not repeatable across metamorphosis, a pattern that was not affected by treatments. The present study provides novel insights on the ecotoxicology of imidacloprid in the presence of a natural stressor, highlighting the importance of including behavioral assays and natural stressors in studies of amphibian ecotoxicology. Environ Toxicol Chem 2022;41:2968-2980. © 2022 SETAC.

Effects of aqueous extracts of wildfire ashes on tadpoles of Pelophylax perezi: Influence of plant coverage

Wildfires have been pointed out as an important source of diffuse contamination to aquatic ecosystems, namely through the input of toxic compounds such as polycyclic aromatic hydrocarbons and metals. However, amphibians' responses to this disturbance have been largely ignored. Hence, this study intended to assess how ashes from Pinus sp. and Eucalyptus sp. plantation forests affect tadpoles of Pelophylax perezi. Tadpoles were exposed 14 days to serial concentrations (26.9 %–100 %) of aqueous extracts of ashes (AEA, with 10 g L−1 of ashes) containing Eucalypt (ELS) and Pine (PLS) ashes. The following endpoints were measured: mortality, malformations, developmental stage, body length and weight. Effects at sub-individual level were also monitored for oxidative stress, neurotoxicity, and energetic metabolism. Chemical characterization of the AEA of ELS showed higher concentrations of As, Cd, Co, Cr, Pb and V, while PLS showed higher concentrations of Cu, Mn, Ni and Zn. Concerning the lethal effects of AEAs on tadpoles, both extracts were able to induce mortality at high concentrations (76.9 and/or 100 % of AEA), although a high variability in the response was found. A significant mortality in tadpoles exposed to ELS was observed at the concentration of 76.9 %. For organisms exposed to PLS, though a mortality above 20 % was registered at the two highest tested concentrations, it was not significantly different from the control. No significant sub-lethal effects were observed in the ELS treatments. Contrasting, exposure to PLS induced a decrease in body length, weight, glutathione-S-transferase activity and an increase in oxygen consumption. Overall, the distinct effects of ELS and PLS suggest an influence of vegetation cover in ash toxicity. In conclusion, exposure to both ash extracts negatively affected sublethal responses of tadpoles of P. perezi. Future research is needed to assess how these effects at individual level may translate into effects at population level.