Runway extinction in terrestrial toads (Rhinella arenarum): Instrumental or Pavlovian?

Instrumental appetitive extinction involves the reduction of a previously reinforced response when its occurrence is no longer rewarded. Two experiments with terrestrial toads (Rhinella arenarum) tested whether the occurrence of a nonreinforced response is necessary for response extinction by varying the time of exposure to nonrewarded goal-box stimuli across groups. In Experiment 1, toads that received the same acquisition training (15 sessions, 1 session/day, 300 s of access to water in the goal box) were randomly assigned to two groups. In Group 600 (n=12), animals spent 600 s in the goal box in 8 daily extinction sessions (water present but inaccessible). In Group 0 (n=11), toads performed the runway response (i.e., walking from the start to the goal box) but were removed as soon as they entered the goal box, thus having minimal exposure to nonrewarded goal-box stimuli. The runway response was weakened in Group 600 across extinction trials, but exhibited little change in Group 0. In Experiment 2, toads were randomly assigned to two groups after the same acquisition training. Group 0 (n=7) was treated the same as Group 0 in the previous experiment. In Group RI (retention interval, n=7), toads remained in their home cage for 13 days. Finally, all animals received 4 extinction sessions with 300 s in the empty goal box. There was little behavioral change in Group 0 during the 13 sessions with minimal exposure to the goal box. In extinction, both groups reduced their runway response at similar rates. Although the procedures were instrumental, extinction of the runway response in toads can be accounted for in terms of a Pavlovian approach response to stimuli paired with reward and nonreward in the goal box.

First evidence of environmental bioaccumulation of pharmaceuticals on adult native anurans (Rhinella arenarum) from Argentina.

The presence of pharmaceutically active compounds (PhACs) in surface water is well known, whereas their natural occurrence in biota is much less explored. The aim of this work was to evaluate the bioaccumulation of PhACs in adult toads of the neotropical species Rhinella arenarum. Three sites were selected in Buenos Aires (Argentina): a reference site (Site 1), a site with direct discharge from a secondary wastewater treatment plant (WWTP) (Site 2) and a site 300 m downstream of the WWTP discharge (Site 3). Surface water samples, as well as muscle, liver and fat bodies of toads were collected, extracted and analyzed by LC-MS/MS. Highly significant differences in total PhACs concentration in surface water (p < 0.005) were detected between Site 2 and the other sites. These concentrations ranged from 0.37 to 52.46 ng/L at Site 1, 0.71-6950.37 ng/L at Site 2, and 0.12-75.45 ng/L at Site 3. In general, bioaccumulation of PhACs in toad tissues was similar between sites and tissues of each site. The highest concentrations were detected in the muscle of toads from Site 3 (1.06-87.24 ng/g dw), followed by liver (1.77-38.10 ng/g dw) and fat bodies (0.68-20.59 ng/g dw) from Site 1. Ibuprofen (6950 ng/L), acetaminophen (3277 ng/L) and valsartan (2504 ng/L) were the compounds with the highest concentrations in surface water from Site 2, whereas acetaminophen (87.2 ng/g dw, muscle from Site 3), desloratadine (38.1 ng/g dw, liver from Site 1), and phenazone (25.9 ng/g dw, liver from Site 1) were the ones that showed the highest concentrations in biota. This is the first time a field study has examined the environmental bioaccumulation of PhACs in anurans, demonstrating their potential for monitoring the status of natural ecosystems.

Direct development or endotrophic tadpole? Morphological aspects of the early ontogeny of the plump toad Osornophryne occidentalis (Anura: Bufonidae).

