Preparation for oxidative stress in Chinese toads (Bufo gargarizans) living under natural conditions along an altitudinal gradient.

Preparation for oxidative stress (POS) has been widely reported in animals under controlled laboratory conditions, but whether this phenomenon is visible in animals under natural conditions remains to be explored. Altitudinal gradients provide a good opportunity to address this question, since environmental conditions become more hostile with increasing altitude. Here, we investigated the levels of oxidative stress, oxidative damage, and antioxidant defenses in Chinese toads (Bufo gargarizans) along an altitudinal gradient (50 m, 1200 m, 2300 m, 3400 m above sea level). The results show that changing altitude led to a significantly lower ratio of oxidized to reduced glutathione in liver, with a higher value at 50 m. This ratio in muscle tissues did not differ significantly between altitudes of 50 m, 2300 m, and 3400 m. However, reduced glutathione content increased significantly along the altitude, with higher values in liver at 2300 m and higher values in skeletal muscle at 3400 m. Malondialdehyde (MDA) content in liver did not change significantly with increasing altitude. Brain and muscle tissues showed a higher MDA content at 50 m than the other three altitudes. The activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase, as well as total antioxidant capacity, also displayed tissue-specific upregulation in heart, skeletal muscle, and brain, but all of these antioxidant enzymes except for glutathione-S-transferase were significantly reduced in liver along the altitudinal gradient. In summary, environmental factors at higher altitude did not lead to higher levels of oxidative stress and oxidative damage in B. gargarizans, mainly due to stronger antioxidant defenses. This study corroborates the occurrence of POS in high-altitude toads living under field conditions and contributes to revealing the biochemical adaptations to extreme environments at higher altitude.

Age structure and body size of two Tibetan toad (Bufo tibetanus) populations from different elevations in China.

Understanding how age and body size vary across elevations can provide insights into the evolution of life-history traits in animals. In the present study, we compared the demographic (using skeletochronology) and morphological traits of the Tibetan toad (Bufo tibetanus) between two populations from different elevational habitats (2650 vs. 3930 m). We found that (1) the mean age and body size of females were significantly greater than those of males in both populations; (2) both sexes of toads from the higher elevation tended to be significantly older in age and larger in body size; (3) there was a significant positive relationship between age and body size within each sex of the toad at both elevations; and (4) growth rates varied between the two populations, with the higher rate observed in the lower-elevation population. Our results suggested that factors other than age, such as elevation-associated temperature, influence the observed differences in body size between the two populations. Future research at a broader range of elevations should focus on these factors and evaluate their influence on animal growth patterns.

Response characteristic and potential molecular mechanism of tail resorption in Bufo gargarizans after exposure to lead and copper, alone or combined.

Tail resorption during amphibian metamorphosis is one of the most dramatic processes that is obligatorily dependent on thyroid hormone (TH). Heavy metals could result in thyroid gland damages and disturb TH homeostasis. Lead (Pb) and copper (Cu) often co-exist in natural aquatic ecosystems. However, there is still little information on how tail resorption responds to alone or combined exposure to Pb and Cu. Our study investigated the effects of Pb and Cu alone or combined exposure on the morphological parameters of the tail, histological changes of thyroid gland and tail, and gene expression programs involved in cell death of the tail in Bufo gargarizans tadpoles at the climax of metamorphosis. Results demonstrated that Pb, Cu and Pb-Cu mixture exposure resulted in a significantly longer tail compared with control. Damages to notochord, muscle, skin and spinal cord of the tail were found in Pb and Cu exposure groups. The colloid area, the height of follicular cells and number of phagocytic vesicles of thyroid gland in Pb-Cu mixture exposure groups were significantly reduced. In addition, the expression levels of TH, apoptosis, autophagy, degradation of cellular components and oxidative stress-related genes in the tail were significantly altered following Pb and Cu exposure. The present work revealed the relationship between environmental pollutants and tail resorption, providing scientific basis for amphibian protection.

[Synthesis and tissue distribution of bufadienolides in Bufo bufo gargarizans].

