Spatially limited pathogen pollution in an invasive tick and host system.

Expansion of global commerce has facilitated pathogen pollution via the transportation and translocation of invasive species and their associated parasites and pathogens. In Florida, imported cane toads (Rhinella horribilis) were accidentally and intentionally released on multiple occasions. Early populations were found to be infested with the invasive tick, Amblyomma rotundatum, yet it is unknown if these ticks dispersed with their hosts as cane toads spread throughout much of the state. The objectives of our investigation were to (1) determine if there are fewer tick infestations on toads at the periphery than at the core of their distribution as predicted by founder effect events, and (2) identify if ticks were infected with exotic pathogens. We captured toads from 10 populations across Florida. We collected ticks, vent tissue, and tick attachment site tissue from each toad, then tested samples for bacteria in the genus, Rickettsia. We found that 3/10 populations had toads that were infested with A. rotundatum, and infested individuals were in the earliest introduced populations at the core of their distribution. Pathogen testing confirmed Rickettisa bellii in ticks, but not in toad tissues. Haplotype networks could not clearly distinguish if R. bellii in Florida was more closely related to North or South American strains, but host-tick associations suggest that the pathogen was exotic to Florida. Our investigation demonstrated that an invasive species facilitated the introduction of parasites and pathogens into Florida, yet the invasive tick species encountered limitations to dispersal on this host species. Supplementary Information: The online version contains supplementary material available at 10.1007/s10530-024-03291-9.

Insights into phylogenetic positions and distribution patterns: Complete mitogenomes of two sympatric Asian horned toads in Boulenophrys (Anura: Megophryidae).

Boulenophrys sangzhiensis and Boulenophrys tuberogranulata, two narrow-distributed toad species within the Megophryidae family in southern China, are experiencing population declines due to habitat loss and degradation. Despite their critical conservation status, the two species remain largely overlooked in public and scientific spheres. This study presented the first sequencing, assembly, and annotation of the complete mitogenomes of both species using next-generation sequencing. The mitogenome of B. sangzhiensis was 16,950 bp, while that of B. tuberogranulata was 16,841 bp, each comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a noncoding control region (D-loop). The gene content, nucleotide composition, and evolutionary rates of each mitogenome were analyzed. Both mitogenomes exhibited negative AT skew and GC skew with high A + T content. ATP8 exhibited the highest evolutionary rate, while COI had the lowest. A phylogenetic analysis based on 28 mitogenomes revealed two major clades of Megophryidae, supporting the classification of two subfamilies, Megophryinae and Leptobrachiinae. Within the subfamily Megophryinae, the genus Boulenophrys was divided into two species groups. Intriguingly, despite coexisting in Zhangjiajie City, B. sangzhiensis and B. tuberogranulata exhibited distinct origins from the two different species groups, underscoring the unique role of the coexisting area Zhangjiajie in driving their speciation and preserving their current populations. A parallel pattern was also identified in the Leptobrachiinae genus Leptobrachium within the same region. This study provided valuable data references and enhanced our understanding of the molecular characteristics of these threatened amphibian species.

Morphology and vocalization comparison of the Houston Toad and the Dwarf American Toad: implications for their historic range.

Documenting changes in the distribution and abundance of a given taxon requires historical data. In the absence of long-term monitoring data collected throughout the range of a taxon, conservation biologists often rely on preserved museum specimens to determine the past or present, putative geographic distribution. Distributional data for the Houston Toad (Anaxyrus houstonensis) has consistently been confounded by similarities with a sympatric congener, the Dwarf American Toad (A. americanus charlesmithi), both in monitoring data derived from chorusing surveys, and in historical data via museum specimens. In this case, misidentification can have unintended impacts on conservation efforts, where the Houston Toad is federally endangered, and the Dwarf American Toad is of least concern. Previously published reports have compared these two taxon on the basis of their male advertisement call and morphological appearance, often with the goal of using these characters to substantiate their taxonomic status prior to the advent of DNA sequencing technology. However, numerous studies report findings that contradict one another, and no consensus on the true differences or similarities can be drawn. Here, we use contemporary recordings of wild populations of each taxon to test for quantifiable differences in male advertisement call. Additionally, we quantitatively examine a subset of vouchered museum specimens representing each taxon to test previously reported differentiating morphometric characters used to distinguish among other Bufonids of East-Central Texas, USA. Finally, we assemble and qualitatively evaluate a database of photographs representing catalogued museum vouchers for each taxon to determine if their previously documented historic ranges may be larger than are currently accepted. Our findings reveal quantifiable differences between two allopatric congeners with respect to their male advertisement call, whereas we found similarities among their detailed morphology. Additionally, we report on the existence of additional, historically overlooked, museum records for the Houston Toad in the context of its putative historic range, and discuss errors associated with the curation of these specimens whose identity and nomenclature have not been consistent through time. These results bookend decades of disagreement regarding the morphology, voice, and historic distribution of these taxa, and alert practitioners of conservation efforts for the Houston Toad to previously unreported locations of occurrence.

