Predicting the potential distribution of the alien invasive American bullfrog (Lithobates catesbeianus) in Brazil

The non-native invasive anuran Lithobates catesbeianus is presently distributed in Brazil, especially in the Atlantic Rainforest biodiversity hotspot. Here, we use a maximum entropy ecological niche modeling algorithm (i) to model the North American native geographic distribution of this species and (ii) to project that model onto the whole of Brazil. After applying a threshold value that balances commission and omission errors, the projection results suggested high probabilities of occurrence mostly in southern and southeastern Brazil. We also present the first report on the species known distribution in Brazil, showing good agreement with model predictions. If the predictive map is interpreted as depicting invasiveness potential of L. catesbeianus, strategies to prevent further invasion in Brazil should be focused especially in the Atlantic Rainforest biodiversity hotspot.

Atividade de fosfomonohidrolases presentes em fígado de girino de rã-touro (Lithobates catesbeianus) durante a metamorfose

Pós-graduação em Biotecnologia - IQ

Characterizing the impact of multiple potential enemies (predators and parasites) on the behaviour of ranid tadpoles

In order to fully understand an organism's behaviours the interactions between multiple enemies or selective pressures need to be considered, as these interactions are usually far more complex than the simple addition of their effects in isolation. In this thesis, I consider the impact of multiple enemies (fish predators and parasites) on the behaviour of three larval anurans (Lithobates sylvaticus, L. clamitans and L. catesbeianus). I also determine whether species that differ in life-histories and habitat preferences possess different antipredator mechanisms and how this affects species responses to multiple enemies. I show that the three Ranid larvae respond differently to the trade-off imposed by the presence of both fish predators and trematode parasites within the environment. The two more permanent pond breeders (L. clamitans and L. catesbeianus) increased activity when in the combined presence of predators and parasites. In contrast, the temporary pond breeder (L. sylvaticus) decreased activity in the combined presence of predator and parasites, in the same manner as they responded to fish alone. Further, the presence of fish along with parasites increased the susceptibility of both L. sylvaticus and L. clamitans to trematode infection, whereas parasite infection in L. catesbeianus was unaffected by the presence of fish. A second experiment to assess palatability of the three anuran species to fish, revealed a range of palatabilities, with L. catesbeianus being least palatable, L. clamitans being somewhat unpalatable, and L. sylvaticus being highly palatable. This result helps to explain the species differences in tthe observed behaviour to the combined presence of fish and parasites. In conclusion, the results from this study highlight the importance of considering multiple selective pressures faced by organisms and how this shapes their behaviour.

Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers-alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor)-and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)β was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERβ expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERβ, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus. © 2012 S. Karger AG, Basel.

Bullfrog hemogram under management stress

Stress is one of the major obstacles in frog culture and can be caused by factors such as inappropriate farming systems; inadequate management among other situations. The objective of the present study was to assess the hemogram, erythrogram and leukogram of bullfrogs (L. catesbeianus) when exposed to stress caused by different types of management: density and handling (manipulation), developed in the laboratory and repeated in the field for the appropriate comparisons in a experimental period of 30 days. The density experiment was conducted with four treatments: 70 animals m(-2) (D70); 100 animals m(-2) (D100), Control; 150 animals m(-2) (D150) and 200 animals m(-2) (D200), with 10, 14, 21 and 28 animals/box in the laboratory, respectively. Each treatment was performed with three simultaneous replicates. The handling experiment was conducted with three treatments: Treatment Without Handling (WH); Treatment with Partial Handling (PH) every 15 days and Treatment with Total Handling (TH) every 15 days. Each treatment was performed with four simultaneous replications. The methodology of the blood analysis followed international recommendations. In the present study we could observe that the animals of the field experiment did not reflect the same stress response observed in the laboratory in both experiment, which demonstrated the plasticity of these animals.

Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors

The aim of this study was to evaluate alterations to the physiological profile (cortisol, glycaemia, and blood parameters) of Lithobates catesbeianus caused by the stressors density and hypoxia. The organisms were in the prometamorphosis stage and exposed to different tadpole densities: 1 tadpole/L (T1), 5 tadpoles/L (T2), and 10 tadpoles/L (T3) for 12 days. The blood was collected through the rupture of the caudal blood vessel and collected under normoxia (immediate collection) and hypoxia (after 15 minutes of air exposure) conditions. Cortisol levels rose on the fourth and eighth days of treatment and returned to basal levels by the end of the experiment. The stressor mechanisms tested did not affect glycaemia. White blood cells (total number of lymphocytes, neutrophils, and eosinophils) showed a significant difference at the twelfth day of the experiment when compared with the start of the experiment. We concluded that, under controlled conditions, a density of up to 10 tadpoles/L and air exposure for 15 minutes did not cause harmful physiological alterations during the experimental period. The answer to these stressors maybe was in another hormonal level (corticosterone).

