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.

Cutaneous shedding in amphibians causes shifts in bacterial microbiomes.

Considerable research has focused on microbes on amphibian skin, as they act as the first line of defense against invading pathogens. This effort has generated substantial data on patterns across species, space, time, and ontogeny, alongside a growing list of beneficial antifungal symbionts. Though there is evidence of stability in amphibian skin microbial communities, there is also an indication that regular skin shedding reduces cultivable bacteria, with regrowth and recolonization in the period between sheds. This suggests that skin communities are in constant flux, and we lack an understanding of how the membership and structure of those communities are affected by shedding events. In this study, we conducted experiments on cane toads (Rhinella marina) to investigate the influence of shedding on skin microbiomes. We first used quantitative PCR to verify a positive correlation between bacterial loads and time in the days after shedding. We then resampled individuals over time to describe changes in community composition in the 38 h after shedding using amplicon sequencing. Similar to trends of bacterial loads, we found increases in alpha diversity over time after shedding, suggesting that shedding reduces bacterial diversity as it knocks down bacterial loads. During the 38-h period, community structure became similar to pre-shed communities in some individuals, but there was no consistent pattern in structural changes among individuals. In light of the amphibian chytridiomycosis pandemic, understanding how physiological events such as skin shedding affect beneficial bacteria and communities on amphibians would provide important insight into amphibian ecology.

Whole-mitogenome analysis unveils previously undescribed genetic diversity in cane toads across their invasion trajectory.

Invasive species offer insights into rapid adaptation to novel environments. The iconic cane toad (Rhinella marina) is an excellent model for studying rapid adaptation during invasion. Previous research using the mitochondrial NADH dehydrogenase 3 (ND3) gene in Hawai'ian and Australian invasive populations found a single haplotype, indicating an extreme genetic bottleneck following introduction. Nuclear genetic diversity also exhibited reductions across the genome in these two populations. Here, we investigated the mitochondrial genomics of cane toads across this invasion trajectory. We created the first reference mitochondrial genome for this species using long-read sequence data. We combined whole-genome resequencing data of 15 toads with published transcriptomic data of 125 individuals to construct nearly complete mitochondrial genomes from the native (French Guiana) and introduced (Hawai'i and Australia) ranges for population genomic analyses. In agreement with previous investigations of these populations, we identified genetic bottlenecks in both Hawai'ian and Australian introduced populations, alongside evidence of population expansion in the invasive ranges. Although mitochondrial genetic diversity in introduced populations was reduced, our results revealed that it had been underestimated: we identified 45 mitochondrial haplotypes in Hawai'ian and Australian samples, none of which were found in the native range. Additionally, we identified two distinct groups of haplotypes from the native range, separated by a minimum of 110 base pairs (0.6%). These findings enhance our understanding of how invasion has shaped the genetic landscape of this species.

The reduction in arterial pH with increased temperature is not affected by hyperoxia in toads (Rhinella marina) and pythons (Python molurus).

It is well established that arterial pH decreases with increased temperature in amphibians and reptiles through an elevation of arterial PCO2, but the underlying regulation remains controversial. The alphastat hypothesis ascribes the pH fall to a ventilatory regulation of protein ionisation, but the pH reduction with temperature is lower than predicted by the pKa change of the imidazole group on histidine. We hypothesised that arterial pH decreases at high, but not at low, temperatures when toads (Rhinella marina) and snakes (Python molurus) are exposed to hyperoxia. In toads, hyperoxia caused similar elevations of arterial PCO2 at 20 and 30°C, indicative of a temperature-independent oxygen-mediated drive to breathing, whereas PCO2 was unaffected by hyperoxia in snakes at 25 and 35°C. These findings do not support our hypothesis of an increased oxygen-mediated drive to breathing as body temperature increases.

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.

Ecology of fear: predator avoidance reduces seed dispersal in an ant.

The ecology of fear refers to the non-fatal cost that predators and parasites impose on prey populations. These non-consumptive effects (NCEs) can influence animal-plant interactions, but evidence thereof comes mainly from vertebrate systems with less focus on invertebrates. Here, I investigated whether the foraging behaviour of the ant Ectatomma ruidum was influenced by its primary predator, the forest toad Rhinella alata. In field tests, the probability of seed removal by the ants was 25% for seeds placed with the forest toad compared to 32% for control seeds, suggesting that toads reduce ant foraging rates. A further experiment revealed that ants which had previously encountered the predator and its faeces were more likely (59%) than inexperienced ants (50%) to avoid the exit with the predator faeces. This outcome suggests that ants are capable of learning cues associated with predation risk, possibly leading to NCEs. This indicates that predators can exert NCEs on invertebrate prey with potential cascading effects on seed dispersal, extending results previously seen only in vertebrate seed dispersal systems.