The role of phenotypic plasticity and corticosterone in coping with pond drying conditions in yellow-bellied toad (Bombina variegata, Linnaeus 1758) tadpoles.

Amphibian larvae inhabiting temporary ponds often exhibit the capacity to accelerate development and undergo metamorphosis in challenging conditions like desiccation. However, not all species exhibit this ability, the yellow-bellied toad (Bombina variegata) is one such example. The underlying mechanisms behind the inability to accelerate development under desiccation remain largely unexplored. The hypothalamic-pituitary-interrenal (HPI) axis and corticosterone (CORT), which act synergistically with thyroid hormone, are thought to facilitate metamorphosis in response to desiccation stress. In this study, we aimed to investigate whether modification in the HPI axis, particularly CORT levels, contributes to the absence of adaptive plasticity in B. variegata under desiccation stress. The study design included four treatments: high water level, high water level with exogenous CORT, low water level, and low water level with metyrapone (a CORT synthesis inhibitor). The main objective was to evaluate the effects of these treatments on whole-body corticosterone levels, life history, morphological traits, and oxidative stress parameters during the prometamorphic and metamorphic climax developmental stages. While low water level had no effect on total corticosterone levels, larval period, body condition index, and metamorphic body shape, it negatively affected metamorph size, mass, and growth rate. Our findings suggest that constant exposure to desiccation stress over generations may have led to modifications in the HPI axis activity in B. variegata, resulting in adaptation to changes in water level, evident through the absence of stress response. Consequently, CORT may not be a relevant stress indicator in desiccation conditions for this species.

Isolation, Identification and Chemical Modification of Bufadienolides from Bufo melanostictus Schneider and Their Cytotoxic Activities against Prostate Cancer Cells.

The traditional Chinese medicine toad venom (Venenum bufonis) has been extensively used to treat various diseases, including cancers, in China and other Southeast Asian countries. The major constituents of toad venom, e.g., bufadienolides and alkaloids, exhibit broad-spectrum pharmacological effects in cancers. Herein, two new bufadienolides (1 and 2), along with eleven known compounds (3-13) were successfully isolated from Bufo melanostictus Schneider. Their structures were elucidated by extensive spectroscopic data and X-ray diffraction analysis. Furthermore, four lactam derivatives were synthesized through the transformation of bufadienolides lactones. The inhibitory effects of these compounds against human prostate cancer cell lines PC-3 and DU145 were evaluated. The outcomes indicated a notable trend, with a substantial subset displaying nanomolar range IC50 values against PC-3 and DU145 cells, underscoring their pronounced cytotoxicity. Moreover, a noteworthy distinction surfaces, wherein lactones consistently outperformed their lactam counterparts, further validating their heightened potency for the treatment of prostate cancer. This study contributes significant preclinical evidence substantiating the therapeutic viability of bufadienolides and toad venom as intervention strategies for prostate cancer.

Evolution of air-borne vocalization: Insights from neural studies in the archeobatrachian species Bombina orientalis.

