Acute stress, steroid plasma levels, and innate immunity in Brazilian toads.

Stress from habitat fragmentation has been shown to impact amphibian declines. Studies from a variety of vertebrates indicate that stressed animals exhibit an acute increase in circulating plasma glucocorticoid (GC) levels and consequent immunomodulation. To further explore the relationship between GCs and immunity, we subjected three species of newly captured Brazilian toads, Rhinella ornata, R. icterica and R. schneideri to restraint with or without movement restriction (maintenance in a moistened cloth bag vs. maintenance in a bin) for 24 h. We compared various parameters from baseline (field conditions) with values after restraint, including those associated with stress (corticosterone [CORT] plasma levels), and the neutrophil/lymphocyte ratio [N:L ratio]), potential reproduction (testosterone [T] plasma levels), and innate immunity (bacterial killing ability [BKA]). General responses to the restraint challenge (baseline vs. restraint) included increased CORT levels and N:L ratio, and decreased T levels and BKA. Additionally, CORT levels and N:L ratio tended to increase more from restraint with movement restriction than to restraint without movement restriction, indicating toads showed increased stress response to the more intense stressor. All variables showed interspecific variation at baseline conditions: R. ornata had higher CORT levels when compared to the other two species, while R. icterica had the highest BKA values. After restraint (with or without movement restriction), R. ornata displayed higher values for T and N:L ratio, and showed higher CORT values after restraint without movement restriction; however, the CORT values were similar among species after restraint with movement restriction. In terms of immunity, in response to restraint, BKA was different among species only after restraint with movement restriction, with R. schneideri showing the lowest BKA values. Our results show that restraint increases common markers of the stress response, and could reduce potential reproduction and innate immune responses in toads from all studied species. Our results also showed variation at the interspecific level, with the amplitude of change in the studied variables being consistent and more pronounced following restraint with movement restriction for the three-studied species.

Buccal jet streaming and dead space determination in the South American lungfish, Lepidosiren paradoxa.

The "jet stream" model predicts an expired flow within the dorsal part of the buccal cavity with small air mixing during buccal pump ventilation, and has been suggested for some anuran amphibians but no other species of air breathing animal using a buccal force pump has been investigated. The presence of a two-stroke buccal pump in lungfish, i.e. expiration followed by inspiration, was described previously, but no quantitative data are available for the dead-space of their respiratory system and neither a detailed description of airflow throughout a breathing cycle. The present study aimed to assess the degree of mixing of fresh air and expired gas during the breathing cycle of Lepidosiren paradoxa and to verify the possible presence of a jet stream during expiration in this species. To do so, simultaneous measurements of buccal pressure and ventilatory airflows were carried out. Buccal and lung gases (PCO2 and PO2) were also measured. The effective ventilation was calculated and the dead space estimated using Bohr equations. The results confirmed that the two-stroke buccal pump is present in lungfish, as it is in anuran amphibians. The present approaches were coherent with a small dead space, with a very small buccal-lung PCO2 difference. In the South American lungfish the dead space (VD) as a percentage of tidal volume (VT) (VD / VT) ranged from 4.1 to 12.5%. Our data support the presence of a jet stream and indicate a small degree of air mixing in the buccal cavity. Comparisons with the literature indicate that these data are similar to previous data reported for the toad Rhinella schneideri.

Thermal sensitivity of innate immune response in three species of Rhinella toads.

Immune activity is temperature-dependent and strongly related to thermal biology in ectotherms. Eurythermic, vs stenothermic, species commonly show a broader range of immune responses. Furthermore, the development of behavioral fever in ectotherms is correlated with improved immune function. Although amphibians generally show restricted capacity for thermoregulation in the field, behavioral fever has been documented in the laboratory for several anurans. However, the match between behavioral fever and improved immune response at fever thermal preferendum has still to be determined in these animals. In this study, we investigate the thermal sensitivity of the innate immune response, as measured by the plasma bacterial killing ability (BKA) against Aeromonas hydrophila, in three species of toads from genus Rhinella (R. schneideri, R. icterica and R. ornata) during their breeding season. Moreover, we show lipopolysaccharide-induced behavioral fever for R. ornata. The three species of toads showed an inverted U-shaped pattern of thermal sensitivity regarding BKA, with a high efficiency of immune response at temperatures around their thermal preferendum. The results partially corroborate the hypothesis that immune function is maximized at fever thermal preferendum, given that two of the species showed a maximal BKA performance temperature closer to fever than their normal thermal preferendum. Toads also showed an eurythermic pattern of immune response (large temperature breadth of BKA performance ≥95%; B95) during the breeding season. This large B95 encompasses much of the ecologically relevant temperatures, with the exception of those exhibited by two species that maintain activity during winter. Lastly, BKA was commonly suppressed at 37 °C, highlighting the importance of choosing ecologically relevant temperatures when conducting in vitro immunological tests.