Duration of spermatogenesis in the bullfrog (Lithobates catesbeianus)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Median lethal concentration of formaldehyde and its genotoxic potential in bullfrog tadpoles (Lithobates catesbeianus)

In order to avoid that contaminated frog farms animals escaping in the environment and become potential vector of emergent diseases, studies with disinfection protocol are strictly necessary. The formaldehyde is one of the compounds tested in fungal disinfection protocols and also used in aquaculture. This study aimed to determine the median lethal concentration (LC50-96h) of formaldehyde in bullfrog tadpoles and to evaluate the possible genotoxic effects in acute exposition. Accordingly, the animals were exposed to formaldehyde in the concentrations of 6, 9, 12, 15, and 18 mg L(-1), and after 96 h blood samples were drawn for the micronucleus (MN) test. The LC50-96h was 10.53 mg L(-1), and the MN frequency increased in proportion to the formaldehyde concentrations, with an estimated frequency in the negative control being 1.35 MN/individual. We concluded that formaldehyde is genotoxic to tadpoles of bullfrogs in the tested concentrations, and the choice of this chemical should be contemplated before its use in animals in captivity.

Duration of spermatogenesis in the bullfrog (Lithobates catesbeianus)

The bullfrog (Lithobates catesbeianus) has substantial economic importance and has also been used as an experimental model for biological studies in the fields of pharmcology, medicine, and reproductive biology, especially studies addressing gametogenesis. However, there is a lack of comprehensive information in the literature regarding testis structure and function in this amphibian. The main objective of the current study was to estimate the duration of the various phases of spermatogenesis in this vertebrate. Sixteen sexually mature bullfrogs received an intracoelomic administration of tritiated thymidine. Testes were analyzed at various times between I h and 33 d after administration to detect the most advanced germ cell types labeled at each interval, as well as labeled preleptotene spermatocytes, which presumably originated from spermatogonial stem cells. The duration of the spermatogonial, spermatocytic, and spermiogenic phases of spermatogenesis in the bullfrog were approximately 18, 14, and 8 d, respectively. Thus, the total duration of the spermatogenesis process from early spermatogonia through to spermatozoa was 40 d in this species, similar to that of most previously investigated mammalian species. To our knowledge, this is the first reliable report on the duration of the full spermatogenic process in any amphibian species. These findings will be very useful for tracking the pace of germ cells in studies involving spermatogonial transplantation in lower vertebrates. (C) 2009 Elsevier B.V. All rights reserved.

Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors

O objetivo deste estudo foi avaliar a resposta fisiológica (cortisol, glicemia e parâmetros sanguineos) de girinos de rã-touro (Lithobates catesbeianus) em diferentes densidades e após exposição aérea. Os animais utilizados no experimento estavam entre os estágios 31 a 39, na fase de pró-metamorfose sendo testados 1 girino/L (Tratamento 1), 5 girinos/L (Tratamento 2) e 10 girinos/L (Tratamento 3), conduzidos em 3 réplicas simultâneas durante 12 dias. O sangue foi retirado por rompimento do vaso caudal na condição de Normóxia - N (tempo zero) e Hipóxia - H (tempo de 15 minutos de exposição ao ar). Foi observado um aumento nos valores de cortisol, aos 4 e 8 dias de exposição aérea retornando aos valores basais ao final do experimento, apesar de não haver diferenças significativas. A glicemia não apresentou diferenças significativas quanto aos estressores aplicados. Os parâmetros hematológicos da série branca, principalmente, o número de linfócitos, neutrófilos e eosinófilos mostraram diferença significativa aos 12 dias de experimentação quando comparados com o momento zero; concluindo-se que, em condições controladas, o adensamento de até 10 girinos/litro e a exposição aérea por 15 minutos não apresentou danos aos girinos de rã-touro durante o período experimental. O padrão de resposta a estes estímulos talvez seja expresso em outro nível hormonal (corticosterona).

Occurrence Of Isoflavonoids In Brazilian Common Bean Germplasm (phaseolus Vulgaris L.).