Andean Toads of the genus Osornophryne are suspected to have direct development on the basis of clutch and egg features. In this work, we describe the morphology of Osornophryne occidentalis embryos from a nest collected in the field. Several traits are similar to those reported in brachycephaloid Eleutherodactylus coqui and other direct-developing lineages. These include the pattern of formation of the body wall and the absence of oral and buccopharyngeal structures. Other features indicate the retention of ancestral larval characters. The development of forelimbs occurs in part within the operculum as in species with biphasic ontogeny; this has been reported in other direct-developing species, and is similar to what is described in African viviparous bufonids. This salient feature, along with the long, low-finned tail and the long and folded intestine early differentiated, gives these embryos a rather tadpole-like appearance. Our data confirm that development in O. occidentalis occurs within terrestrial eggs until advanced stages, and this would satisfy current definitions of direct development. At the same time, morphological differences regarding other species with comparable breeding reinforce interpretations about the wide anatomical and ontogenetic variations associated with endotrophic nutrition.

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.

Evidence of successive negative contrast in terrestrial toads (Rhinella arenarum): central or peripheral effect?

Prior research with terrestrial toads (Rhinella arenarum) in a water-reinforced instrumental situation indicated a direct relationship between acquisition rate and reward magnitude. However, a reward downshift produced a gradual adjustment of instrumental performance and a rapid adjustment of consummatory performance, rather than the abrupt and transient deterioration of behavior typical of a successive negative contrast effect. In Experiment 1, using a two-chamber box, a downshift from deionized water (which supports maximal rehydration) to 250-mM sodium chloride solution (which supports a lower rehydration), also yielded a gradual adjustment of instrumental behavior. In this experiment, animals received one trial per day and were allowed 300 s of access to the reward in the goal box. Experiment 2 used the same procedure, except that animals were allowed access to the solution in the goal box for 600 s. Under these conditions, reward downshift led to longer latencies (instrumental) and lower rehydration levels (consummatory) than those of unshifted controls, providing evidence for successive negative contrast. Unlike in similar experiments with mammals, the effect was not transient, but persisted relatively unmodified over twelve daily postshift trials. In this case, the possibility of adaptation of the peripheral mechanisms for water uptake is considered. The comparative relevance of these results is discussed in terms of habit formation versus expectancy-guided behavior in vertebrate learning.

5-HT neurons of the medullary raphe contribute to respiratory control in toads.

Air-breathing vertebrates undergo respiratory adjustments when faced with disturbances in the gas composition of the environment. In mammals, the medullary raphe nuclei are involved in the neuronal pathway that mediates the ventilatory responses to hypoxia and hypercarbia. We investigate whether the serotoninergic neurons of the medullary raphe nuclei of toads (Rhinella diptycha) play a functional role in respiratory control during resting conditions (room air), hypercarbia (5% CO2), and hypoxia (5% O2). The raphe nuclei were located and identified based on the location of the serotoninergic neurons in the brainstem. We then lesioned the medullary raphe (raphe pallidus, obscurus and magnus) with anti-SERT-SAP and measured ventilation in both control and lesioned groups and we observed that serotonin (5-HT) specific chemical lesions of the medullary raphe caused reduced respiratory responses to both hypercarbia and hypoxia. In summary, we report that the serotoninergic neurons of the medullary raphe of the cururu toad Rhinella diptycha participate in the chemoreflex responses during hypercarbia and hypoxia, but not during resting conditions. This current evidence in anurans, together with the available data in mammals, brings insights to the evolution of brain sites, such as the medullary raphe, involved in the ventilatory chemoreflex in vertebrates.

Proteins from Rhinella jimi parotoid gland secretion: A comprehensive analytical approach.