Vocalization of tetrapods evolved as an air-driven mechanism. Thus, it is conceivable that the underlaying neural network might have evolved from more ancient respiratory circuits and be made up of homologous components that generate breathing rhythms across vertebrates. In this context, the extant species of stem anurans provide an opportunity to analyze the connection of the neural circuits of lung ventilation and vocalization. Here, we analyzed the fictive lung ventilation and vocalization behavior of isolated brains of the Chinese fire-bellied toad Bombina orientalis during their mating season by nerve root recordings. We discovered significant differences in durations of activation of male brains after stimulation of the statoacoustic nerve or vocalization-relevant forebrain structures in comparison to female brains. The increased durations of motor nerve activities in male brains can be interpreted as fictive calling, as male's advertisement calls in vivo had the same general pattern compared to lung ventilation, but longer duration periods. Female brains react to the corresponding stimulations with the same shorter activity pattern that occurred spontaneously in both female and male brains and thus can be interpreted as fictive lung ventilations. These results support the hypothesis that vocal circuits evolved from ancient respiration networks in the anuran caudal hindbrain. Moreover, we could show that the terrestrial stem archeobatrachian Bombina spec. is an appropriate model to study the function and evolution of the shared network of lung ventilation and vocal generation.

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.

Integrated post-acquisition data processing strategy for rapid steroid sulfate characterization in Toad gall-bladder.

LC-MS serves as a workhorse for chemical profile characterization of Chinese medicinal materials (CMMs) attributing to the ability of measuring fruitful MS/MS spectral information. However, it is laborious to extract the information belonging to the compounds-of-interest from the massive data matrixes even employing those well-defined post-acquisition data processing strategies. Here, efforts were devoted to propose an integrated strategy allowing rapid chemical homologs-focused data filtering through integrating the fit-for-purpose existing strategies, such as molecular weight imprinting (MWI), diagnostic fragment ion filtering (DFIF), neutral loss filtering (NLF), and isotope pattern filtering (IPF). Homologs-focused chemical characterization of a precious CMM namely Toad gall-bladder (Chinese name: Chandan) that is rich of diverse effective steroid sulfates, particularly bufogenin sulfates, bile acid sulfates and bilichol sulfates, was employed as a proof-of-concept. Recombinant human SULT2A1-catalyzed in vitro metabolism was undertaken to generate eight bufogenin sulfates to facilitate summarizing MS/MS spectral behaviors. After in-house data library construction and MS1 and MS2 spectral acquisition, data filtering was conducted as follows: 1) MWI and IPF was utilized in combination to capture deprotonated molecular ions and the 34S isotopic ions for the sulfates of those reported steroids; 2) m/z 79.9568 (SO3-·) and 96.9596 (HSO4-) were applied to DFIF; and 3) SO3 (79.9568 Da) served as the feature to achieve NLF. Those captured MS/MS information subsequently participated in tentatively structural annotation through applying those empirical mass fragmentation rules. As a result, 71 compounds including 7 bufogenin sulfates, 17 bile acid sulfates, 13 bilichol sulfates and a C-23 steroid sulfate were detected from Toad gall-bladder and thereof, 39 ones received plausible identities assignment. Above all, the steroid sulfates in Toad gall-bladder were profiled in depth, and more importantly, the proposed strategy should be a meaningful option for, but not limited to, submetabolome characterization in CMMs.

Exploring peptides from toad venom for source identification by LC-MS/MS using MRM method.

Toad venom is a traditional Chinese medicine (TCM) with various sources and wide-ranging preparations. Previous quality assessment studies primarily concentrated on small molecular compounds like toad dienolactones and indole alkaloids, studies on macromolecular peptides and proteins as quality assessment standards remained at the qualitative stage, lacking the development of practical and convenient quantitative methods. In this study, to explore the peptides from toad venom as a new method for identifying and evaluating its source, a complete scan of the water extract of peptides from toad venom was conducted using HPLC-Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF) 5600, leading to the identification of peptides based on mass spectrometry data. Subsequently, HPLC- Quadrupole-Linear Ion Trap Mass Spectrometer (Q-Trap) 5500 employing Multiple Reaction Monitoring (MRM) mode was utilized to quantitatively analyze peptides in various sources of toad venom, followed by Partial Least Squares Discriminant Analysis (PLS-DA) to further analyze the data and evaluate the effectiveness. This study highlights the importance of exploring macromolecular substance in natural products research and provides a foundation for further studies on toad venom.

