Effects of hibernation on two important contractile tissues in tibetan frogs, Nanorana parkeri: a perspective from transcriptomics and metabolomics approaches.

BACKGROUND: In response to seasonal cold and food shortage, the Xizang plateau frogs, Nanorana parkeri (Anura: Dicroglossidae), enter a reversible hypometabolic state where heart rate and oxygen consumption in skeletal muscle are strongly suppressed. However, the effect of winter hibernation on gene expression and metabolic profiling in these two tissues remains unknown. In the present study, we conducted transcriptomic and metabolomic analyses of heart and skeletal muscle from summer- and winter-collected N. parkeri to explore mechanisms involved in seasonal hibernation. RESULTS: We identified 2407 differentially expressed genes (DEGs) in heart and 2938 DEGs in skeletal muscle. Enrichment analysis showed that shared DEGs in both tissues were enriched mainly in translation and metabolic processes. Of these, the expression of genes functionally categorized as "response to stress", "defense mechanisms", or "muscle contraction" were particularly associated with hibernation. Metabolomic analysis identified 24 and 22 differentially expressed metabolites (DEMs) in myocardium and skeletal muscle, respectively. In particular, pathway analysis showed that DEMs in myocardium were involved in the pentose phosphate pathway, glycerolipid metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis. By contrast, DEMs in skeletal muscle were mainly involved in amino acid metabolism. CONCLUSIONS: In summary, natural adaptations of myocardium and skeletal muscle in hibernating N. parkeri involved transcriptional alterations in translation, stress response, protective mechanisms, and muscle contraction processes as well as metabolic remodeling. This study provides new insights into the transcriptional and metabolic adjustments that aid winter survival of high-altitude frogs N. parkeri.

Are novel or locally adapted pathogens more devastating and why? Resolving opposing hypotheses.

There is a rich literature highlighting that pathogens are generally better adapted to infect local than novel hosts, and a separate seemingly contradictory literature indicating that novel pathogens pose the greatest threat to biodiversity and public health. Here, using Batrachochytrium dendrobatidis, the fungus associated with worldwide amphibian declines, we test the hypothesis that there is enough variance in "novel" (quantified by geographic and phylogenetic distance) host-pathogen outcomes to pose substantial risk of pathogen introductions despite local adaptation being common. Our continental-scale common garden experiment and global-scale meta-analysis demonstrate that local amphibian-fungal interactions result in higher pathogen prevalence, pathogen growth, and host mortality, but novel interactions led to variable consequences with especially virulent host-pathogen combinations still occurring. Thus, while most pathogen introductions are benign, enough variance exists in novel host-pathogen outcomes that moving organisms around the planet greatly increases the chance of pathogen introductions causing profound harm.

Combined effect of seasons and life history in an anuran strengthens the response and relationship between their physiology and gut microbiota.

Gut microbiota impact host physiology, though simultaneous investigations in ectothermic vertebrates are rare. Particularly, amphibians may exhibit more complex interactions between host physiology and the effects of gut microbiota due to the combination of seasonal changes and complex life histories. In this study, we assessed the relationships among food resources, gut bacterial communities, and host physiology in frogs (Phelophylax nigromaculatus), taking into account seasonal and life history variations. We found that food sources were not correlated with physiological parameters but had some relationships with the gut bacterial community. Variations in gut bacterial community and host physiology were influenced by the combined effects of seasonal differences and life history, though mostly driven by seasonal differences. An increase in Firmicutes was associated with higher fat content, reflecting potential fat storage in frogs during the non-breeding season. The increase in Bacteroidetes resulted in lower fat content in adult frogs and decreased immunity in juvenile frogs during the breeding season, demonstrating a direct link. Our results suggest that the gut microbiome may act as a link between food conditions and physiological status, and that the combined effect of seasons and life history could reinforce the relationship between gut microbiota and physiological status in ectothermic animals. While food sources may influence the gut microbiota of ectotherms, we contend that temperature-correlated seasonal variation, which predominately influences most ectotherms, is a significant factor.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Impacts of a Simulated Infection on the Locomotor Behavior of Invasive and Noninvasive Species of Congeneric Anurans.