Toad skin secretions are sources of complex mixtures of bioactive compounds, such as proteins and peptides. Rhinella jimi species is a common toad in the Brazilian northeast, considered by only a few known studies. The experimental design was applied to optimize the protein extraction method from R. jimi parotoid gland secretions. The optimum condition was using 100 mmol L-1 Tris-HCl buffer pH 7.2 under vortexing for 5 min. The FTIR analysis combined with PCA revealed high-protein purity of the extracts, confirming the success of the proposed extraction method. The total protein concentration by the Bradford method was 102.4 and 66.5 mg g-1 on toad poisons from Teresina and Picos, respectively. The comparative proteomic analysis using HPLC-SEC-DAD and 1D SDS-PAGE revealed significant differences in protein abundance. HMW biomolecules showed greater abundance in toads from Teresina, while LMW protein species were more abundant in toads from Picos. The significant difference in amphibian proteome can be attributed to the edaphoclimatic conditions of their habitat. The cytotoxicity of the protein extract from Teresina was higher on the tumor cell lines 4T1 and CT26.WT. These new findings are fundamental for future studies the on identity and biological activity of biomolecules from this noble sample.

Differences between poison and venom: an attempt at an integrative biological approach

We discuss the use of the terms venom and poison, in the context of integrative biology, with particular emphasis on behaviour and natural history. Our purpose is to reach a broad scientific audience, especially that dedicated to zoology. The meaning of the two terms is reviewed from the secretory perspective, mainly focussed on the reptiles and amphibians. We justify the use of the two words, based on biological and behavioural differences.

Anurans of the Caparaó National Park and surroundings, southeast Brazil / Anuros do Parque Nacional do Caparaó e arredores, sudeste brasileiro

Abstract The Atlantic Forest (AF) is one of the biodiversity hotspots of the world, and the most fragmented biome of Brazil. This biome includes different phytophysiognomies, as riparian, slope, cloudy forests, and grasslands. Such complexity, allied to huge latitudinal and high elevational range, provides diverse habitats and conditions for amphibian speciation. As a result, there are over 600 amphibian species known to occur in the AF. Within this biome the Caparaó National Park (CNP) is relevant, as it includes the highest peak of the biome, the Pico da Bandeira at almost 3,000 m above sea level, as well as different phytophysiognomies as rocky fields and humid forests. In spite of that, its amphibian fauna is still poorly described. We inventoried amphibians at the CNP and surrounding areas from 2016 to 2018 and recorded 47 anuran species, of which two are locally endemic and at least six have not been described yet. Additionally, we compiled data from previous surveys (2004 to 2008) and secondary data from scientific collections. All together, we registered a total of 61 anuran species from 12 families for the CNP and surroundings, placing this area among the 10 amphibian richest sites in the AF. Some of these species are represented by only one or two collected specimens and have not been registered in the CNP since the 1980's, such as Thoropa lutzi and Hylodes vanzolinii. These species could be examples of population declines or even past local extinctions, highlighting the need of further sampling efforts in that highly biodiverse site.

Resumo A Mata Atlântica (MA) é considerada um dos hotspots de biodiversidade do mundo, sendo o bioma mais degradado no Brasil. Este bioma compreende muitas fitofisionomias, como floresta ombrófila, semidecidual, e campos de altitude. Esta complexidade, aliada à enorme variação latitudinal e altitudinal, disponibiliza habitats e condições diversas à especiação dos anfíbios. Como resultado, atualmente são conhecidas mais de 600 espécies de anfíbios que ocorrem na MA. Dentro deste bioma o Parque Nacional do Caparaó (PNC) é relevante, pois inclui o ponto mais alto da Mata Atlântica, o Pico da Bandeira com cerca de 3000 m de elevação, assim como diferentes fitofisionomias, como campos rupestres e florestas úmidas. Apesar disso, sua fauna de anfíbios é ainda pobremente descrita. Realizamos inventários de anfíbios no PNC e entorno de 2016 a 2018 e registramos 47 espécies de anuros, das quais duas são endêmicas e pelo menos seis ainda não foram descritas. Adicionalmente, compilamos dados de coletas anteriores (2004 a 2008) e dados secundários de coleções científicas. Ao juntar todos os dados registramos um total de 61 espécies de anuros de 12 famílias para o PNC e arredores, colocando esta área entre as 10 áreas mais ricas em anfíbios da MA. Algumas destas espécies estão representadas apenas por um ou dois exemplares em coleções e não foram registradas no PNC desde a década de 1980, como Thoropa lutzi e Hylodes vanzolinii. Estas espécies podem ser exemplos de declínios populacionais ou mesmo de extinções locais, destacando a necessidade de maiores esforços amostrais neste local altamente biodiverso.