Morphological and biochemical characterization of the cutaneous poison glands in toads (Rhinella marina group) from different environments.

BACKGROUND: Amphibian defence against predators and microorganisms is directly related to cutaneous glands that produce a huge number of different toxins. These glands are distributed throughout the body but can form accumulations in specific regions. When grouped in low numbers, poison glands form structures similar to warts, quite common in the dorsal skin of bufonids (toads). When accumulated in large numbers, the glands constitute protuberant structures known as macroglands, among which the parotoids are the most common ones. This work aimed at the morphological and biochemical characterization of the poison glands composing different glandular accumulations in four species of toads belonging to group Rhinella marina (R. icterica, R. marina, R. schneideri and R. jimi). These species constitute a good model since they possess other glandular accumulations together with the dorsal warts and the parotoids and inhabit environments with different degrees of water availability. RESULTS: We have observed that the toads skin has three types of poison glands that can be differentiated from each other through the morphology and the chemical content of their secretion product. The distribution of these different glands throughout the body is peculiar to each toad species, except for the parotoids and the other macroglands, which are composed of an exclusive gland type that is usually different from that composing the dorsal warts. Each type of poison gland presents histochemical and biochemical peculiarities, mainly regarding protein components. CONCLUSIONS: The distribution, morphology and chemical composition of the different types of poison glands, indicate that they may have different defensive functions in each toad species.

Associations of water balance and thermal sensitivity of toads with macroclimatic characteristics of geographical distribution.

Interspecific variation in patterns of geographical distribution of phylogenetically related species of amphibians might be related to physiological adaptation to different climatic conditions. In this way, a comparative study of resistance to evaporative water loss, rehydration rates and sensitivity of locomotor performance to variations on hydration level and temperature was performed for five species of Bufonidae toads (Rhinella granulosa, R. jimi, R. ornata, R. schneideri and R. icterica) inhabiting different Brazilian biomes. The hypotheses tested were that, when compared to species inhabiting mesic environments, species living at hot and dry areas would show: (1) greater resistance to evaporative water loss, (2) higher rates of water uptake, (3) lower sensitivity of locomotor performance to dehydration and (4) lower sensitivity of locomotor performance at higher temperatures and higher sensitivity of locomotor performance at lower temperatures. This comparative analysis showed relations between body mass and interspecific variation in rehydration rates and resistance to evaporative water loss in opposite directions. These results might represent a functional compensation associated with relatively lower absorption areas in larger toads and higher evaporative areas in smaller ones. Moreover, species from the semi-arid Caatinga showed locomotor performance less sensitive to dehydration but highly affected by lower temperatures, as well greater resistance to evaporative water loss, when compared to the other species from the mesic Atlantic Forest and the savannah-like area called Cerrado. These results suggest adaptation patterns to environmental conditions.

Baroreflex regulation affects ventilation in the cururu toad Rhinella schneideri.