AbstractLocomotion is essential for survival, but it requires resources such as energy and metabolites and therefore may conflict with other physiological processes that also demand resources, particularly expensive processes such as immunological responses. This possible trade-off may impose limits on either the magnitude of immune responses or the patterns of activity and performance. Previous studies have shown that invasive species may have a depressed immune response, allowing them to maintain locomotor function and reproduction even when sick. This may contribute to the ecological success of invasive species in colonization and dispersal. In contrast, noninvasive species tend to reduce activity as a response to infection. Here, we studied the impact of a simulated infection on locomotor performance and voluntary movement in the anurans Xenopus laevis (a globally invasive species) and Xenopus allofraseri (a noninvasive congeneric). We found that a simulated infection reduces locomotor performance in both species, with an accentuated effect on X. allofraseri. Voluntary movement was marginally different between species. Our data suggest that a simulated infection leads to behavioral depression and reduced locomotor performance in anurans and show that this effect is limited in the invasive X. laevis. Contrasting responses to an immune challenge have been reported in the few amphibian taxa analyzed to date and suggest relationships between ecology and immunology that deserve further investigation. Specifically, a depressed immune response may underlie a propension to invasion in some species. Whether this is a general trend for invasive species remains to be tested, but our data add to the growing body of work documenting depressed immune systems in invasive species.

Testes size seen through the glass of amphibian care.

Despite the great diversity of parental care types found in amphibians, studies linking them to post-copulatory sexually selected traits are scarce, presumably due to a lack of data. Valencia-Aguilar et al. used fieldwork and museum collections to show that paternal care appears to trade-off with testes size in glass frogs.

Population origin and heritable effects mediate road salt toxicity and thermal stress in an amphibian.

Human impacts on wild populations are numerous and extensive, degrading habitats and causing population declines across taxa. Though these impacts are often studied individually, wild populations typically face suites of stressors acting concomitantly, compromising the fitness of individuals and populations in ways poorly understood and not easily predicted by the effects of any single stressor. Developing understanding of the effects of multiple stressors and their potential interactions remains a critical challenge in environmental biology. Here, we focus on assessing the impacts of two prominent stressors associated with anthropogenic activities that affect many organisms across the planet - elevated salinity (e.g., from road de-icing salt) and temperature (e.g. from climate change). We examined a suite of physiological traits and components of fitness across populations of wood frogs originating from ponds that differ in their proximity to roads and thus their legacy of exposure to pollution from road salt. When experimentally exposed to road salt, wood frogs showed reduced survival (especially those from ponds adjacent to roads), divergent developmental rates, and reduced longevity. Family-level effects mediated these outcomes, but high salinity generally eroded family-level variance. When combined, exposure to both temperature and salt resulted in very low survival, and this effect was strongest in roadside populations. Taken together, these results suggest that temperature is an important stressor capable of exacerbating impacts from a prominent contaminant confronting many freshwater organisms in salinized habitats. More broadly, it appears likely that toxicity might often be underestimated in the absence of multi-stressor approaches.

An Overview of Frog Skin-Derived Esc Peptides: Promising Multifunctional Weapons against Pseudomonas aeruginosa-Induced Pulmonary and Ocular Surface Infections.

Antimicrobial resistance is a silent pandemic harming human health, and Pseudomonas aeruginosa is the most common bacterium responsible for chronic pulmonary and eye infections. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. In this review, the in vitro/in vivo activities of the frog skin-derived AMP Esc(1-21) are shown. Esc(1-21) rapidly kills both the planktonic and sessile forms of P. aeruginosa and stimulates migration of epithelial cells, likely favoring repair of damaged tissue. However, to undertake preclinical studies, some drawbacks of AMPs (cytotoxicity, poor biostability, and limited delivery to the target site) must be overcome. For this purpose, the stereochemistry of two amino acids of Esc(1-21) was changed to obtain the diastereomer Esc(1-21)-1c, which is more stable, less cytotoxic, and more efficient in treating P. aeruginosa-induced lung and cornea infections in mouse models. Incorporation of these peptides (Esc peptides) into nanoparticles or immobilization to a medical device (contact lens) was revealed to be an effective strategy to ameliorate and/or to prolong the peptides' antimicrobial efficacy. Overall, these data make Esc peptides encouraging candidates for novel multifunctional drugs to treat lung pathology especially in patients with cystic fibrosis and eye dysfunctions, characterized by both tissue injury and bacterial infection.