This study explored the biosynthesis of bufadienolides(BDs) in Bufo bufo gargarizans to solve the dilemma of the decreasing resources of B. bufo gargarizans and provide a theoretical basis for the sustainable utilization of the resources. Ultra-high performance liquid chromatography-Orbitrap-mass spectrometry(UHPLC-Orbitrap-MS) was employed to detect the synthesis sites of BDs in B. bufo gargarizans, and the results were verified by desorption electrospray ionization-mass spectrometry imaging(DESI-MSI) and homogenate incubation experiments. BDs in B. bufo gargarizans had the highest content in the liver and the highest concentration in the gallbladder, in addition to the parotid gland and skin, which suggested that the liver could synthesize BDs. The results of DESI-MSI also showed that BDs were mainly enriched in the liver rather than the immature parotid gland. The incubation experiment of liver homogenates demonstrated the liver of B. bufo gargarizans had the ability to synthesize BDs. This study showed that the liver was a major organ for the synthesis of BDs in B. bufo gargarizans during metamorphosis, development, and growth, which provided strong theoretical support for the biosynthesis of BDs and the sustainable utilization of B. bufo gargarizans resources.

Spatio-temporal analysis of toxigenic genes expression in the growing Bufo gargarizans based on RNA sequencing data.

BACKGROUND: Bufo gargarizans Cantor, a widely distributed amphibian species in Asia, produces and releases toxins through its retroauricular and granular glands. Although various tissues have been sequenced, the molecular mechanisms underlying the toxin production remain unclear. To elucidate these mechanisms, abdominal skin (non-toxic secretory glands) and retroauricular gland (toxic secreting glands) samples were collected at different time points (3, 6, 12, 24, and 36 months) for RNA sequencing (RNA-seq) and analysis. RESULTS: In comparison to the S group during the same period, a total of 3053, 3026, 1516, 1028, and 2061 differentially expressed genes (DEGs) were identified across five developmental stages. Gene Ontology (GO) analysis revealed that DEGs were primarily enriched in biological processes including cellular processes, single-organism processes, metabolic processes, and biological regulation. In terms of cellular components, the DEGs were predominantly localized in the cell and cell parts, whereas molecular function indicated significant enrichment in binding and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the metabolism and synthesis of various substances, such as lipid metabolism, cofactor and vitamin metabolism, tryptophan metabolism, steroid biosynthesis, and primary bile acid biosynthesis, were accompanied by the development of toads. Additionally, using trend analysis, we discovered candidate genes that were upregulated in the retroauricular glands during development, and the abundance of these genes in the abdominal skin was extremely low. Finally, we identified 26 genes that are likely to be involved in toxin production and that are likely to be involved in toxin anabolism. CONCLUSION: Overall, these results provide new insights into the genes involved in toxin production in B. gargarizans, which will improve our understanding of the molecular mechanisms underlying toxigenic gene expression.

Is developmental plasticity triggered by DNA methylation changes in the invasive cane toad (Rhinella marina)?

Many organisms can adjust their development according to environmental conditions, including the presence of conspecifics. Although this developmental plasticity is common in amphibians, its underlying molecular mechanisms remain largely unknown. Exposure during development to either 'cannibal cues' from older conspecifics, or 'alarm cues' from injured conspecifics, causes reduced growth and survival in cane toad (Rhinella marina) tadpoles. Epigenetic modifications, such as changes in DNA methylation patterns, are a plausible mechanism underlying these developmental plastic responses. Here we tested this hypothesis, and asked whether cannibal cues and alarm cues trigger the same DNA methylation changes in developing cane toads. We found that exposure to both cannibal cues and alarm cues was associated with local changes in DNA methylation patterns. These DNA methylation changes affected genes putatively involved in developmental processes, but in different genomic regions for different conspecific-derived cues. Genetic background explains most of the epigenetic variation among individuals. Overall, the molecular mechanisms triggered by exposure to cannibal cues seem to differ from those triggered by alarm cues. Studies linking epigenetic modifications to transcriptional activity are needed to clarify the proximate mechanisms that regulate developmental plasticity in cane toads.