High frequency of larval abnormalities of the toad Rhinella arenarum (Anura: Bufonidae) in an Argentinian agroecosystem

Introduction: The frequent use of pesticides is currently considered a cause of environmental pollution due to the high rate of entry of these substances into agroecosystems. This constitutes a risk for the species that inhabit these ecosystems, in particular anurans whose characteristics make them prone to exposure to and interaction with environmental pollutants. Objective: To report the occurrence of abnormalities in larvae of the common toad Rhinella arenarum inhabiting ponds surrounded by agroecosystems. Methods: In two consecutive springs (2015 and 2016), reproductive events of common toads were monitored in temporary pond systems in agricultural and non-agricultural areas, located near the city of La Plata (Buenos Aires, Argentina). The physicochemical parameters of the ponds were measured, and the stage of each reproductive event was recorded, such as the numbers of adult toads, amplexus and clutches. In the laboratory, the larvae were measured and photographed, their stage of development was recorded, and their morphology was examined under a stereomicroscope. Representative samples (normal and abnormal) from each pond studied were processed for histopathological analysis. Results: In the field studies carried out on a population of R. arenarum collected in an agroecosystem, a lower number of reproductive adults and clutches were observed in relation to the population of a non-agricultural pond. A total of 1 910 larvae were collected: 529 and 1 381 larvae from ponds located in non-agricultural and agricultural areas, respectively. Larvae from the agroecosystem showed two types of abnormalities: severe tail flexure and abdominal bloating. In addition, five degrees of severity could be determined in relation to abdominal bloating. Conclusions: This work reports the high frequency and severity of abnormalities observed in the early stages of R. arenarum larvae living within an agroecosystem, providing evidence of the negative impact that agricultural activities cause on aquatic ecosystems surrounded by farming areas.

Introducción: El uso frecuente de plaguicidas es considerado actualmente una causa de contaminación ambiental debido a las altas tasas de ingreso de estas sustancias a los agroecosistemas. Esta situación es un riesgo para las especies que habitan en estos ecosistemas, en particular los anuros cuyas características los hacen propensos a la exposición e interacción con contaminantes ambientales. Objetivo: Informar la presencia de anormalidades en larvas del sapo común Rhinella arenarum que habitan en estanques rodeados por un agroecosistema. Métodos: En dos primaveras consecutivas (2015 y 2016), se monitorearon los eventos reproductivos del sapo común proveniente de sistemas de estanques temporales ubicados en zonas agrícolas y no agrícolas, cerca de la ciudad de La Plata (Buenos Aires, Argentina). Se midieron los parámetros fisicoquímicos de los estanques y se registraron las etapas de cada evento reproductivo como el número de sapos adultos, amplexos y nidadas. En el laboratorio, las larvas fueron medidas y fotografiadas, se registró su estado de desarrollo y se examinó la morfología de cada una bajo microscopio estereoscópico. Se procesaron muestras representativas (normales y anormales) de cada estanque estudiado para análisis histopatológico. Resultados: En la población de R. arenarum que vive dentro de un agroecosistema, se observó un menor número de adultos reproductores y puestas en relación con la del estanque en la zona no agrícola. Se recolectaron un total de 1 910 larvas: 529 y 1 381 larvas de estanques ubicados en zonas no agrícolas y agrícolas, respectivamente. Las larvas del agroecosistema mostraron dos tipos de anormalidades: severa flexión de la cola y distensión abdominal. Además, se pudo determinar cinco grados de gravedad en relación con la distensión abdominal. Conclusiones: Una alta frecuencia y severidad de anormalidades en los estadios tempranos de larvas de R. arenarum que viven dentro de un agroecosistema proporciona evidencia del impacto negativo que las actividades agrícolas causan en los ecosistemas acuáticos rodeados por áreas de cultivo.

Geographical Variation in Body Size in the Asian Common Toad (Duttaphrynus melanostictus).

The geographic variation in life-history traits of organisms and the mechanisms underlying adaptation are interesting ideas in evolutionary biology. This study investigated age and body size of the Asian common toad (Duttaphrynus melanostictus) among five populations along a geographical gradient. We found that geographical variation in age was non-significant among populations but there was a significant and positive correlation between mean age and body size. Although the body size values at 1043 m are quite different from other sites, after controlling for age effects, there was a significant positive correlation between altitude and body size. Our findings followed the predictions of Bergmann's rule, suggesting that the body size of D. melanostictus is potentially influenced by the low air temperatures at higher altitudes.

Neoplasia in an assurance population of Puerto Rican crested toads (Peltophryne lemur).