A Nonbactericidal Anionic Antimicrobial Peptide Provides Prophylactic and Therapeutic Efficacies against Bacterial Infections in Mice by Immunomodulatory-Antithrombotic Duality.

Although bactericidal cationic antimicrobial peptides (AMPs) have been well characterized, less information is available about the antibacterial properties and mechanisms of action of nonbactericidal AMPs, especially nonbactericidal anionic AMPs. Herein, a novel anionic antimicrobial peptide (Gy-CATH) with a net charge of -4 was identified from the skin of the frog Glyphoglossus yunnanensis. Gy-CATH lacks direct antibacterial effects but exhibits significantly preventive and therapeutic capacities in mice that are infected with Staphylococcus aureus, Enterobacteriaceae coli, methicillin-resistant Staphylococcus aureus (MRSA), or carbapenem-resistant E. coli (CREC). In vitro and in vivo investigations proved the regulation of Gy-CATH on neutrophils and macrophages involved in the host immune defense against infection. Moreover, Gy-CATH significantly reduced the extent of pulmonary fibrin deposition and prevented thrombosis in mice, which was attributed to the regulatory role of Gy-CATH in physiological anticoagulants and platelet aggregation. These findings show that Gy-CATH is a potential candidate for the treatment of bacterial infection.

Comparative transcriptomic analysis delineates adaptation strategies of Rana kukunoris toward cold stress on the Qinghai-Tibet Plateau.

BACKGROUND: Cold hardiness is fundamental for amphibians to survive during the extremely cold winter on the Qinghai-Tibet plateau. Exploring the gene regulation mechanism of freezing-tolerant Rana kukunoris could help us to understand how the frogs survive in winter. RESULTS: Transcriptome of liver and muscle of R. kukunoris collected in hibernation and spring were assisted by single molecule real-time (SMRT) sequencing technology. A total of 10,062 unigenes of R. kukunoris were obtained, and 9,924 coding sequences (CDS) were successfully annotated. Our examination of the mRNA response to whole body freezing and recover in the frogs revealed key genes concerning underlying antifreeze proteins and cryoprotectants (glucose and urea). Functional pathway analyses revealed differential regulated pathways of ribosome, energy supply, and protein metabolism which displayed a freeze-induced response and damage recover. Genes related to energy supply in the muscle of winter frogs were up-regulated compared with the muscle of spring frogs. The liver of hibernating frogs maintained modest levels of protein synthesis in the winter. In contrast, the liver underwent intensive high levels of protein synthesis and lipid catabolism to produce substantial quantity of fresh proteins and energy in spring. Differences between hibernation and spring were smaller than that between tissues, yet the physiological traits of hibernation were nevertheless passed down to active state in spring. CONCLUSIONS: Based on our comparative transcriptomic analyses, we revealed the likely adaptive mechanisms of R. kukunoris. Ultimately, our study expands genetic resources for the freezing-tolerant frogs.

Fossilized anuran soft tissues reveal a new taphonomic model for the Eocene Geiseltal Konservat-Lagerstätte, Germany.

The Eocene Geiseltal Konservat-Lagerstätte (Germany) is famous for reports of three dimensionally preserved soft tissues with sub-cellular detail. The proposed mode of preservation, direct replication in silica, is not known in other fossils and has not been verified using modern approaches. Here, we investigated the taphonomy of the Geiseltal anurans using diverse microbeam imaging and chemical analytical techniques. Our analyses confirm the preservation of soft tissues in all body regions but fail to yield evidence for silicified soft tissues. Instead, the anuran soft tissues are preserved as two layers that differ in microstructure and composition. Layer 1 comprises sulfur-rich carbonaceous microbodies interpreted as melanosomes. Layer 2 comprises the mid-dermal Eberth-Katschenko layer, preserved in calcium phosphate. In addition, patches of original aragonite crystals define the former position of the endolymphatic sac. The primary modes of soft tissue preservation are therefore sulfurization of melanosomes and phosphatization of more labile soft tissues, i.e., skin. This is consistent with the taphonomy of vertebrates in many other Konservat-Lagerstätten. These findings emphasize an emerging model for pervasive preservation of vertebrate soft tissues via melanosome films, particularly in stagnation-type deposits, with phosphatization of more labile tissues where tissue biochemistry is favorable.