Anurans regulate short-term oscillations in blood pressure through changes in heart rate (fH), vascular resistance and lymphatic fH Lung ventilation in anurans is linked to blood volume homeostasis by facilitating lymph return to the cardiovascular system. We hypothesized that the arterial baroreflex modulates pulmonary ventilation in the cururu toad Rhinella schneideri, and that this relationship is temperature dependent. Pharmacologically induced hypotension (sodium nitroprusside) and hypertension (phenylephrine) increased ventilation (25°C: 248.7±25.7 ml kg-1 min-1; 35°C: 351.5±50.2 ml kg-1 min-1) and decreased ventilation (25°C: 9.0±6.6 ml kg-1 min-1; 35°C: 50.7±15.6 ml kg-1 min-1), respectively, relative to control values from Ringer solution injection (25°C: 78.1±17.0 ml kg-1 min-1; 35°C: 137.7±15.5 ml kg-1 min-1). The sensitivity of the ventilatory response to blood pressure changes was higher during hypotension than during hypertension [25°C: -97.6±17.1 versus -23.6±6.0 breaths min-1 kPa-1; 35°C: -141.0±29.5 versus -28.7±6.4 breaths min-1 kPa-1, respectively; negative values indicate an inverse relationship between blood pressure and ventilation (or breathing frequency), i.e. as blood pressure increases, ventilation decreases, and vice versa], while temperature had no effect on these sensitivities. Hyperoxia (30%; 25°C) diminished ventilation, but did not abolish the ventilatory response to hypotension, indicating a response independent of peripheral chemoreceptors. Although there are previous data showing increased fH baroreflex sensitivity from 15 to 30°C in this species, further increases in temperature (35°C) diminished fH baroreflex gain (40.5±5.62 versus 21.6±4.64% kPa-1). Therefore, besides an involvement of pulmonary ventilation in matching O2 delivery to demand at higher temperatures in anurans, it also plays a role in blood pressure regulation, independent of temperature, possibly owing to an interaction between baroreflex and respiratory areas in the brain, as previously suggested for mammals.

Temperature effects on baroreflex control of heart rate in the toad, Rhinella schneideri.

For an adequate blood supply to support metabolic demands, vertebrates regulate blood pressure to maintain sufficient perfusion to avoid ischemia and other tissue damage like edema. Using a pharmacological approach (phenylephrine and sodium nitroprusside) we investigated baroreflex sensitivity at 15, 25, and 30°C in toads Rhinella schneideri. Baroreflex sensitivity presented a high thermal dependence (Q10=1.9-4.1), and the HR-baroreflex curve was shifted up and to the right as temperature increased from 15 to 30°C. Baroreflex variables, namely, HR range, gain50 (maximal gain) and normalized gain50 increased 206, 235, and 160% from 15 to 30°C, respectively. The cardiac limb of the baroreflex response to pharmacological treatments was significantly blunted after full autonomic blockade. In addition, there was a clear baroreflex-HR response mainly to hypotension at all three temperatures tested. These findings indicate that toads present temperature dependence for cardiac limb of the barostatic response and the cardiac baroreflex response in R. schneideri is primarily hypotensive rather than hypertensive as well as crocodilians and mammals. Thus, the cardiac baroreflex compensation to changes in arterial pressure might present different patterns among amphibian species, since the previously reported bradycardic compensation to hypertension in some anurans was not observed in the toad used in the present study.

Checklist of nematode parasites of amphibians from Argentina.

This review includes information about 47 taxa of nematode parasites reported from 34 species of Argentinean amphibians, all belonging to order Anura (33 native species and 1 introduced species). Thirty four nematode species have been reported as adults and 13 species were reported as larvae (10 taxa) or juveniles (3 taxa). Two species, Cosmocerca parva and C. podicipinus (Cosmocercidae), collected as adults, are the most commonly occurring adult nematodes in Argentinean amphibians; each of them parasitize 14 amphibian species. The bufonid Rhinella schneideri and the leptodactylid Leptodactylus bufonius present the highest species richness of parasitic nematodes (9 species); followed by Rhinella fernandezae, R. arenarum and Leptodactylus chaquensis, each of which is parasitized by 8 nematode species. Mean species richenss was highest for the family Bufonidae (4.5±3.4; range: 1-9); followed by the Leptodactylidae (3.5±2.8; range: 1-9). Data on hosts, geographical distribution, site of infection, location of deposited materials, and information about life cycles are provided. This is the first compilation of information on nematode parasites of amphibians in Argentina.

Life cycle and behavior of Amblyomma rotundatum (Acari: Ixodidae) under laboratory conditions and remarks on parasitism of toads in Brazil.

The life cycle and behavior of Amblyomma rotundatum were evaluated under laboratory conditions. The experiment started with four engorged females collected from toads (Rhinella schneideri) naturally infested at the Pirapitinga Ecological Station in the state of Minas Gerais, Brazil. Developmental periods of free-living stages were assessed in an incubator at 27 ± 1 °C, >80 % RH and darkness. The complete life cycle, including pre-attachment periods for each parasitic stage, ranged from 126 to 228 days. The pre-attachment, feeding and molting periods increased as the life cycle progressed from larva to adult female. Oviposition lasted about 20 days, with the peak occurring on days 4 and 5. Longevity of nymphs and adult females was quite similar (approximately 250 and 240 days, respectively) and slightly longer than that of larvae. Lesions caused by tick feeding are discussed and a list of known hosts, including new host records for A. rotundatum, is offered.