3α-Hydroxybufadienolides in Bufo gallbladders: structural insights and biotransformation.

Bufadienolides, naturally occurring steroids primarily found in toads, have garnered attention for their pharmacological properties and ecological significance. In this study, we isolated and identified 21 bufadienolides from the gallbladders of Bufo gargarizans, comprising four new compounds and 17 known ones. Notably, the predominance of 15 bufadienolides with a 3α-OH configuration in toad bile differs significantly from the 3ß-OH bufadienolides found in venom secreted by toad glands. Moreover, our investigation into the biotransformation of 3ß-OH and 3α-OH bufadienolides in the liver and kidney tissues of toads revealed an irreversible conversion from 3ß-OH to 3α-OH bufadienolides, suggesting a crucial role in toad self-detoxification. These findings provide valuable insights into the structural diversity of bufadienolides and advance our understanding of their medical and ecological significance.

Comprehensive Analysis of Bufadienolide and Protein Profiles of Gland Secretions from Medicinal Bufo Species.

Toad Venom (TV) is the dried product of toxic secretions from Bufo bufo gargarizans Cantor (BgC) or B. melanostictus Schneider (BmS). Given the increasing medical demand and the severe depletion of wild toads, a number of counterfeit TVs appeared on the market, posing challenges to its quality control. In order to develop an efficient, feasible, and comprehensive approach to evaluate TV quality, a thorough analysis and comparison of chemical compounds among legal species BgC and BmS, as well as the main confusion species B. andrewsi Schmidt (BaS) and B. raddei Strauch (BrS), were conducted by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), high performance liquid chromatography (HPLC), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Nano LC-MS/MS analyses. We identified 126 compounds, including free or conjugated bufadienolides, indole alkaloids and amino acids, among the four Bufo species. The content of main bufadienolides, such as gamabufotalin, bufotalin, bufalin, cinobufagin, and resibufogenin, and the total protein contents varied widely among 28 batches of TV due to their origin species. The sum of the five bufadienolides within the BgC, BmS, BaS, and BrS samples were 8.15-15.93%, 2.45-4.14%, 11.15-13.50%, and 13.21-14.68%, respectively. The total protein content of BgC (6.9-24.4%) and BaS (19.1-20.6%) samples were higher than that of BmS (4.8-20.4%) and BrS (10.1-13.7%) samples. Additionally, a total of 1357 proteins were identified. There were differences between the protein compositions among the samples of the four Bufo species. The results indicated that BgC TV is of the highest quality; BaS and BrS TV could serve as alternative resources, whereas BmS TV performed poorly overall. This research provides evidence for developing approaches to evaluate TV quality and selecting the proper Bufo species as the origin source of TV listed in the Chinese pharmacopoeia.

A new bufadienolide from Bufo gargarizans Cantor.

Bufo gargarizans Cantor (B. gargarizans) is the most widely distributed and abundant species of toad in China. Bufadienolides and indole alkaloids have cardiotonic and anti-tumor activities and are important pharmacological components of B. bufo gargarizans. In this experiment, a novel compound (1) and two known compounds (2 and 3) were isolated and identified from the dry skin of B. bufo gargarizans, both of which are bufadienolides. Various column chromatography methods were used to separate and purify the extract from the dried skin of B. bufo gargarizans. Accurate molecular weights were measured by HR-ESI-MS, and the chemical structure of the compounds was determined by NMR spectrometers. The structures were named as (2ß,5ß,16α)-2,5,16-trihydroxide bufa-14,20,22 dienolide (1), gamabufotalin (2) and desacetylbufotalin (3). In vitro cytotoxic activity assay indicated that compound 1 showed a moderate cytotoxicity against A549 cells with IC50 value of 12.65 µM.

Population declines of a widespread amphibian in agricultural landscapes.