A morphological database for Colombian anuran species from conservation-priority ecosystems.

Species traits provide a strong link between an organism's fitness and processes at community and ecosystem levels. However, such data remain scarce for amphibians in the Neotropics. Colombia is the country with the highest number of threatened amphibians and the second greatest number of amphibian species worldwide. We present a data set containing eight morphological traits for 4,623 museum specimens of the seven largest collections in the country corresponding to 293 species of 14 families. The number of measured specimens per species ranged from 1 to 118 individuals with a median of 8 individuals per species. Overall, this database gathered morphological information for 37.6% of Colombian anuran diversity. Species measured were mainly distributed in the high Andean forest, the páramo, and wetland ecosystems, and was part of a national initiative led by the Instituto Alexander von Humboldt. The morphological traits were selected on the basis of their role in species' responses to environmental variability and their contributions to ecosystem processes. These traits were related to habitat use, (forearm length, tibia length, femur length, foot length, and foot webbing), predation and food chains (head width and mouth width), and nutrient recycling (snout-vent length). We expect this data set will be used in studies on functional diversity in amphibians and the development of conservation planning for these taxa. No copyright or proprietary restrictions are associated with the use of this data set other than citation of this Data Paper.

Duration of extinction trials as a determinant of instrumental extinction in terrestrial toads (Rhinella arenarum).

Instrumental learning guides behavior toward resources. When such resources are no longer available, approach to previously reinforced locations is reduced, a process called extinction. The present experiments are concerned with factors affecting the extinction of acquired behaviors in toads. In previous experiments, total reward magnitude in acquisition and duration of extinction trials were confounded. The present experiments were designed to test the effects of these factors in factorial designs. Experiment 1 varied reward magnitude (900, 300, or 100 s of water access per trial) and amount of acquisition training (5 or 15 daily trials). With total amount of water access equated in acquisition, extinction with large rewards was faster (longer latencies in 900/5 than 300/15), but with total amount of training equated, extinction with small rewards was faster (longer latencies in 100/15 than 300/15). Experiment 2 varied reward magnitude (1200 or 120 s of water access per trial) while holding constant the number of acquisition trials (5 daily trials) and the duration of extinction trials (300 s). Extinction performance was lower with small, rather than large reward magnitude (longer latencies in 120/300 than in 1200/300). Thus, instrumental extinction depends upon the amount of time toads are exposed to the empty goal compartment during extinction trials.

The Paraguayan Rhinella toad venom: Implications in the traditional medicine and proliferation of breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Toads belonging to genus Rhinella are used in Paraguayan traditional medicine to treat cancer and skin infections. AIM OF THE STUDY: The objective of the study was to determine the composition of venoms obtained from three different Paraguayan Rhinella species, to establish the constituents of a preparation sold in the capital city of Paraguay to treat cancer as containing the toad as ingredient, to establish the effect of the most active Rhinella schneideri venom on the cell cycle using human breast cancer cells and to assess the antiprotozoal activity of the venoms. METHODS: The venom obtained from the toads parotid glands was analyzed by HPLC-MS-MS. The preparation sold in the capital city of Paraguay to treat cancer that is advertised as made using the toad was analyzed by HPLC-MS-MS. The effect of the R. schneideri venom and the preparation was investigated on human breast cancer cells. The antiprotozoal activity was evaluated on Leishmania braziliensis, L. infantum and murine macrophages. RESULTS: From the venoms of R. ornata, R. schneideri and R. scitula, some 40 compounds were identified by spectroscopic and spectrometric means. Several minor constituents are reported for the first time. The preparation sold as made from the toad did not contained bufadienolides or compounds that can be associated with the toad but plant compounds, mainly phenolics and flavonoids. The venom showed activity on human breast cancer cells and modified the cell cycle proliferation. The antiprotozoal effect was higher for the R. schneideri venom and can be related to the composition and relative ratio of constituents compared with R. ornata and R. scitula. CONCLUSIONS: The preparation sold in the capital city of Paraguay as containing the toad venom, used popularly to treat cancer did not contain the toad venom constituents. Consistent with this, this preparation was inactive on proliferation of human breast cancer cells. In contrast, the toad venoms of Rhinella species altered the cell cycle progression, affecting the proliferation of malignant cells. The findings suggest that care should be taken with the providers of the preparation and that the crude drug present a strong activity towards human breast cancer cell lines. The antiprotozoal effect of the R. schneideri venom was moderate while the venom of R. ornata was devoid of activity and that of R. scitula was active at very high concentration.