Internal oral morphology in larvae of the genus Rhinella Fitzinger, 1826 (Amphibia, Anura, Bufonidae) .

From the 86 species allocated in the genus Rhinella, 25 have their tadpoles described and only R. arenarum, R. chrysophora, R. icterica, R. ornata, R. schneideri and R. spinulosa have aspects of the internal oral morphology evidenced. Herein, the internal oral morphology from 12 species of Rhinella distributed between the morphological groups of R. crucifer, R. granulosa, R. marina and R margaritifera is described and compared. The internal oral morphology of Rhinella is little variable in many aspects. Despite the many similarities found between the tadpoles of Rhinella, the study showed that there are characteristics that exhibit interspecific variation that can be used in the taxonomy of the genus. Important features to distinguish species were: number of infrarrostral projections; number and shape of the infralabial papillae; size, arrangement, shape and apex of the lingual papillae; shape of the buccal floor arena papillae; number of projections of the ventral velum; shape of the prenarial ridge; choanae arrangement; number and apex of the postnarial papillae; number and shape of the secondary branches on the lateral ridge papilla; buccal roof arena papillae arrangement. 

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.

Untangling interactions between Bitis vipers and their prey using coagulotoxicity against diverse vertebrate plasmas.

Venom is a key evolutionary innovation which plays a primary role in prey subjugation by venomous snakes. However, while there is a growing body of literature indicating the composition and activity of snake venoms is under strong natural selection driven by differences in prey physiology, the majority of studies have historically focussed on the activity of snake venoms with regards only towards human or mammalian physiologies. This study aimed to use clotting assays measuring both time and strength of clotting to characterise the coagulotoxic activity of venoms from a taxonomically, morphologically, and ecologically diverse range of Bitis spp. of viperid snakes upon the plasma of model species: amphibian (Cane Toad, Rhinella marina); lizard (Blue-tongue Skink, Tiliqua scincoides); avian (Domestic Chicken, Gallus gallus); and rodent (Brown Rat, Rattus norvegicus). Significant variation in coagulotoxic activity across the different plasmas was observed between species and compared to the known affects upon human plasma. Bitis caudalis was notable in being active on all four plasmas, but in extremely divergent manners: accelerating clotting times and producing strong, stable clots upon amphibian plasma (consistent with true procoagulation); accelerating clotting time but producing weak, unstable clots upon lizard plasma (consistent with pseudo-procoagulation); delaying avian clotting time beyond machine maximum reading time (strong anticoagulation consistent with either inhibition of clotting enzymes or total destruction of fibrinogen, or both); and delaying clotting of rodent plasma (consistent with inhibition of clotting enzymes) and with only weak clots formed (consistent with destruction of fibrinogen). In contrast, the sister species B. peringueyi and B. schneideri displayed activity only upon the lizard plasma, slightly accelerating the clotting times to produce weak, unstable clots (consistent with pseudo-procoagulation). The other dwarf species, B. cornuta, displayed strong anticoagulation upon avian and rodent plasmas, delaying clotting beyond the machine maximum reading time (strong anticoagulation consistent with either inhibition of clotting enzymes or total destruction of fibrinogen, or both). In contrast, the giant species studied (B. gabonica) showed only a very weak pseudo-procoagulant activity upon lizard plasma. The wide range of variation seen within this study highlights the importance of studying venom activity on relevant models when making conclusions about the ecological role of venoms and the extreme limitation in extrapolating animal results to predict potential human clinical effects.

Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers.