Modern agricultural practices are suspected to play a major role in the ongoing erosion of biodiversity. In order to assess whether this biodiversity loss is linked to past habitat modifications (e.g. land consolidation) or to current consequences of modern agriculture (e.g. use of agrochemicals), it remains essential to monitor species that have persisted in agricultural landscapes to date. In this study, we assessed the presence, abundance and recent population trends of one such species, the spined toad (Bufo spinosus) along a gradient of habitats from preserved (forests) to highly agricultural sites in rural Western France. Our results showed that both presence and abundance of spined toads were markedly lower in reproductive ponds surrounded by intensive agriculture. The most salient result of our study is the ongoing decline of this species in farmland habitats. Indeed, this result suggests that unknown factors are currently affecting a widespread terrestrial amphibian previously thought to persist in agricultural landscapes. These factors have recently induced strong population declines over the course of a few years. Future investigations are required to identify these factors at a time when anthropogenic activities are currently leading to unprecedented rates of biodiversity loss.

Unraveling the antimicrobial potential of Lactiplantibacillus plantarum strains TE0907 and TE1809 sourced from Bufo gargarizans: advancing the frontier of probiotic-based therapeutics.

Introduction: In an era increasingly defined by the challenge of antibiotic resistance, this study offers groundbreaking insights into the antibacterial properties of two distinct Lactiplantibacillus plantarum strains, TE0907 and TE1809, hailing from the unique ecosystem of Bufo gargarizans. It uniquely focuses on elucidating the intricate components and mechanisms that empower these strains with their notable antibacterial capabilities. Methods: The research employs a multi-omics approach, including agar diffusion tests to assess antibacterial efficacy and adhesion assays with HT-29 cells to understand the preliminary mechanisms. Additionally, gas chromatography-mass spectrometry (GC-MS) is employed to analyze the production of organic acids, notably acetic acid, and whole-genome sequencing is utilized to identify genes linked to the biosynthesis of antibiotics and bacteriocin-coding domains. Results: The comparative analysis highlighted the exceptional antibacterial efficacy of strains TE0907 and TE1809, with mean inhibitory zones measured at 14.97 and 15.98 mm, respectively. A pivotal discovery was the significant synthesis of acetic acid in both strains, demonstrated by a robust correlation coefficient (cor ≥ 0.943), linking its abundance to their antimicrobial efficiency. Genomic exploration uncovered a diverse range of elements involved in the biosynthesis of antibiotics similar to tetracycline and vancomycin and potential regions encoding bacteriocins, including Enterolysin and Plantaricin. Conclusion: This research illuminates the remarkable antibacterial efficacy and mechanisms intrinsic to L. plantarum strains TE0907 and TE1809, sourced from B. gargarizans. The findings underscore the strains' extensive biochemical and enzymatic armamentarium, offering valuable insights into their role in antagonizing enteric pathogens. These results lay down a comprehensive analytical foundation for the potential clinical deployment of these strains in safeguarding animal gut health, thereby enriching our understanding of the role of probiotic bacteria in the realm of antimicrobial interventions.

[Molecular identification of animal species of Bufonis Venenum from secretions].

The animal species is one of the key factors affecting the quality of Bufonis Venenum. The quality of Bufonis Venenum derived from Bufo bufo gargarizans is significantly higher than that from B. melanostictus. Since Bufonis Venenum is from secretions, the conventional identification methods are difficult to identify the animal species due to the lack of the appearance and morphology of the animals. The rapid development of molecular identification technology has provided new methods for the identification of Bufonis Venenum. However, because of the low content and serve degradation of residual DNA in secretions, the research on the molecular identification of Chinese medicinal materials from secretions remains to be carried out. To understand the animal species of Bufonis Venenum, this study collected 83 samples of Bufonis Venenum, including 7 commercially available samples, 5 reference medicinal materials, and 71 animal samples from which Bufonis Venenum was prepared according to the method in the 2020 edition of the Chinese Pharmacopoeia. Different DNA extraction methods were used and compared, and the mitochondrial 16S rRNA gene fragments were amplified, on the basis of which the phylogenetic trees were built. Finally, molecular identification of the animal species of the samples was performed. The results showed that the DNA extracted from Bufonis Venenum by the reagent kit had good quality, and 16S rRNA sequences were successfully amplified from 80 out of the 83 samples. In addition, 71 16S rRNA sequences of the animal species of Bufonis Venenum were downloaded from GenBank. The phylogenetic trees constructed based on the neighbor-joining(NJ) method and the Bayesian inference(BI) method showed that the samples derived from B. bufo gargarizans and B. melanostictus were clustered into separate monophyletic clades, with the support of 100%(NJ) and 1.00(BI), respectively. The animal species of both commercially available samples and reference medicinal materials were B. bufo gargarizans. In conclusion, DNA can be extracted from Bufonis Venenum derived from secretions, and the 16S rRNA gene sequences can be amplified, which can be used for molecular identification of the animal species of Bufonis Venenum. The findings provide a reference for the quality control of Bufonis Venenum and the identification of animal species of medicinal materials derived from secretions.