Anuros del norte de los Andes: patrones de riqueza de especies y estado de conservación

El conocimiento de los anuros andinos está en constante aumento, razón por la cual se necesita una actualización. Revisamos la literatura y museos registros científicos con el fin de determinar la riqueza de especies de anuros del norte de los Andes con base en las categorías de la UICN. El norte de los Andes alberga 740 especies de anuros, pertenecientes a 13 familias y 63 géneros. La familia con el mayor número de especies fue craugastoridae (334 spp.), seguida por Bufonidae (92 spp.) y Centrolenidae (81 spp.). La Cordillera Central de Colombia alberga la mayor riqueza de especies (202 spp.), mientras que la Cordillera de la Costa Oriental de Venezuela contiene el número más bajo de especies (17 spp.). La mayoría de las especies (74%) es exclusiva del norte de los Andes, pero ninguna familia es endémica del norte de los Andes. Alrededor del 50% de las especies está amenazada y cerca del 20% con datos deficientes. Los patrones de riqueza de especies en el norte de los Andes podrían ser causados, entre otras razones, por factores históricos, y a su vez por a la falta de muestreo en algunas zonas.(AU)

Knowledge of Andean anurans is constantly increasing; for this reason, it needs an update. We reviewed scientific literature and museum records to determine the species richness of anurans in the northern Andes and their conservation status based on the IUCN categories. The northern Andes harbor 740 species of anurans, belonging to 13 families and 63 genera. The family with the highest species number was Craugastoridae (334 spp.), followed by Bufonidae (92 spp.), and Centrolenidae (81 spp.). The Cordillera Central of Colombia harbors the highest species richness (199 spp.), while the Cordillera de la Costa Oriental de Venezuela contains the lowest species number (17 spp.). Most species (74%) is endemic to the northern Andes, but no family is endemic to the northern Andes. About 50% species is threatened and 20% is included in the category of deficient data. The species richness patterns in northern Andes might be caused, among other reasons, by historical factors, but also result of sampling lack in some areas.(AU)

Anuros del norte de los Andes: patrones de riqueza de especies y estado de conservación

El conocimiento de los anuros andinos está en constante aumento, razón por la cual se necesita una actualización. Revisamos la literatura y museos registros científicos con el fin de determinar la riqueza de especies de anuros del norte de los Andes con base en las categorías de la UICN. El norte de los Andes alberga 740 especies de anuros, pertenecientes a 13 familias y 63 géneros. La familia con el mayor número de especies fue craugastoridae (334 spp.), seguida por Bufonidae (92 spp.) y Centrolenidae (81 spp.). La Cordillera Central de Colombia alberga la mayor riqueza de especies (202 spp.), mientras que la Cordillera de la Costa Oriental de Venezuela contiene el número más bajo de especies (17 spp.). La mayoría de las especies (74%) es exclusiva del norte de los Andes, pero ninguna familia es endémica del norte de los Andes. Alrededor del 50% de las especies está amenazada y cerca del 20% con datos deficientes. Los patrones de riqueza de especies en el norte de los Andes podrían ser causados, entre otras razones, por factores históricos, y a su vez por a la falta de muestreo en algunas zonas.