An overview of the osseous palmar sesamoid in Anura, with the particular case of some Rhinella species.

Background: Sesamoids are generally regarded as structures that are not part of the tetrapod body plan. The presence of a palmar sesamoid is assumed to serve as a distribution point for the forces of the flexor digitorum communis muscle to the flexor tendons of the digits, which are embedded in the flexor plate. It has been considered that the palmar sesamoid is present in most anuran groups, and it has been suggested that it acts by inhibiting the closing of the palm, preventing grasping. Typical arboreal anuran groups lack a palmar sesamoid and flexor plate, a pattern shared with other tetrapod groups, which can retain a reduced sesamoid and flexor plate. We focus on the anatomical structure of the Rhinella group, which includes species that present an osseous palmar sesamoid and climb bushes or trees to avoid depredation or escape dangerous situations, and can exhibit scansorial and arboreal behaviors. We also add data on the bony sesamoids of 170 anuran species to study the anatomy and evolution of the osseous palmar sesamoid within this amphibian group. Our objective is to bring an overview of the osseous palmar sesamoid in anurans, unveiling the relationship between this element of the manus, its phylogeny, and the anuran habitat use. Methods: Skeletal whole-mount specimens of Rhinella were cleared and double-dyed to describe the sesamoid anatomy and related tissues. We review and describe the palmar sesamoid of 170 anuran species from CT images downloaded from Morphosource.org, representing almost all Anuran families. We performed an standard ancestral state reconstruction by optimizing two selected characters (osseous palmar sesamoid presence, distal carpal palmar surface) along with the habitat use of the sampled taxa, using parsimony with Mesquite 3.7. Results: Our primary finding is that sesamoid optimization in the anuran phylogeny revealed that its presence is associated with certain clades and not as widespread as previously anticipated. Additionally, we will also be delving into other important outcomes of our study that are relevant to those working in the field of anuran sesamoids. The osseous palmar sesamoid is present in the clade Bufonidae-Dendrobatidae-Leptodactylidae-Brachicephalidae that we named as PS clade, and also in the archeobatrachian pelobatoid Leptobranchium, all strongly terrestrial and burrowing species, though with exceptions. The osseous palmar sesamoid is always present in Bufonidae, but varies in form and size, depending on the mode that they use their manus, such as in the Rhinella margaritifera which has a cylindrical one and also grasping abilities that involve closing the manus. The scattered presence of the bony palmar sesamoid among anuran clades raises the question whether this sesamoid can be present with a different tissular composition in other groups.

Preliminary ionome of the parotoid gland secretion from Rhinella jimi toad.

The cururu toad (Rhinella jimi) is an anuran belonging to the fauna of the Brazilian northeast region, which releases a secretion with toxins from your parotoid glands. Although it has some information about secondary metabolites and proteins, the elemental composition of the released secretion is unknown. Therefore, this is the first report on the ionome of the secretion of the parotoid glands from R. jimi, investigating the influences of abiotic factors such as biome, seasonality, and gender. ICP-MS was used for measurements combined with principal component analysis (PCA). A screening of the secretion sample detected 68 elements which the total concentration of 18 elements was determined. PCA revealed that biome and seasonality factors have a greater influence on the ionomic profile of parotoid secretion. The presence of toxic metals in the secretion samples indicates that the R. jimi toad can be considered a potential bioindicator. These findings may contribute to understanding the metabolism, lifestyle, and interaction of the R. jimi toad with environmental factors as well as open new perspectives to investigate the relationships of the ionome with other biomolecules, for example, metalloproteins and their physiological functions.