Distribution and diversification of Adelphobates, emblematic poison frogs from Brazilian Amazonia.

Adelphobates contains three species, and the inaccurate identification of A. quinquevittatus and the scarcity of records of A. castaneoticus complicate inference of their distributions; the latter species occurs in sympatry with A. galactonotus. Our objective was to revise the distributions of Adelphobates by compiling data and modeling habitat suitability, as range limits may be shaped by landscape features and biotic interactions. We initially analyzed the existence of operational taxonomic units within the nominal species and subsequently inferred the observed and potential distributions, taking into account the possible independent lineages for the three species, and we also generated a molecular timetree to understand the chronology of interspecific diversification events. Adelphobates quinquevittatus was found to have a more easterly distribution than previously described, and specimens with phenotypic variation were found to occur in areas inconsistent with the modeling, and A. castaneoticus was concentrated in the Tapajós-Xingu interfluve, surrounded by A. galactonotus. Models indicated that the right bank of the Xingu River is suitable for both species, indeed, both were found there. Despite Adelphobates species having their distributions delimited by major Amazonian rivers, estimated divergence times predate the formation of the modern river network, suggesting that other mechanisms were involved in their diversification.

Marine introgressions and Andean uplift have driven diversification in neotropical Monkey tree frogs (Anura, Phyllomedusinae).

The species richness in the Neotropics has been linked to environmental heterogeneity and a complex geological history. We evaluated which biogeographic processes were associated with the diversification of Monkey tree frogs, an endemic clade from the Neotropics. We tested two competing hypotheses: the diversification of Phyllomedusinae occurred either in a "south-north" or a "north-south" direction in the Neotropics. We also hypothesized that marine introgressions and Andean uplift had a crucial role in promoting their diversification. We used 13 molecular markers in a Bayesian analysis to infer phylogenetic relationships among 57 species of Phyllomedusinae and to estimate their divergence times. We estimated ancestral ranges based on 12 biogeographic units considering the landscape modifications of the Neotropical region. We found that the Phyllomedusinae hypothetical ancestor range was probably widespread throughout South America, from Western Amazon to Southern Atlantic Forest, at 29.5 Mya. The Phyllomedusines' ancestor must have initially diverged through vicariance, generally followed by jump-dispersals and sympatric speciation. Dispersal among areas occurred mostly from Western Amazonia towards Northern Andes and the South American diagonal of dry landscapes, a divergent pattern from both "south-north" and "north-south" diversification hypotheses. Our results revealed a complex diversification process of Monkey tree frogs, occurring simultaneously with the orogeny of Northern Andes and the South American marine introgressions in the last 30 million years.

A hundred-year-old mystery-the reproductive mode and larval morphology of the enigmatic frog genus Allophryne (Amphibia; Anura; Allophrynidae).

Frogs of the Allophrynidae are an enigmatic family from South America. To date, published information is lacking regarding this group's reproductive biology and larval morphology. Here, we provide the first detailed description of the reproductive mode, developmental mode, and tadpole morphology for Allophryne ruthveni. We developed a captive breeding and rearing protocol for this species and then conducted a series of observations to describe aspects of its reproductive biology. In captivity, this species exhibits aquatic oviposition, where single eggs are laid ungrouped within a simple jelly capsule and are scattered free in the water column before sinking to develop on benthic substrates. We did not observe parental care nor any parental interactions with eggs post-fertilization. Tadpoles are characterized by an oval body, anteroventral oral disc, a labial tooth row formula of 2(2)/3, and a dextral vent tube. The buccopharyngeal cavity is marked by the presence of two pairs of infralabial papilla and four lingual papillae. Cranial morphology is characterized by the presence of the commissura quadratoorbital. This species possesses an additional slip of the m. rectus cervicis and of the m. levator arcuum branchialium III. We discuss our results in comparison with glassfrogs (Centrolenidae).

Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

Vocal repertoire of Microhyla nilphamariensis from Delhi and comparison with closely related M. ornata populations from the western coast of India and Sri Lanka.