3D Morphological Scanning and Environmental Correlates of Bufo gargarizans in the Yellow River Basin.

Morphology plays a crucial role in understanding the intricacies of biological forms. Traditional morphometric methods, focusing on one- or two-dimensional geometric levels, often fall short of accurately capturing the three-dimensional (3D) structure of organisms. The advent of 3D scanning techniques has revolutionized the study of organismal morphology, enabling comprehensive and accurate measurements. This study employs a 3D structured light scanning system to analyze the morphological variations in the Chinese toad (Bufo gargarizans Cantor, 1842) along the Yellow River Basin. The 3D digital model obtained from the scan was used to calculate various morphological parameters including body surface area, volume, fractal dimensions, and limb size. The research explores geographic variability patterns and identifies environmental drivers affecting the 3D phenotypic variation of B. gargarizans. Results reveal a bimodal pattern of variation in the toad population, with higher elevations exhibiting smaller body sizes, greater appendage proportions, and more complex body structures. Linear regression analyses highlight the influence of elevation and annual mean temperature on the morphological variation of B. gargarizans, with elevation playing a significant role. This study underscores the significance of 3D morphometric analysis in unraveling the intricacies of organismal morphology and understanding the adaptive strategies of species in diverse environments.

A biological invasion modifies the dynamics of a host-parasite arms race.

By imposing novel selection pressures on both participants, biological invasions can modify evolutionary 'arms races' between hosts and parasites. A spatially replicated cross-infection experiment reveals strong spatial divergence in the ability of lungworms (Rhabdias pseudosphaerocephala) to infect invasive cane toads (Rhinella marina) in Australia. In areas colonized for longer than 20 years, toads are more resistant to infection by local strains of parasites than by allopatric strains. The situation reverses at the invasion front, where super-infective parasites have evolved. Invasion-induced shifts in genetic diversity and selective pressures may explain why hosts gain advantage over parasites in long-colonized areas, whereas parasites gain advantage at the invasion front.

Using genome-wide data to ascertain taxonomic status and assess population genetic structure for Houston toads (Bufo [= Anaxyrus] houstonensis).

The Houston toad (Bufo [= Anaxyrus] houstonensis) is an endangered amphibian with a small geographic range. Land-use changes have primarily driven decline in B. houstonensis with population supplementation predominant among efforts to reduce its current extinction risk. However, there has been historic uncertainty regarding the evolutionary and conservation significance of B. houstonensis. To this end, we used 1170 genome-wide nuclear DNA markers to examine phylogenetic relationships between our focal taxon, representatives of the Nearctic B. americanus group, and B. nebulifer, a sympatric Middle American species. Phylogenetic analyses indicate B. houstonensis is a taxon that is distinct from B. americanus. We corroborated such genetic distinctiveness with an admixture analysis that provided support for recent reproductive isolation between B. americanus and B. houstonensis. However, ABBA-BABA tests for ancient admixture indicated historic gene flow between Nearctic species while no signal of historic gene flow was detected between Nearctic and Middle-American species. We used an admixture analysis to recognize four Management Units (MU) based on observed genetic differentiation within B. houstonensis and recommend captive propagation, population supplementation, and habitat restoration efforts specific to each MU. Our results re-affirm the evolutionary novelty of an endangered relict.