Common bean (Phaseolus vulgaris) is present in the daily diet of various countries and, as for other legumes, has been investigated for its nutraceutical potential. Thus, 16 genotypes from different gene pools, representing seven types of seed coats and different responses to pathogens and pests, were selected to verify their isoflavone contents. The isoflavonoids daidzein and genistein and the flavonols kaempferol, myricetin, and quercetin were found. Grains of the black type showed the highest concentrations of isoflavonoids and were the only ones to exhibit daidzein. IAC Formoso, with high protein content and source of resistance to anthracnose, showed the greatest concentration of genistein, representing around 11% of the content present in soybean, as well as high levels of kaempferol. Arc 1, Raz 55, and IAC Una genotypes showed high content of coumestrol. The results suggest the use of IAC Formoso to increase the nutraceutical characteristics in common bean.

Influence Of The Stage Of Ripeness On The Composition Of Iridoids And Phenolic Compounds In Genipap (genipa Americana L.).

Genipap fruits, native to the Amazon region, were classified in relation to their stage of ripeness according to firmness and peel color. The influence of the part of the genipap fruit and ripeness stage on the iridoid and phenolic compound profiles was evaluated by HPLC-DAD-MS(n), and a total of 17 compounds were identified. Geniposide was the major compound in both parts of the unripe genipap fruits, representing >70% of the total iridoids, whereas 5-caffeoylquinic acid was the major phenolic compound. In ripe fruits, genipin gentiobioside was the major compound in the endocarp (38%) and no phenolic compounds were detected. During ripening, the total iridoid content decreased by >90%, which could explain the absence of blue pigment formation in the ripe fruits after their injury. This is the first time that the phenolic compound composition and iridoid contents of genipap fruits have been reported in the literature.

Auxin And Physical Constraint Exerted By The Perianth Promote Androgynophore Bending In Passiflora Mucronata L. (passifloraceae).

The androgynophore column, a distinctive floral feature in passion flowers, is strongly crooked or bent in many Passiflora species pollinated by bats. This is a floral feature that facilitates the adaptation to bat pollination. Crooking or bending of plant organs are generally caused by environmental stimulus (e.g. mechanical barriers) and might involve the differential distribution of auxin. Our aim was to study the role of the perianth organs and the effect of auxin in bending of the androgynophore of the bat-pollinated species Passiflora mucronata. Morpho-anatomical characterisation of the androgynophore, including measurements of curvature angles and cell sizes both at the dorsal (convex) and ventral (concave) sides of the androgynophore, was performed on control flowers, flowers from which perianth organs were partially removed and flowers treated either with auxin (2,4-dichlorophenoxyacetic acid; 2,4-D) or with an inhibitor of auxin polar transport (naphthylphthalamic acid; NPA). Asymmetric growth of the androgynophore column, leading to bending, occurs at a late stage of flower development. Removing the physical constraint exerted by perianth organs or treatment with NPA significantly reduced androgynophore bending. Additionally, the androgynophores of plants treated with 2,4-D were more curved when compared to controls. There was a larger cellular expansion at the dorsal side of the androgynophores of plants treated with 2,4-D and in both sides of the androgynophores of plants treated with NPA. This study suggests that the physical constraint exerted by perianth and auxin redistribution promotes androgynophore bending in P. mucronata and might be related to the evolution of chiropterophily in the genus Passiflora.

Supercritical Carbon Dioxide Extraction Of Capsaicinoids From Malagueta Pepper (capsicum Frutescens L.) Assisted By Ultrasound.

Extracts from malagueta pepper (Capsicum frutescens L.) were obtained using supercritical fluid extraction (SFE) assisted by ultrasound, with carbon dioxide as solvent at 15MPa and 40°C. The SFE global yield increased up to 77% when ultrasound waves were applied, and the best condition of ultrasound-assisted extraction was ultrasound power of 360W applied during 60min. Four capsaicinoids were identified in the extracts and quantified by high performance liquid chromatography. The use of ultrasonic waves did not influence significantly the capsaicinoid profiles and the phenolic content of the extracts. However, ultrasound has enhanced the SFE rate. A model based on the broken and intact cell concept was adequate to represent the extraction kinetics and estimate the mass transfer coefficients, which were increased with ultrasound. Images obtained by field emission scanning electron microscopy showed that the action of ultrasonic waves did not cause cracks on the cell wall surface. On the other hand, ultrasound promoted disturbances in the vegetable matrix, leading to the release of extractable material on the solid surface. The effects of ultrasound were more significant on SFE from larger solid particles.