Estudo da influência da secreção cutânea do anfíbio Rhinella diptycha (Rhinella schneideri) sobre o processo de formação in vitro de osteoclastos

Osteoclasts (OCs) are multinucleated cells, specialized in reabsorbing calcified bone matrix. OCs are derived from hematopoietic precursor cells and can be generated in vitro by stimulation of peripheral blood mononuclear cells (PBMC) with the cytokines M-CSF and RANKL. Abnormal activation of OCs occurs in pathologies such as osteoporosis, rheumatoid arthritis, osteoarthritis, periodontal disease, Paget's disease, metastatic cancers and others, causing morbidity and promoting physical limitations. Chronic inflammation leads to the secretion of many pro-inflammatory cytokines and RANKL among them. These cytokines are primarily responsible for osteoclast activation and subsequent bone destruction. Poisoning, caused by different animal species (bees, frogs, scorpions, snakes, etc.), is also known to induce a local and acute inflammatory process. The effect of the venoms of these animals on the differentiation, maturation and degeneration of OCs is unknown. To improve the understanding of the physiology of OCs, and a possible interference in their formation and function processes, we used a model of differentiation from human PBMCs. PBMCs were induced for osteogenic differentiation using the routine protocol. Then we treated the PBMCs with the cutaneous secretion of the amphibian Rhinella dypticha (Rhinella schneideri) to evaluate its effect on the OC differentiation process, which can be divided into three stages, pre-osteclasts, OCs and mature multinucleated OCs. When we used amphibian skin secretion at concentrations of 25 ng/mL and 2.5 ng/mL, we observed a statistically significant decrease in the viability of these cells. Additionally, we showed that the lower concentrations of 0.25 ng/mL and 0.125 ng/mL, although not toxic, induced a decrease in the formation of OCs, which was evidenced by a positive Tartrate-Resistant Acid Phosphatase (TRAP), which is a marker of OCs. mature. With the labeling with Phalloidin conjugated, we saw that the concentration of 0.25 ng/mL affected the formation of multinucleated OCs due to the decrease in the number of cells with 2 and 3 - 5 nuclei. The results obtained suggest the presence of molecules in the cutaneous secretion of Rhinella diptycha that influence the differentiation of OCs in vitro, decreasing the formation of TRAP-positive OCs – mature and affecting the process of formation of multinucleated OCs with 2 to 5 nuclei.

Os osteoclastos (OCs) são células multinucleadas, especializadas em reabsorver a matriz óssea calcificada. OCs se derivam de células precursoras hematopoiéticas e podem ser gerados in vitro pela estimulação de células mononucleares do sangue periférico (PBMC) com as citocinas M-CSF e RANKL. Ativação anormal dos OCs ocorre em patologias como osteoporose, artrite reumatoide, osteoartrite, doença periodontal, doença de Paget, cânceres metastáticos e outras, causando morbidade e promovendo limitações físicas. A inflamação crônica leva à secreção de muitas citocinas pró-inflamatórias e RANKL entre elas. Estas citocinas são as principais responsáveis pela ativação dos osteoclastos e subsequente destruição óssea. O envenenamento, causado por diferentes espécies animais (abelhas, rãs, escorpiões, cobras etc.), é conhecido também por induzir um processo inflamatório local e agudo. O efeito dos venenos destes animais na diferenciação, maturação e degeneração dos OCs é desconhecido. Para melhorar a compreensão da fisiologia dos OCs, e de uma possível interferência em seus processos de formação e função, utilizamos um modelo de diferenciação a partir das PBMCs humanas. As PBMCs foram induzidas para diferenciação osteogênica utilizando o protocolo de rotina. Em seguida tratamos as PBMCs com a secreção cutânea do anfíbio Rhinella dypticha (Rhinella schneideri) para avaliar o seu efeito no processo de diferenciação dos OCs que pode ser dividida em três fases, pré- osteclastos, OCs e OCs maduros multinucleados. Quando utilizamos a secreção cutânea do anfíbio nas concentrações de 25 ng/mL e 2,5 ng/mL observamos a diminuição estatisticamente significativa na viabilidade dessas células. Adicionalmente mostramos que, as concentrações mais baixas de 0,25 ng/mL e 0,125 ng/mL embora não tenham sido tóxicas induziram a diminuição de formação dos OCs que foi evidenciado pela Fosfatase Acida Resistente ao Tartarato (TRAP) positivo que é um marcador de OCs maduros. Já com a marcação com Faloidina conjugada, vimos que a concentração de 0,25 ng/mL afetou a formação dos OCs multinucleadas devido a diminuição do número de células de 2 e de 3 - 5 núcleos. Os resultados obtidos sugerem a presença de moléculas na secreção cutânea de Rhinella diptycha que influenciam a diferenciação de OCs in vitro diminuindo a formação dos OCs TRAP positivas – maduras e afetam o processo de formação das OCs multinucleadas de 2 a 5 núcleos.