BACKGROUND: The classical perspective that interspecific hybridization in animals is rare has been changing due to a growing list of empirical examples showing the occurrence of gene flow between closely related species. Using sequence data from cyt b mitochondrial gene and three intron nuclear genes (RPL9, c-myc, and RPL3) we investigated patterns of nucleotide polymorphism and divergence between two closely related toad species R. marina and R. schneideri. By comparing levels of differentiation at nuclear and mtDNA levels we were able to describe patterns of introgression and infer the history of hybridization between these species. RESULTS: All nuclear loci are essentially concordant in revealing two well differentiated groups of haplotypes, corresponding to the morphologically-defined species R. marina and R. schneideri. Mitochondrial DNA analysis also revealed two well-differentiated groups of haplotypes but, in stark contrast with the nuclear genealogies, all R. schneideri sequences are clustered with sequences of R. marina from the right Amazon bank (RAB), while R. marina sequences from the left Amazon bank (LAB) are monophyletic. An Isolation-with-Migration (IM) analysis using nuclear data showed that R. marina and R. schneideri diverged at ≈ 1.69 Myr (early Pleistocene), while R. marina populations from LAB and RAB diverged at ≈ 0.33 Myr (middle Pleistocene). This time of divergence is not consistent with the split between LAB and RAB populations obtained with mtDNA data (≈ 1.59 Myr), which is notably similar to the estimate obtained with nuclear genes between R. marina and R. schneideri. Coalescent simulations of mtDNA phylogeny under the speciation history inferred from nuclear genes rejected the hypothesis of incomplete lineage sorting to explain the conflicting signal between mtDNA and nuclear-based phylogenies. CONCLUSIONS: The cytonuclear discordance seems to reflect the occurrence of interspecific hybridization between these two closely related toad species. Overall, our results suggest a phenomenon of extensive mtDNA unidirectional introgression from the previously occurring R. schneideri into the invading R. marina. We hypothesize that climatic-induced range shifts during the Pleistocene/Holocene may have played an important role in the observed patterns of introgression.

Water balance and locomotor performance in three species of neotropical toads that differ in geographical distribution.

Water availability in the environment is a fundamental factor in determining the limits of geographical distribution and the evolution of the physiological characters associated to water balance in anurans. In this paper, we compare some aspects of water balance and the sensitivity of locomotor performance to dehydration at different temperatures for three species of toads from the genus Rhinella, with different levels of dependence on forested environments. Results show patterns associated to interspecific differences in both geographical distribution and time of seasonal reproduction. Sensitivity of locomotor performance to dehydration was lower at low temperatures for R. icterica, the species that are reproductively active during winter and lower at intermediate temperatures for R. schneideri, the species that reproduces mostly during spring, suggesting a pattern of thermal adaptation of locomotor performance for these species. Otherwise, R. ornata, a species with broader reproductive season, shows high sensitivity of locomotor performance to dehydration at all temperatures tested, suggesting a stronger relation of breeding activity with patterns of rainfall than temperature variation. Furthermore, the low rates of water uptake of R. ornata may pose restrictions on the occupation of open areas by this species.

Reassessment of a fossil specimen of Rhinella marina (Linnaeus, 1758) (Anura: Bufonidae), from Early Pleistocene of Bolivia.

Bufonidae is a cosmopolite and speciose clade that is currently hypothesized to have originated in Gondwana around 78-99 Ma (Pramuk et al. 2008). The systematics of the family was assessed using morphological and molecular data, alone or in a total evidence analysis (Pramuk 2006; Pramuk et al. 2008; Bocxlaer et al. 2010; Pyron Wiens 2011). Due to taxonomic changes, most of the South American species of Bufo Garsault were relocated to the genus Rhinella Fitzinger, currently the second most speciose genus with 92 scientifically named and valid species (Frost 2020). The species in the genus are arranged in six taxonomic groups (crucifer, granulosa, margaritifera, marina, spinulosa, veraguensis species groups [Frost 2020]); the Rhinella marina group is characterized by specimens with well-ossified and exostosed skull, ornamented with deep striations, pits, and rugosities (Maciel et al. 2010). The Rhinella marina group includes eleven living species, distributed in two main clades named after their geographical distribution: the north-central clade and south-central clade. The south-central clade bears the species: R. achavali (Maneyro, Arrieta de Sá), R. arenarum (Hensel), R. icterica (Spix), and R. rubescens (Lutz). The north-central clade bears the species: R. cerradensis Maciel, Brandão, Campos Sebben, R. horribilis (Wiegmann), R. jimi (Stevaux), R. marina (Linnaeus), R. poeppigii (Tschudi), R. schneideri (Werner), and R. veredas (Brandão, Maciel Sebben).

The Insecticidal Activity of Rhinella schneideri (Werner, 1894) Paratoid Secretion in Nauphoeta cinerea Cocroaches.