Advertisement calls in frogs have evolved to be species-specific signals of recognition and are therefore considered an essential component of integrative taxonomic approaches to identify species and delineate their distribution range. The species rich genus Microhyla is a particularly challenging group for species identification, discovery and conservation management due to the small size, conserved morphology and wide distribution of its members, necessitating the need for a thorough description of their vocalization. In this study, we provide quantitative description of the vocal behaviour of Microhyla nilphamariensis, a widely distributed south Asian species, from Delhi, India, based on call recordings of 18 individuals and assessment of 21 call properties. Based on the properties measured acrossed 360 calls, we find that a typical advertisement call of M. nilphamariensis lasts for 393.5 ±  57.5 ms, has 17 pulses on average and produce pulses at rate of 39 pulses/s. The overall call dominant frequency was found to be 2.8 KHz and the call spectrum consisted of two dominant frequency peaks centered at 1.6 KHz and 3.6 KHz, ranging between 1.5-4.1 KHz. Apart from its typical advertisement call, our study also reveals the presence of three 'rare' call types, previously unreported in this species. We describe variability in call properties and discuss their relation to body size and temperature. We found that overall dominant frequency 1 (spectral property) was found to be correlated with body size, while first pulse period (temporal property) was found to be correlated with temperature. Further, we compare the vocal repertoire of M. nilphamariensis with that of the congener Microhyla ornata from the western coast of India and Sri Lanka and also compare the call properties of these two populations of M. ornata to investigate intra-specific call variation. We find statistically significant differentiation in their acoustic repertoire in both cases. Based on 18 call properties (out of 20), individuals of each locality clearly segregate on PCA factor plane forming separate groups. Discriminant function analysis (DFA) using PCA factors shows 100% classification success with individuals of each locality getting classified to a discrete group. This confirms significant acoustic differentiation between these species as well as between geographically distant conspecifics. The data generated in this study will be useful for comparative bioacoustic analysis of Microhyla species and can be utilized to monitor populations and devise conservation management plan for threatened species in this group.

Ontogenetic Expression of Aquaporins in the Kidney and Urinary Bladder of the Japanese Tree Frog, Dryophytes japonicus.

For adult anuran amphibians, the kidney and urinary bladder play important osmoregulatory roles through water reabsorption. In the present study, we have examined ontogenetic expression of aquaporins, i.e., AQP2, AQPamU (AQP6ub, AQPa2U), and AQP3, in these organs using the Japanese tree frog, Dryophytes japonicus. Immunohistochemistry using the metamorphosing larvae at stages 40-43 localized AQP2 protein to the collecting ducts in the dorsal zone of the mesonephric kidney. At prometamorphic stages 40 and 41, labelling of AQP2 protein was observed in the apical/ subapical regions of the collecting duct cells. At climax stages 42 and 43, labels for AQP2 and AQP3 became observed in the apical/subapical regions and basolateral membrane of the collecting duct cells, respectively, as seen in the adults. As for the urinary bladder, immuno-positive labels for AQPamU were localized to the apical/subapical regions of granular cells in the mucosal epithelium at stages 40-43. On the other hand, AQP3 immunoreactivity was hardly observed in the urinary bladder at stage 40, and weakly appeared in many granular cells at stage 41. Thereafter, labels for AQP3 became evident along the basolateral membrane of granular cells at stages 42 and 43, together with AQPamU in the apical/subapical regions. These results suggest that the kidney and urinary bladder might be capable of water reabsorption, via AQP2, AQPamU, and AQP3, at stage 42, contributing to the acclimation of the tree frogs to terrestrial environments.

Toxic to the touch: The makings of lethal mantles in pitohui birds and poison dart frogs.