Estudo da secreção cutânea da Rhinella schneideri em novos mecanismos e tratamentos para a Doença de Alzheimer

Alzheimer’s Disease (AD) is a global public health priority, accounting for 70% of dementia diagnoses. Abnormal processing of β amyloid precursor protein generates peptides that oligomerize and accumulate in brain tissue. Failures occurring in the removal process of these peptides lead to neurodegeneration, with synaptic and neuronal losses. In addition, changes in the lysosomal-autophagic system contribute to the enhancement of the activity of lysosomal cathepsins. Moreover, in AD, tau protein is hyperphosphorylated, which results in its dissociation from microtubules, self-oligomerization, and neurofibrillary tangles formation. In this context, the discovery of new molecules that improve the comprehension of the mechanisms involved in AD pathogenesis, and possible intervention in disease progression are extremely relevant. In the present work, bioactive molecules from Rhinella schneideri skin secretion (SS), were investigated towards: (I) inhibition of caspases and cathepsins B and D; (II) improvement of neuron-like cells neuronal network; (III) reduction of tau protein expression and (IV) reduction of βA-42 cytotoxicity. SS fractions and subfractions were obtained by liquid-liquid partition, followed by RP-HPLC fractioning, and molecules identification by mass spectrometry. SS, in 250 μg/mL, inhibited cathepsin B enzyme activity, but not cathepsin D, caspase -1, and -6 ones. High Content Screening analysis showed that polar subfraction 5 (PsF5) was able to reduce tau protein expression and to improve neuron-like cells neuronal network. MTT assay demonstrated that SS, polar and apolar fractions, and PsF5 did not revert βA-42 cytotoxicity in neuron-like cells. Taken together, the results obtained indicate that SS contains molecules with the potential to aid in the comprehension of AD physiopathology and in the development of new therapeutical strategies.

A Doença de Alzheimer (DA) é uma prioridade global de saúde pública, sendo responsável por aproximadamente 70% dos diagnósticos de demência. Anomalias no processamento da proteína precursora β amiloide (PPA), por secretases, geram peptídeos que oligomerizam e se acumulam no tecido cerebral. Falhas nos processos de remoção desses peptídeos levam à neurodegeneração, com perda sináptica e neuronal. Além disso, ocorrem alterações no sistema lisossomal-autofágico que contribuem para o aumento da atividade das catepsinas lisossomais. Ainda, no desenvolvimento da DA ocorre a hiperfosforilação da proteína tau, resultando na sua dissociação dos microtúbulos, interligação entre si e formação de emaranhados neurofibrilares. Diante disso, a descoberta de novas moléculas que possam melhorar a compreensão dos mecanismos envolvidos na patogênese da DA e uma possível intervenção na progressão da doença, são de extrema relevância. Neste trabalho, moléculas bioativas provenientes da secreção cutânea da Rhinella schineideri (SCB) foram investigadas, com o objetivo de buscar: (I) atividade inibidora de caspases e catepsinas B e D; (II) Efeito no aumento da rede neuronal das células neuron-like; (III) Redução da expressão proteica da proteína tau e (IV) Redução da citotoxicidade causada pelo peptídeo βA-42. Para obtenção das frações e subfrações da SCB, foi realizada partição líquido-líquido, seguido de fracionamento, por RP-HPLC, e identificação das moléculas, por espectrometria de massas. A SCB, na concentração de 250 μg/mL, inibiu a atividade enzimática da catepsina B, mas não das enzimas catepsina D, caspases -1 e -6. Em concentrações menores, a SCB e suas subfrações, causaram uma redução parcial da atividade da catepsina B. A subfração polar 5 foi capaz de reduzir a expressão proteica da tau e aumentar a rede neuronal das células neuron-like, analisadas pela técnica de High Content Screening. A SCB, FP, FA e a SfP5 não foram capazes de reverter a citotoxicidade causada pelo peptídeo βA-42 nas células neuron-like, analisada pelo ensaio de MTT. Em conjunto, os resultados obtidos demonstram que a SCB contém moléculas com potencial para auxiliar na compreensão da fisiopatologia da DA e no desenvolvimento de novas estratégicas terapêuticas.