In silico molecular characterization of TGF-ß gene family in Bufo bufo: genome-wide analysis.

Bufo bufo is a living example of evolutionary processes due to its numerous physiological and ecological adaptations. This is the first study to genetically characterize the TGF-ß gene family in B. bufo at the genome-wide level, and a total of 28 TGF-ß gene family homologs are identified. Physicochemical characteristics of TGF-ß homologs exhibit a basic nature except for BMP1, BMP4, BMP10, BMP15, AMH, INHA, NODAL Modulator and TGFB1. Phylogenetic analysis divided the TGF-ß gene family homologs into 2 major clades along with other vertebrate species. In domain and motif composition analysis, the gene structure for all TGF-ß homologs exhibited homogeneity except BMP1. We have identified the TGF-ß propeptide domain together with the TGF-ß in all family homologs of TGF-ß superfamily. Gene structure comparisons indicated that the TGF-ß gene family have arisen by gene duplications. We also identified 10 duplicated gene pairs, all of which were detected to be segmental duplications. The Ka/Ks test ratio findings for every pair of genes revealed that none of the ratios surpassed 1 except for one gene pair (INHA/BMP1), indicating that these proteins are under positive selection. Circos analysis showed that TGF-ß gene family homologs are arranged in 11 dispersed clusters and all were segmentally arrayed in the genome. This study provides a molecular basis for TGF-ß ligand protein functional analysis and may serve as a reference for in-depth phylogenomics and may promote the development of novel strategies.Communicated by Ramaswamy H. Sarma.

Elevational variation in metabolic rate, feeding capacity and their associations in the Asiatic toad Bufo gargarizans.

Foraging behavior is known to place demands on the metabolic characteristics of anurans. Active foragers feeding on sedentary prey typically have high aerobic capacity and low anaerobic capacity, whereas sit-and-wait foragers feeding on active and mobile prey have the opposite pattern. Thus, the energetic demands of foraging may influence their metabolic adaptations to harsh environments, such as high elevations. Anurans that engage in active foraging have been found to increase maximum metabolic rate (MMR) and aerobic scope (AS, the difference between MMR and resting metabolic rate, RMR) at high elevations. However, data are lacking in amphibian ambush foragers. In this study, we examined the RMR, MMR, AS, and feeding capacity of a sit-and-wait forager ─the Asiatic toad (Bufo gargarizans), from two populations that are in close geographic proximity but differ by 1350 m in elevation. Our results show that there is no elevational variation in RMR and feeding capacity in either males or females. However, there are sex-specific variations in MMR and AS along an elevational gradient; females from high elevations have lower MMR and smaller net AS than their counterparts from low elevations while males maintain similar MMR and net AS across elevations. Furthermore, aerobic performances do not appear to be associated with feeding capacity at either the individual or population level. Our results support the hypothesis that sit-and-wait foragers may not increase their aerobic capacity as a strategy in hypoxic and low food availability environments and the role of sex in these adaptive adjustments should not be overlooked.

An amphibian-derived cathelicidin accelerates cutaneous wound healing through its main regulatory effect on phagocytes.