Disease monitoring of amphibian assurance populations is an important buffer against ongoing global extinctions. This study documents a high incidence of neoplasia in a zoo-managed assurance population of Puerto Rican crested toads (Peltophryne lemur; PRCTs). Over 5 years, neoplasia was diagnosed in 17/49 (35%) submitted adult PRCTs and was the cause of death or euthanasia in 13/17 (72%). Most toads were male (16/17; 94%) and 6 to 11-years-old (average 8.1 years). Notably, seven toads (41%) had multiple neoplasms. Of the 29 neoplasms identified, 17 (59%) were cutaneous or subcutaneous. The most common neoplasms included mast cell tumors (MCTs; 8/29; 28%), histiocytic sarcomas (6/29; 21%), lymphoma/leukemia (4/29; 14%), and squamous cell carcinomas (3/29; 10%). Distant metastases were documented in 6/8 (75%) toads with MCTs. Causes for neoplasia in this population were not determined though may include genetic or environmental factors. Continued investigations of managed endangered amphibians will help elucidate mechanisms of carcinogenesis.

DO Toxic Invasive Prey Become a Toxin Source for Native Consumers?

Toxic organisms can become food that potentially harms consumers. When these organisms become invasive species, the harm often turns to a serious threat that disrupts native ecosystems. On the other hand, there are consumers that can exploit toxic organisms for food and sequester intact toxins from them for the consumers' own chemical defense. Therefore, it can be expected that toxic invasive prey can become a toxin source for native consumers. Here, we focused on the relationship between toads, which are one of the major toxic invasive organisms and possess bufadienolides (BDs), and Rhabdophis snakes, which sequester BDs from toads. On Sado Island, Japan, R. tigrinus is native, but no toads had inhabited this island until Bufo japonicus formosus was introduced as a domestic invasive species in 1963 and 1964. At present, invasive toads are distributed only in the southwestern part of the island. We collected a total of 25 and 24 R. tigrinus from areas allopatric and sympatric with toads, respectively. Then, we investigated the possession of BDs and the BD profile of these snakes. We found that only R. tigrinus sympatric with toads possessed BDs, whereas all snakes allopatric with toads lacked BDs. Based on the characteristics of the BD profile, the toxin source was identified as B. j. formosus. Our findings show that a new case of impact caused by toxic invasive species, i.e., "toxin supply to native consumers from invasive prey", could occur.

Thimet oligopeptidase (THOP 1) distribution in cane toad (Bufo Marinus, Linnaeus, 1758) brain.

Thimet oligopeptides (THOP 1) is a metal-dependent peptidase involved in the metabolism of neuropeptides and the presentation of peptides via MHC-1. It has been shown to play a role in the regulation of protein-protein interactions and the metabolism of intracellular peptides. THOP 1 is associated with important biological processes such as metabolism and neurodegenerative diseases, among others. The objective of this study is to elucidate the distribution of THOP 1 in the Bufo marinus brain. The analysis of THOP 1 amino acid sequences indicates that they have been conserved throughout evolution, with significant homology observed across various phyla. When comparing amphibians with other species, more than 70% identity can be identified. Immunohistochemistry analysis of the toad's brain has demonstrated that the enzyme has a ubiquitous distribution, consistent with previous findings in mammals. THOP 1 can be found in important areas of the brain, such as bulb, thalamic nuclei, striatum, hypothalamus, and among others. Nonetheless, THOP 1 is consistently localized within the nucleus, a pattern also observed in the rat brain. Therefore, based on these results, the toad appears to be an excellent model for studying the general biology of THOP 1, given the substantial homology of this enzyme with mammals and its similarity in distribution within the brain.

Gamete Collection, Artificial Fertilization and Captive-Rearing of Eggs in a Terrestrial-Breeding Anuran with Parental Care: Alytes obstetricans.

The genus Alytes consists of six primitive terrestrial-breeding species (five of them endangered or present in restricted distributions) with unique male parental care. Alytes obstetricans was used as a model for the development of assisted reproductive technologies for gamete collection, artificial fertilization and captive-rearing of embryos. Sperm collection was achieved with human chorionic gonadotropin (hCG), but only 17 to 50% of males responded to the hormone. In addition, an effect of captivity on sperm quality was observed. A combination of gonadotropin-releasing hormone agonist (GnRHa) and hCG was tested for induction of ovulation, and 100% of females responded to the treatment. Recently collected sperm was used in artificial fertilization, and 10% of eggs showed complete embryo development. To design a method for captive-rearing of embryos, natural clutches (n = 13) were removed from males and divided into two sub-clutches. One was kept with all eggs together and in the other, the egg string was cut and eggs were split individually. All sub-clutches with eggs kept separated presented embryo survival (ranging from 27 to 61%) that hatched normally, while only one sub-clutch with embryos maintained together had some embryo survival (36%). These results may contribute to the management of this and related species.