Anti-Trypanosomal Bufadienolides from the Oocytes of the Toad Rhinella alata (Anura, Bufonidae).

Amphibians are widely known as a prolific source of bioactive metabolites. In this work, we isolated and characterized compounds with antiparasitic activity from the oocytes of the toad Rhinella alata collected in Panama. Bio-guided isolation and structural elucidation were carried out using chromatographic and spectroscopic techniques, respectively. The organic extract was subjected to solid phase extraction followed by HPLC purification of the fraction with in vitro activity against Trypanosoma cruzi trypomastigotes. Seven steroids (1-7) of the bufadienolide family were isolated, and their structures were determined using NMR and MS analyses; of these 19-formyl-dyscinobufotalin, (3) is reported as a new natural product. Compounds 1 and 3-7 resulted in a good anti-trypanosomal activity profile. Among these, 16ß-hydroxyl-hellebrigenin (1) and bufalin (7) showed significant selectivity values of >5 and 2.69, respectively, while the positive control benznidazole showed a selectivity of 18.81. Furthermore, molecular docking analysis showed compounds 1, 3 and 7 interact through H-bonds with the amino acid residues GLN-19, ASP-158, HIS-159 and TRP-177 from cruzipain at the catalytic site. Given the lack of therapeutic options to treat American trypanosomiasis, this work can serve as the basis for further studies that aim for the development of bufadienolides or their derivatives as drugs against Chagas disease.

Uncovering the antimalarial potential of toad venoms through a bioassay-guided fractionation process.

Malaria remains to date one of the most devastating parasitic diseases worldwide. The fight against this disease is rendered more difficult by the emergence and spread of drug-resistant strains. The need for new therapeutic candidates is now greater than ever. In this study, we investigated the antiplasmodial potential of toad venoms. The wide array of bioactive compounds present in Bufonidae venoms has allowed researchers to consider many potential therapeutic applications, especially for cancers and infectious diseases. We focused on small molecules, namely bufadienolides, found in the venom of Rhinella marina (L.). The developed bio-guided fractionation process includes a four solvent-system extraction followed by fractionation using flash chromatography. Sub-fractions were obtained through preparative TLC. All samples were characterized using chromatographic and spectrometric techniques and then underwent testing on in vitro Plasmodium falciparum cultures. Two strains were considered: 3D7 (chloroquine-sensitive) and W2 (chloroquine-resistant). This strategy highlighted a promising activity for one compound named resibufogenin. With IC50 values of (29 ± 8) µg/mL and (23 ± 1) µg/mL for 3D7 and W2 respectively, this makes it an interesting candidate for further investigation. A molecular modelling approach proposed a potential binding mode of resibufogenin to Plasmodium falciparum adenine-triphosphate 4 pump as antimalarial drug target.

Vasoconstrictor and hemodynamic effects of a methanolic extract from Rhinella marina toad poison.

Rhinella marina toad is abundant in Brazil. Its poison contains cardiac glycosides called bufadienolides, which are extensively investigated for their bioactivity. Our aim was to characterize the vasoactivity of Rhinella marina poison (RmP) on the aorta of male Wistar rats. For this, the RmP was first collected and processed to obtain an alcoholic extract. To determine cardiovascular effects of RmP, we performed in vivo tests by administering RmP intravenously in doses of 0.1-0.8 mg/kg. Vascular reactivity was also performed through concentration-response curves to RmP (10 ng/mL to 200 µg/mL) in aortic segments with and without endothelium. RmP induced a concentration-dependent contraction in rat aorta which was partly endothelium-mediated. Nitric oxide contributes with this response in view that incubation with L-NAME increased the contractile response. Additionally, treatment with indomethacin [cyclooxygenase, (COX) inhibitor], nifedipine (L-type voltage-gated calcium channels blocker), and BQ-123 (ETA receptors antagonist) decreased maximum response, and ketanserin (5-HT2 receptors antagonist) decreased pEC50, suggesting active participation of these pathways in the contractile response. On the other hand, apocynin (NADPH oxidase inhibitor) did not alter contractility. Incubation with prazosin (α1-adrenergic receptor antagonist) abolished the contractile response, suggesting that the RmP-induced contraction is dependent on the adrenergic pathway. In the Na+/K+ ATPase protocol, a higher Emax was observed in the RmP experimental group, suggesting that RmP potentiated Na+/K+ATPase hyperpolarizing response. When this extract was injected (i.v.) in vivo, increase in blood pressure and decrease in heart rate were observed. The results were immediate and transitory, and occurred in a dose-dependent manner. Overall, these data suggest that the poison extract of R. marina toad has an important vasoconstrictor action and subsequent vasopressor effects, and its use can be investigated to some cardiovascular disorders.