Rhinella schneideri is a common toad found in South America, whose paratoid toxic secretion has never been explored as an insecticide. In order to evaluate its insecticidal potential, Nauphoeta cinerea cockroaches were used as an experimental model in biochemical, physiological and behavioral procedures. Lethality assays with Rhinella schneideri paratoid secretion (RSPS) determined the LD50 value after 24 h (58.07µg/g) and 48 h exposure (44.07 µg/g) (R2 = 0.882 and 0.954, respectively). Acetylcholinesterase activity (AChE) after RSPS at its highest dose promoted an enzyme inhibition of 40%, a similar effect observed with neostigmine administration (p < 0.001, n= 5). Insect locomotion recordings revealed that RSPS decreased the distance traveled by up to 37% with a concomitant 85% increase in immobile episodes (p < 0.001, n = 36). RSPS added to in vivo cockroach semi-isolated heart preparation promoted an irreversible and dose dependent decrease in heart rate, showing a complete failure after 30 min recording (p < 0.001, n ≥ 6). In addition, RSPS into nerve-muscle preparations induced a dose-dependent neuromuscular blockade, reaching a total blockage at 70 min at the highest dose applied (p < 0.001, n ≥ 6). The effect of RSPS on spontaneous sensorial action potentials was characterized by an increase in the number of spikes 61% (p < 0.01). Meanwhile, there was 42% decrease in the mean area of those potentials (p < 0.05, n ≥ 6). The results obtained here highlight the potential insecticidal relevance of RSPS and its potential biotechnological application.

Assessment of neuropharmacological potential of low molecular weight components extracted from Rhinella schneideri toad poison.

BACKGROUND: Studies on toad poison are relevant since they are considered a good source of toxins that act on different biological systems. Among the molecules found in the toad poison, it can be highlighted the cardiotonic heterosides, which have a known mechanism that inhibit Na+/K+-ATPase enzyme. However, these poisons have many other molecules that may have important biological actions. Therefore, this work evaluated the action of the low molecular weight components from Rhinella schneideri toad poison on Na+/K+-ATPase and their anticonvulsive and / or neurotoxic effects, in order to detect molecules with actions of biotechnological interest. METHODS: Rhinella schneideri toad (male and female) poison was collected by pressuring their parotoid glands and immediately dried and stored at -20 °C. The poison was dialysed and the water containing the low molecular mass molecules (< 8 kDa) that permeate the dialysis membrane was collected, frozen and lyophilized, resulting in the sample used in the assays, named low molecular weight fraction (LMWF). Na+/K+ ATPase was isolated from rabbit kidneys and enzyme activity assays performed by the quantification of phosphate released due to enzyme activity in the presence of LMWF (1.0; 10; 50 and 100 µg/mL) from Rhinella schneideri poison. Evaluation of the L-Glutamate (L-Glu) excitatory amino acid uptake in brain-cortical synaptosomes of Wistar rats was performed using [3H]L-glutamate and different concentration of LMWF (10-5 to 10 µg/µL). Anticonvulsant assays were performed using pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA) to induce seizures in Wistar rats (n= 6), which were cannulated in the lateral ventricle and treated with different concentration of LMWF (0.25; 0.5; 1.0; 2.0; 3.0 and 4.0 µg/µL) 15 min prior to the injection of the seizure agent. RESULTS: LMWF induced a concentration-dependent inhibition of Na+/K+-ATPase (IC50% = 107.5 µg/mL). The poison induces an increased uptake of the amino acid L-glutamate in brain-cortical synaptosomes of Wistar rats. This increase in the L-glutamate uptake was observed mainly at the lowest concentrations tested (10-5 to 10-2 µg/µL). In addition, this fraction showed a very relevant central neuroprotection on seizures induced by PTZ and NMDA. CONCLUSIONS: LMWF from Rhinella schneideri poison has low molecular weight compounds, which were able to inhibit Na+/K+-ATPase activity, increase the L-glutamate uptake and reduced seizures induced by PTZ and NMDA. These results showed that LMWF is a rich source of components with biological functions of high medical and scientific interest.