How do chemically defended animals resist their own toxins? This intriguing question on the concept of autotoxicity is at the heart of how species interactions evolve. In this issue of Molecular Ecology (Molecular Ecology, 2024, 33), Bodawatta and colleagues report on how Papua New Guinean birds coopted deadly neurotoxins to create lethal mantles that protect against predators and parasites. Combining chemical screening of the plumage of a diverse collection of passerine birds with genome sequencing, the researchers unlocked a deeper understanding of how some birds sequester deadly batrachotoxin (BTX) from their food without poisoning themselves. They identified that birds impervious to BTX bear amino acid substitutions in the toxin-binding site of the voltage-gated sodium channel Nav1.4, whose function is essential for proper contraction and relaxation of vertebrate muscles. Comparative genetic and molecular docking analyses show that several of the substitutions associated with insensitivity to BTX may have become prevalent among toxic birds through positive selection. Intriguingly, poison dart frogs that also co-opted BTX in their lethal mantles were found to harbour similar toxin insensitivity substitutions in their Nav1.4 channels. Taken together, this sets up a powerful model system for studying the mechanisms behind convergent molecular evolution and how it may drive biological diversity.

Metal nanoparticle-induced effects on green toads (Amphibia, Anura) under climate change: conservation implications.

The toxicity of aluminum oxide (Al2O3), copper oxide (CuO), iron oxide (Fe3O4), nickel oxide (NiO), zinc oxide (ZnO), and titanium dioxide (TiO2) nanoparticles (NPs) on amphibians and their interaction with high temperatures, remain unknown. In this study, we investigated the survival, developmental, behavioral, and histological reactions of Bufotes viridis embryos and larvae exposed to different NPs for a duration of 10 days, using lethal concentrations (LC25%, LC50%, and LC75% mg/L) under both ambient (AT: 18 °C) and high (HT: 21 °C) temperatures. Based on LC, NiONPs > ZnONPs > CuONPs > Al2O3NPs > TiO2NPs > Fe3O4NPs showed the highest mortality at AT. A similar pattern was observed at HT, although mortality occurred at lower concentrations and Fe3O4NPs were more toxic than TiO2NPs. The results indicated that increasing concentrations of NPs significantly reduced hatching rates, except for TiO2NPs. Survival rates decreased, abnormality rates increased, and developmental processes slowed down, particularly for NiONPs and ZnONPs, under HT conditions. However, exposure to low concentrations of Fe3O4NPs for up to 7 days, CuONPs for up to 72 h, and NiO, ZnONPs, and TiO2NPs for up to 96 h did not have a negative impact on survival compared with the control group under AT. In behavioral tests with larvae, NPs generally induced hypoactivity at AT and hyperactivity at HT. Histological findings revealed liver and internal gill tissue lesions, and an increase in the number of melanomacrophage centers at HT. These results suggest that global warming may exacerbate the toxicity of metal oxide NPs to amphibians, emphasizing the need for further research and conservation efforts in this context.

Broad-scale pesticide screening finds anticoagulant rodenticide and legacy pesticides in Australian frogs.

Pesticide contamination poses a significant threat to non-target wildlife, including amphibians, many of which are already highly threatened. This study assessed the extent of pesticide exposure in dead frogs collected during a mass mortality event across eastern New South Wales, Australia between July 2021 and March 2022. Liver tissue from 77 individual frogs of six species were analysed for >600 legacy and contemporary pesticides, including rodenticides. More than a third (36 %) of the liver samples contained at least one of the following pesticides: brodifacoum, dieldrin, DDE, heptachlor/heptachlor epoxide, fipronil sulfone, and 2-methyl-4-chlorophenoxyacetic acid (MCPA). Brodifacoum, a second-generation anticoagulant rodenticide, was found in four of the six frog species analysed: the eastern banjo frog (Limnodynastes dumerilii), cane toad (Rhinella marina), green tree frog (Litoria caerulea) and Peron's tree frog (Litoria peronii). This is the first report of anticoagulant rodenticide detected in wild amphibians, raising concerns about potential impacts on frogs and extending the list of taxa shown to accumulate rodenticides. Dieldrin, a banned legacy pesticide, was also detected in two species: striped marsh frog (Limnodynastes peronii) and green tree frog (Litoria caerulea). The toxicological effects of these pesticides on frogs are difficult to infer due to limited comparable studies; however, due to the low frequency of detection the presence of these pesticides was not considered a major contributing factor to the mass mortality event. Additional research is needed to investigate the effects of pesticide exposure on amphibians, particularly regarding the impacts of second-generation anticoagulant rodenticides. There is also need for continued monitoring and improved conservation management strategies for the mitigation of the potential threat of pesticide exposure and accumulation in amphibian populations.