The integrated biomarker response in three anuran species larvae at sublethal concentrations of cypermethrin, chlorpyrifos, glyphosate, and glufosinate-ammonium.

The aim of the present study was to evaluate the response in larvae of the anuran species Rhinella arenarum, Rhinella dorbignyi and Odontophrynus americanus exposed to glyphosate (GLY, 2.5 mg L-1), cypermethrin (CYP, 0.013 mg L-1), chlorpyrifos (CP, 0.1 mg L-1) and glufosinate-ammonium (GLU, 15 mg L-1) using two behavioral endpoints: mean speed (MS) and total distance moved (TD); and two enzymatic biomarkers: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In order to assess a global response and to determine the most sensitive species, an integrated biomarker response (IBR) index was calculated. Behavioral biomarkers were tested at 1 and 60 min, and the enzymes at 60 min after exposure. The results showed that: (1) there were statistical differences between species in a series of responses in swimming behavior, and cholinesterase activities within the first-hour of exposure to CYP, GLY, and CP at environmentally relevant concentrations (ERC); (2) IBR determined that Rhinella species were the most sensitive of the species tested and (3) IBR provided a comprehensive assessment of the health status of species exposed to ERC of a wide variety of agrochemicals globally and frequently used.

Comparative study of composition of methanolic extracts of the paratoid gland secretions (PGS) of Rhinella jimi (cururu toad) from northeastern Brazil: Gender, seasonality and geographic occurrence.

Toads belonging to the Bufonidae family have a pair of paratoid glands that store highly toxic a biological secretion with varied chemical composition, that act as a chemical defense against microbial infections and predators. The paratoid gland secretion (PGS) of bufonids is rich in bioactive steroids, alkaloids, proteins, bufadienolides and bufotoxins. In the present investigation we performed a systematic analysis of the chemical profile of PGS obtained from the Bufonidae toad Rhinella jimi ("Cururu" toad) collected at three different regions of Piauí state, Northeastern Brazil. Our aim was to investigate the PGS variation related to the season of animals collection, geographic distribution and gender of the animals. The methanolic extracts of PGS were analyzed by UPLC-QToF-MS/MS. Principal component analysis (PCA) were applied to the data set obtained by the UPLC-QToF-MS/MS analyses. Among 23 compounds identified, dehydrobufotenine, suberoyl arginine, 3-(N-suberoyl-argininyl) telocinobufagin, 3-(N-suberoyl-argininyl) marinobufagin, telocinobufagin, marinobufagin and 3-(N-suberoyl-argininyl) bufalin were detected in all PGS. Minimal variations in the composition of paratoid secretions of R. jimi were observed related to distinct geographical and seasonal parameters. R. jimi female animals presented the most diverse chemical composition in its PGS. With this comparative study, unprecedented for the species, it was possible to observe that the secretions of the paratoid glands produced by R. jimi from different regions of the state of Piauí, at different times of the year, presented consistent chemical composition, with discrete particularities in the number and nature chemistry of its constituents.