Cathelicidins are an important family of antimicrobial peptides (AMPs) involved in the innate immunity in vertebrates. The mammalian cathelicidins have been well characterized, but the relationship between structure and function in amphibian cathelicidins is still not well understood. In this study, a novel 29-residue cathelicidin antimicrobial peptide (BugaCATH) was identified from the skin of Bufo gargarizans. Unlike other AMPs, BugaCATH does not display any direct antimicrobial effects in vitro. However, it effectively promotes full-thickness wound repair in mice. Following injury, BugaCATH initiates and expedites the inflammatory stage by recruiting neutrophils and macrophages to the wound site. BugaCATH not only regulates neutrophil phagocytic activity but also stimulates the generation of cytokines (TNF-α, IL-6, and IL-1ß) and chemokines (CXCL1, CXCL2, CCL2, and CCL3) in macrophages and in mice. Furthermore, it promotes macrophage M2 polarization that facilitates the conversion from a pro-inflammatory macrophage-dominated wound environment to an anti-inflammatory one during the mid to late stages, which is crucial for reducing inflammation and effective wound repair. The MAPK (ERK, JNK, and p38) and NF-κB-NLRP3 signaling pathways are involved in the activity. Moreover, BugaCATH directly enhances the migration of keratinocytes and vascular endothelial cells without affecting their proliferation. Notably, BugaCATH significantly improves the proliferation of keratinocytes and endothelial cells in the presence of macrophages. The current study revealed that in addition to proliferation of keratinocytes and endothelial cells, BugaCATH possesses the ability to modulate inflammatory processes during skin injury through its regulatory effect on phagocytes. The combination of these capabilities makes BugaCATH a potent candidate for skin wound therapy.

Larval Body Shape and Eye Size Variation in Japanese Toads (Anura: Bufonidae: Bufo): An Ecological Implication for Lentic and Lotic Habitats.

Variation in morphological traits of anurans has evolved due to the pressures imposed by inhabiting different environments. The Japanese stream toad, Bufo torrenticola, breeds in running water, such as mountain streams, where the larvae grow. This lotic-breeding habit of B. torrenticola was suggested to have evolved from a lentic-breeding ancestor. Evolutionary shift of breeding habit from lentic- to lotic-breeding caused larval morphological changes to adapt to the stream habitat. However, morphological adaptation associated with the larval habitat of these three types of Japanese toads has not been explored well. In this study, we applied geometric morphometrics and distance measurements to compare body and eye characters among the tadpoles of three Japanese toads. The results showed that B. torrenticola has more dorsally and posteriorly positioned eyes, and a longer and wider rostrum because of having a larger mouth and more-developed oral muscles than its close relatives. These characters might be related to lotic lifestyle. Meanwhile, tadpoles of B. torrenticola and its sister taxon B. japonicus japonicus showed similar eye growth pattern, supporting their close phylogenetic relationship. Some of the lotic-adapted characters have also been reported in other lotic tadpoles, which is indicative of convergent evolution among stream-adapted tadpoles.

Keep the ball rolling: sexual differences in conglobation behavior of a terrestrial isopod under different degrees of perceived predation pressure.

Background: Antipredator behaviors are theoretically subjected to a balance by which their display should be minimized when their benefits do not outweigh their costs. Such costs may be not only energetic, but also entail a reduction in the time available for other fitness-enhancing behaviors. However, these behaviors are only beneficial under predation risk. Therefore, antipredator behaviors are predicted to be maximized under strong predation risk. Moreover, predation pressure can differ among individuals according to traits such as sex or body size, if these traits increase vulnerability. Antipredator behaviors are expected to be maximized in individuals whose traits make them more conspicuous to predators. However, how sex, body size and antipredator behaviors interact is not always understood. Methods: In this work, I tested the interaction between sex, body size and antipredator behavior in the common pill woodlouse (Armadillidium vulgare), which conglobate (i.e., they roll up their bodies almost conforming a sphere that conceals their appendages) in response to predator attacks. Specifically, I tested whether latency to unroll after a standardized mechanical induction was greater in animals exposed to predator chemical cues (toad feces) than in conspecifics exposed to cues of non-predatory animals (rabbits) or no chemical cues whatsoever (distilled water), incorporating sex and body mass in the analyses. Results: In agreement with my prediction, latency to unroll was greater in individuals exposed to predator chemical cues. In other words, these animals engage in conglobation for longer under perceived predator vicinity. However, this result was only true for males. This sexual dimorphism in antipredator behavior could result from males being under greater predation risk than females, thus having evolved more refined antipredator strategies. Indeed, males of this species are known to actively search for females, which makes them more prone to superficial ground mobility, and likely to being detected by predators. Body size was unrelated to latency to unroll. As a whole, these results support the hypothesis that antipredator behavior is tuned to predator cues in a way consistent with a balance between costs and benefits, which might differ between the sexes.