Knowledge of Andean anurans is constantly increasing; for this reason, it needs an update. We reviewed scientific literature and museum records to determine the species richness of anurans in the northern Andes and their conservation status based on the IUCN categories. The northern Andes harbor 740 species of anurans, belonging to 13 families and 63 genera. The family with the highest species number was Craugastoridae (334 spp.), followed by Bufonidae (92 spp.), and Centrolenidae (81 spp.). The Cordillera Central of Colombia harbors the highest species richness (199 spp.), while the Cordillera de la Costa Oriental de Venezuela contains the lowest species number (17 spp.). Most species (74%) is endemic to the northern Andes, but no family is endemic to the northern Andes. About 50% species is threatened and 20% is included in the category of deficient data. The species richness patterns in northern Andes might be caused, among other reasons, by historical factors, but also result of sampling lack in some areas.

Telencephalic neural activation following passive avoidance learning in a terrestrial toad.

The present study explores passive avoidance learning and its neural basis in toads (Rhinella arenarum). In Experiment 1, two groups of toads learned to move from a lighted compartment into a dark compartment. After responding, animals in the experimental condition were exposed to an 800-mM strongly hypertonic NaCl solution that leads to weight loss. Control animals received exposure to a 300-mM slightly hypertonic NaCl solution that leads to neither weight gain nor loss. After 10 daily acquisition trials, animals in the experimental group showed significantly longer latency to enter the dark compartment. Additionally, 10 daily trials in which both groups received the 300-mM NaCl solution after responding eliminated this group effect. Thus, experimental animals showed gradual acquisition and extinction of a passive avoidance respond. Experiment 2 replicated the gradual acquisition effect, but, after the last trial, animals were sacrificed and neural activation was assessed in five brain regions using AgNOR staining for nucleoli-an index of brain activity. Higher activation in the experimental animals, relative to controls, was observed in the amygdala and striatum. Group differences in two other regions, lateral pallium and septum, were borderline, but nonsignificant, whereas group differences in the medial pallium were nonsignificant. These preliminary results suggest that a striatal-amygdala activation could be a key component of the brain circuit controlling passive avoidance learning in amphibians. The results are discussed in relation to the results of analogous experiments with other vertebrates.

Seven new microendemic species of Brachycephalus (Anura: Brachycephalidae) from southern Brazil.

Brachycephalus (Anura: Brachycephalidae) is a remarkable genus of miniaturized frogs of the Brazilian Atlantic Rainforest. Many of its species are highly endemic to cloud forests, being found only on one or a few mountaintops. Such level of microendemism might be caused by their climatic tolerance to a narrow set of environmental conditions found only in montane regions. This restriction severely limits the chance of discovery of new species, given the difficulty of exploring these inaccessible habitats. Following extensive fieldwork in montane areas of the southern portion of the Atlantic Rainforest, in this study we describe seven new species of Brachycephalus from the states of Paraná and Santa Catarina, southern Brazil. These species can be distinguished from one another based on coloration and the level of rugosity of the skin in different parts of their body. These discoveries increase considerably the number of described species of Brachycephalus in southern Brazil.

Functional assessment of toad parotoid macroglands: a study based on poison replacement after mechanical compression.

Toads have a pair of parotoid macroglands behind the eyes that secrete poison used in passive defence against predators. These macroglands are composed of juxtaposed alveoli, each one bearing a syncytial gland, all connected to the exterior by ducts. When the parotoids are bitten, the poison is expelled on the predator oral mucosa in the form of jets, causing several pharmacological actions. After poison release, the empty secretory syncytia immediately collapse in the interior of their respective alveoli and gradually start refilling. After parotoid manual compression, simulating a predator's bite, we studied, by means of morphological methods, the replacement of the poison inside the alveoli. The results showed that after compression, a considerable number of alveoli remained intact. In the alveoli that were effectively affected the recovery occurs in different levels, from total to punctual and often restrict to some areas of the syncytia. The severely affected alveoli seem not recover their original functional state. The fact that only a part of the parotoid alveoli is compressed during an attack seems to be crucial for toad survival, since the amphibian, after being bitten by a predator, do not lose all its poison stock, remaining protected in case of new attacks.