A paraguayan toad Rhinella schneideri preparation based on Mbya tradition increases mitochondrial bioenergetics with migrastatic effects dependent on AMPK in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In Paraguay, healers from the Mbya culture treat cancer with a recipe prepared with the native toad Rhinella schneideri. However, the chemical composition and biological effects of the recipe remain unknown. AIM OF THE STUDY: The aim is to determine the composition of the traditional preparation made using the toad R. schneideri and to evaluate its effect on human breast cancer (BC) cells. MATERIALS AND METHODS: The metabolites contained in the preparation were concentrated using XAD-7 resin, and the concentrate was analyzed by HPLC-MS/MS. The effect of the preparation was assessed in normal (MCF10F) and BC cells (MDA-MB-231 and MCF7). The mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) levels, and cell cycle progression were determined by flow cytometry. The oxygen consumption rate (OCR) was measured by Clark electrode, and fibronectin-dependent migration in normoxia and hypoxia-like conditions were evaluated by transwell assay. RESULTS: From the Amberlite-retained extract from the preparation, 24 compounds were identified, including alkaloids, amino acids, bufadienolides, and flavonoids, among others. The crude extract (CE) did not affect cell cycle progression and viability of BC cell lines. Moreover, it did not make cancer cells more sensitive to the cytotoxic effect of the chemotherapeutics doxorubicin and teniposide. On the other hand, the CE reduced the menadione-induced ROS production and increased NADH, Δψm, and the OCR. Respiratory complexes I and III as well as ATP synthase levels were increased in an AMPK-dependent manner. Moreover, the CE inhibited the migration of BC cells in normoxia and a hypoxia-like condition using CoCl2 as a HIF1α-stabilizing agent. This latter effect involved an AMPK-dependent reduction of HIF1α levels. CONCLUSIONS: The Paraguayan toad recipe contains metabolites from the toad ingredient, including alkaloids and bufadienolide derivatives. The CE lacks cytotoxic effects alone or in combination with chemotherapeutics. However, it increases mitochondrial bioenergetics and inhibits the cancer cell migration in an AMPK-dependent manner in BC cells. This is the first report of the in vitro anticancer effect of a traditional Rhinella sp. toad preparation based on Mbya tradition.

Natural multi-target modulators of pathological pathways in Alzheimer's disease isolated from the Rhinella arenarum skin.

The skin of anuran amphibians is a rich source of compounds with great medicinal potential. Alzheimer's disease (AD) is a complex disease associated with numerous pathological pathways, making their simultaneous modulation necessary. Nowadays the development of anti-AD drugs is focused on a Multi-Target Directed Ligands strategy. Herein we report the bioactivity of the skin extracts of the toad Rhinella arenarum obtained by an invasive and non-invasive methods, against five AD pathological targets (AChE, BChE, MAO-B, antioxidant and chelating activities). The extract derived from the non-invasive technique showed the highest biological activity, being capable of acting on all or almost all the pathological targets of AD, while also avoiding harm to the animal.

Phylogenomics, introgression, and demographic history of South American true toads (Rhinella).

The effects of genetic introgression on species boundaries and how they affect species' integrity and persistence over evolutionary time have received increased attention. The increasing availability of genomic data has revealed contrasting patterns of gene flow across genomic regions, which impose challenges to inferences of evolutionary relationships and of patterns of genetic admixture across lineages. By characterizing patterns of variation across thousands of genomic loci in a widespread complex of true toads (Rhinella), we assess the true extent of genetic introgression across species thought to hybridize to extreme degrees based on natural history observations and multilocus analyses. Comprehensive geographic sampling of five large-ranged Neotropical taxa revealed multiple distinct evolutionary lineages that span large geographic areas and, at times, distinct biomes. The inferred major clades and genetic clusters largely correspond to currently recognized taxa; however, we also found evidence of cryptic diversity within taxa. While previous phylogenetic studies revealed extensive mitonuclear discordance, our genetic clustering analyses uncovered several admixed individuals within major genetic groups. Accordingly, historical demographic analyses supported that the evolutionary history of these toads involved cross-taxon gene flow both at ancient and recent times. Lastly, ABBA-BABA tests revealed widespread allele sharing across species boundaries, a pattern that can be confidently attributed to genetic introgression as opposed to incomplete lineage sorting. These results confirm previous assertions that the evolutionary history of Rhinella was characterized by various levels of hybridization even across environmentally heterogeneous regions, posing exciting questions about what factors prevent complete fusion of diverging yet highly interdependent evolutionary lineages.