Cytotoxicity and antimitotic activity of Rhinella schneideri and Rhinella marina venoms.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhinella schneideri and Rhinella marina are toad venoms distributed in different parts of the world, including Brazil, Columbia and amazon. Venoms extracted from different species have many clinical applications such as antimicrobial cardiotonics and treatment of cancer. Aim of the study; In this study, we aim to investigate the effect of venoms extracted from R. schneideri and R. marina on cancer cells and verify possible mechanism of action. MATERIAL AND METHOD: Cytotoxicity analyses was performed using the resazurin reduction assay, where different concentrations of venoms were tested against sensitive CCRF-CEM and P-gp overexpressing ADR/CEM5000 leukemia cells. Programmed cell death was investigated using the flow cytometric annexin V/propidium iodide apoptosis assay. Furthermore, we analyzed flow cytometric cell cycle analyses of CCRF-CEM cells. Effect on tubulin formation was tested using molecular docking and fluorescence microscopy of U2OS-GFP-α-tubulin osteosarcoma cells treated for 24 h with venoms. RESULTS: Cytotoxicity assays revealed a strong activity towards wild-type CCRF-CEM cells (IC50 values of 0.202 ±â€¯0.005 µg/ml and 0.18 ±â€¯0.007 µg/ml for R. schneideri and R. marina, respectively) and multidrug-resistant CEM/ADR5000 cells (IC50 0.403 ±â€¯0.084 µg/ml and 0.32 ±â€¯0.077 µg/ml for R. schneideri and R. marina, respectively). The venoms induced apoptosis as major mechanism of cell death. The venoms induced strong G2/M cell arrest in CCRF-CEM cells. We suggested tubulin as a major target for the venoms. In silico molecular docking of the major constituents of the venoms, i.e. bufalin, marinobufagin, telocinbufagin, hellebrigenin, showed strong binding affinities to tubulin. This result was verified in vitro. The venoms dysregulated microtubule arrangement of U2OS cells expressing GFP-labeled tubulin. Toxicity predictions by QSAR methodology highlighted the toxic features of bufadienolides. CONCLUSION: Our study demonstrated the importance of toad venoms as source of cytotoxic compounds that may serve as lead compounds for the development of novel anticancer drugs.

Biomarkers at different levels of organisation after atrazine formulation (SIPTRAN 500SC®) exposure in Rhinella schineideri (Anura: Bufonidae) Neotropical tadpoles.

Brazil is an important consumer of herbicides. In sugarcane cultivation-the country's most extensive agricultural crop-atrazine-based formulations are the principal form of weed control. Several studies have investigated adverse effects of atrazine or their formulations on anurans, but not specifically on Brazilian species. Our aim was therefore to investigate the lethal and sublethal effects of an atrazine-based herbicide in Rhinella schneideri tadpoles and, in particular, effects on the pigmentation system as a new endpoint in ecotoxicological studies. Rhinella schneideri tadpoles at the Gosner-30 stage were exposed to the atrazine-based herbicide formulation, SIPTRAN 500 SC®, in acute bioassays at concentrations of 1.5-25 mg/L. The lethal and sublethal effects induced were analysed at different ecotoxicological levels: organismal level (alterations in behaviour, growth, development, and body mass; morphologic abnormalities), histological level (liver histopathology), the pigmentation system (melanomacrophages and dermal-melanophores), and cellular level (erythrocyte micronucleus formation and other nuclear-abnormalities). This herbicide induced sublethal effects at the organismal level with alterations in swimming and growth and morphologic abnormalities. These results demonstrated that, in anuran tadpoles, the atrazine-based agrochemical increased the frequency of micronucleus formation and other nuclear-abnormalities in erythrocytes and caused liver damage. In addition, we demonstrated for the first time effects of an atrazine-based formulation on the pigmentation system of anuran tadpoles, specifically an increase in the number of melanomacrophages and dermal melanophores. This study is the first to use several widely differing endpoints at different ecotoxicological levels in a comprehensive manner for assessment of the effects of environmental stressors in order to determine the health status of Neotropical anuran species. In doing so, this study establishes a foundation for future ecological assessments.

New species of Aplectana (Ascaridida: Cosmocercidae) in the toads, Rhinella granulosa and Rhinella schneideri (Anura: Bufonidae) from northern Argentina.

Aplectana adaechevarriae n. sp., from the large intestine of Rhinella granulosa and Rhinella schneideri, is described and illustrated. Of the 43 nominal species of Aplectana, A. adaechevarriae n. sp. represents the 23rd Neotropical species and the third species reported from Argentina. Of the Neotropical species, only 4, Aplectana elenae, Aplectana hylambatis, Aplectana raillieti, and A. adaechevarriae n. sp., have 8 pairs of preanal papillae. Aplectana adaechevarriae lacks a median preanal papilla; the other 3 species possess a median preanal papilla.