Vulnerability of long-term memory to temporal delays in amphibians.

Two experiments with toads were designed to test the memory-decay hypothesis that extinction (i.e., nonreinforced) performance is a function of time since the last reinforcement. In Experiment 1, toads (Rhinella arenarum, formerly Bufo arenarum) received 15 daily acquisition trials each reinforced with access to water during 300 s in a runway and were then randomly assigned to one of 6 retention intervals (RIs): 1, 4, 8, 16, 32, and 64 days. Extinction started after the RI and lasted 8 additional daily trials. Overall extinction performance was a logarithmic function of the RI. Although 4 extinction trials produced similar performance than 4 days of RI (consistent with memory decay), 8 extinction trials produced lower performance than 8 days of RI (consistent with a decremental effect of nonreinforcement). In Experiment 2, two groups of toads received 15 daily acquisition trials each reinforced with access to water for either 30 or 600 s, thus producing two reward magnitudes. After an 8-day RI, extinction performance was weaker after training with the small, than with the large reward magnitude. These results suggest that, at least in early extinction, the instrumental performance of toads is strongly influenced by the time since the last reinforcement.

Alkaloids from single skins of the Argentinian toad Melanophryniscus rubriventris (ANURA, BUFONIDAE): An unexpected variability in alkaloid profiles and a profusion of new structures.

GC-MS analysis of single-skins of ten Melanophryniscus rubriventris toads (five collections of two toads each) captured during their breeding season in NW Argentina has revealed a total of 127 alkaloids of which 56 had not been previously detected in any frog or toad. Included among these new alkaloids are 23 new diastereomers of previously reported alkaloids. What is particularly distinguishing about the alkaloid profiles of these ten collections is the occurrence of many of the alkaloids, whether known or new to us, in only one of the ten skins sampled, despite two skins being obtained from each breeding site of the five populations. Many of the alkaloids are of classes known to have structures with branched-chains (e.g. pumiliotoxins and tricyclic structures) that are considered to derive from dietary mites. A large number of previously reported and new alkaloids are also of unclassified structures. Only a very few 3,5-disubstituted-indolizidine or -pyrrolizidine alkaloids are observed that have a straight-chain carbon skeleton and are likely derived from ant prey. The possible relationship of these collections made during the toad's brief breeding episodes to sequestration of dietary arthropods and individual alkaloid profiles is discussed.

Ocorrência de Amblyomma fuscum Neumann, 1899 e Amblyomma humerale Koch, 1844 (Acari: Ixodidae) em Bufo arenalis no estado de São Paulo, Brasil / Occurence of Amblyomma fuscum Neumann, 1899 and Amblyomma humerale Koch, 1844 (Acari: Ixodidae) in Bufo arenalis in the state of São Paulo, Brazil

O objetivo deste trabalho é relatar a ocorrência do parasitismo monoespecífico de A. fuscum NEUMANN, 1899 e A. humerale KOCH, 1844 em sapos (Bufo arenalis) no Município de Rosana, Estado de São Paulo, Brasil. Este relato caracteriza um novo hospedeiro e uma nova localização geográfica para estas duas espécies de carrapatos.

The objective of this work is to describe the occurence of the monoespecific parasitism of A. fuscum NEUMANN, 1899 and A. humerale KOCH, 1844 in toads (Bufo arenalis) from the County of Rosana, State of São Paulo, Brazil. This is the description of a new host and new geographic site for those two species.