A cane toad (Rhinella marina) N-methyltransferase converts primary indolethylamines to tertiary psychedelic amines.

Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include 'magic mushrooms' (Psilocybe spp.), plants used to prepare ayahuasca, and toads. The skin and parotid glands of certain toads accumulate a variety of specialized metabolites including toxic guanidine alkaloids, lipophilic alkaloids, poisonous steroids, and hallucinogenic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin. The occurrence of psychedelics has contributed to the ceremonial use of toads, particularly among Mesoamerican peoples. Yet, the biosynthesis of psychedelic alkaloids has not been elucidated. Herein, we report a novel indolethylamine N-methyltransferase (RmNMT) from cane toad (Rhinella marina). The RmNMT sequence was used to identify a related NMT from the common toad, Bufo bufo. Close homologs from various frog species were inactive, suggesting a role for psychedelic indolethylamine biosynthesis in toads. Enzyme kinetic analyses and comparison with functionally similar enzymes showed that recombinant RmNMT was an effective catalyst and not product inhibited. The substrate promiscuity of RmNMT enabled the bioproduction of a variety of substituted indolethylamines at levels sufficient for purification, pharmacological screening, and metabolic stability assays. Since the therapeutic potential of psychedelics has been linked to activity at serotonergic receptors, we evaluated binding of derivatives at 5-HT1A and 5-HT2A receptors. Primary amines exhibited enhanced affinity at the 5-HT1A receptor compared with tertiary amines. With the exception of 6-substituted derivatives, N,N-dimethylation also protected against catabolism by liver microsomes.

Quantification of chlamydiae in the endangered Houston toad (Anaxyrus houstonensis).

Two primer set/probe combinations targeting variable regions on the 23S rRNA gene were designed to detect and quantify chlamydiae in DNA extracted from brain swabs of the endangered Houston toad (Anaxyrus houstonensis) using SYBRGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Prevalence and abundance values for samples were generally different between SYBRGreen- and Taqman-based detection methods, with higher specificity observed for Taqman-based detection. Of the 314 samples analyzed, initial screening with SYBRGreen-based qPCR retrieved 138 positive samples, of which 52 were confirmed by Taqman-based analyses as chlamydiae. All of these samples were subsequently identified as Chlamydia pneumoniae by specific qPCR and confirmed by comparative sequence analyses of 23S rRNA gene amplicons. These results demonstrate the usefulness of our developed qPCR methods to screen for and verify prevalence of chlamydiae in DNA of brain swabs, and ultimately specifically identify and quantify chlamydiae, specifically C. pneumoniae in these samples.

Increasingly cautious sampling, not the black colouration of unpalatable prey, is used by fish in avoidance learning.

The efficiency of aposematic colouration of prey is based on the innate bias or facilitation of avoidance learning of predators. In many toxic bufonids, larvae are uniformly black, which is considered a warning signal. We compared fish predation on normal (black) and 'transient albino' (greyish) common toad Bufo bufo tadpoles that did not differ in toxicity or activity. In a two-stage experiment, each fish was presented with tadpoles of one colour in the first trial and the other colour in a subsequent trial. While tadpoles sampled by fish were typically not ingested, some died from injuries. The attack rate did not differ between tadpole phenotypes nor trials, irrespective of which phenotype was the first exposed to the fish. However, during the second trial, the sampled tadpoles, independent of colouration, were mouthed by fish for shorter periods and tadpole mortality decreased. The duration of mouthing also declined with an increasing number of attacks during subsequent trials. We conclude that in single-species prey populations, black tadpole colouration is not a warning signal as it does not accelerate predator learning about prey unprofitability. Our results indicate that with growing experience, predators sample potentially toxic prey more cautiously. This may explain why natural selection does not eliminate aposematic morphs even if predators continuously sample conspicuous prey.