Uma atenção crescente tem sido dada aos efeitos da introgressão genética nos limites das espécies e como eles afetam a integridade e a persistência das espécies ao longo do tempo evolutivo. A crescente disponibilidade de dados genômicos revelou padrões contrastantes de fluxo gênico entre regiões do genoma, o que impõe desafios às inferências de relações evolutivas e de padrões de mistura genética entre linhagens. Com base em padrões de variação em milhares de marcadores genômicos em um complexo amplamente distribuído de sapos (Rhinella), avaliamos a extensão de introgressão genética entre espécies que, acredita-se, hibridizam amplamente com base em observações de história natural e análises multi-locus. Nossa amostragem geográfica abrangente de cinco táxons neotropicais revelou várias linhagens evolutivas distintas que abrangem grandes áreas geográficas e, por vezes, biomas distintos. Os principais clados e grupos genéticos inferidos correspondem em grande parte aos táxons atualmente reconhecidos; no entanto, também encontramos evidência de diversidade críptica. De acordo com estudos filogenéticos anteriores que revelaram extensa discordância mitonuclear, nossas análises de agrupamento genético revelaram vários indivíduos geneticamente misturados. Adicionalmente, análises demográficas históricas sugerem que a história evolutiva desses sapos envolveu fluxo gênico entre táxons tanto em épocas antigas quanto recentes. Por fim, testes ABBA-BABA revelaram amplo compartilhamento de alelos entre espécies, um padrão que pode ser atribuído à introgressão genética ao invés de sorteamento incompleto de alelos entre linhagens. Esses resultados confirmam sugestões anteriores de que a história evolutiva de Rhinella foi caracterizada por vários níveis de hibridização, mesmo entre ambientes distintos, levantando questões sobre quais fatores impedem a fusão completa de linhagens evolutivas divergentes porém altamente interdependentes.

Migration and Histologic Effects of Visible Implant Elastomer (VIE) and Passive Integrated Transponder (PIT) Tags in the Marine Toad (Rhinella marina).

Passive integrated transponder (PIT) and visible implant elastomer (VIE) tags are commonly used to identify reptiles, amphibians, and fish. Despite reports of good retention rates and little to no negative effect on survival time, migration remains a concern and histologic changes have not been widely evaluated. Fifty-six wild-caught marine toads (Rhinella marina) were marked with a PIT tag in the left caudal thigh and a VIE tag over the right gastrocnemius muscle prior to transport to the North Carolina Zoo. Fourteen toads were then humanely euthanized on day 9, 15, 32, and 62 for postmortem examination and histopathology which were compared to 10 control toads with no tags. All PIT tags were functional and 95% remained at the insertion site with minimal to no histologic changes. Externally, 48% of VIE tags were visible through the skin at the original site of injection under fluorescent or UV light. Upon gross examination of the tissues, VIE tags had an overall retention rate of 62% at the injection site, with similar retention rates across time points. Migrated VIE material was visible grossly and histologically in the kidneys of 98% of toads and along the right leg, proximally and distally, in 25% of toads. VIE material was also found sporadically in mesentery, colon, and free in the coelomic cavity. Histologically, VIE material in the skin was surrounded by minimal to mild granulomatous inflammation while in the kidney it was associated with dilation of the small vessels, edema, and granulomatous nephritis that progressed in severity over time. Based on these findings, the authors recommend the use of PIT tags over VIE tags for identification of adult anurans, when feasible.

Discovery of Novel Viruses Associated With the Invasive Cane Toad (Rhinella marina) in Its Native and Introduced Ranges.

Cane toads (Rhinella marina) are notoriously successful invaders: from 101 individuals brought to Australia in 1935, poisonous toads now cover an area >1.2 million km2 with adverse effects on native fauna. Despite extensive research on the role of macroparasites in cane toad invasion, viral research is lagging. We compared viral prevalence and diversity between toads in their native range (French Guiana, n=25) and two introduced ranges: Australia (n=151) and Hawai'i (n=10) with a metatranscriptomic and metagenomic approach combined with PCR screening. Australian toads almost exclusively harbor one of seven viruses detected globally. Rhimavirus-A (Picornaviridae) exhibited low genetic diversity and likely actively infected 9% of sampled Australian toads extending across ~2,000km of Northern Australia and up to the current invasion front. In native range cane toads, we identified multiple phylogenetically distinct viruses (Iridoviridae, Picornaviridae, Papillomaviridae, and Nackedna-like virus). None of the same viruses was detected in both ranges, suggesting that Australian cane toads have largely escaped the viral infection experienced by their native range counterparts. The novel native range viruses described here are potential biocontrol agents, as Australian toads likely lack prior immunological exposure to these viruses. Overall, our evidence suggests that there may be differences between viruses infecting cane toads in their native vs. introduced ranges, which lays the groundwork for further studies on how these viruses have influenced the toads' invasion history.