Water-balance response of Rhinella arenarum (Hensel, 1867) tadpoles to graduated increase in environmental osmolarity / Resposta de Rhinella arenarum larvae a meio electrolítico e não electrolítico com aumento escalonado da osmolaridade

The water balance and the upper limit of osmotic tolerance of premetamorphic Rhinella arenarum larvae (Gosner's stage 26) was evaluated after semistatic incubation in electrolyte (NaCl) and non-electrolyte (mannitol) media following a protocol of progressively increased osmotic pressure. Wet and dry weights were measured to calculate the water content as a derived variable indicative of the hydric balance. Statistical analysis was performed using univariate and integrated multivariate analysis. Tadpoles survived in electrolyte and non-electrolyte solutions up to 200 mOsm. The discriminant function was the best tool to describe the responses of the animals to external environmental stress under experimental conditions. The results were compared with those obtained in previous studies using a protocol of acute exposure to the same media used in this study. It was concluded that a) multivariate analysis is an appropriate approach to describe the responses of tadpoles to changes in the environmental physicochemical parameters, and b) progressive and acute acclimation to the experimental solutions induced similar responses.

Avaliou-se o balanço hídrico e o limite superior de tolerância osmótica em larvas pré-metamórficas do Rhinella arenarum (etapa 26 de Gosner) sob condições de incubação semiestáticas, num meio eletrolítico (NaCl) e não eletrolítico (manitol), seguindo um protocolo de aumento progressivo da pressão osmótica do meio. A quantificação das respostas se efetuou por meio da medição dos valores de peso úmido e seco e do cálculo, a partir destes, do conteúdo de água, como variável derivada indicativa do equilíbrio hídrico. A análise estatística foi realizada usando análise univariada e multivariada. As larvas conseguiram sobreviver em soluções eletrolíticas e não eletrolíticas até 200 mOsm. A função discriminante foi a melhor ferramenta para descrever as respostas dos animais ao estresse osmótico ambiental. Os resultados foram comparados com os obtidos em estudos anteriores, usando um protocolo de exposição aguda aos mesmos meios de incubação usados neste estudo. Concluiu-se: a) a análise multivariada é a aproximação adequada para descrever as respostas das larvas às mudanças nos parâmetros físico-químicos do seu meio; e b) tanto a aclimatação progressiva, como as exposições agudas às soluções experimentais induziram as respostas semelhantes.

Hemoglobin affinity for oxygen in three subspecies of toads (Bufo sp.) living at different altitudes

Blood oxygen affinity and red blood cell properties were measured in three subspecies of genus Bufo: Bufo spinulosus limensis, collected at sea level and at an average day temperature of 20§C; Bufo spinulosus trifolium, from 3100 m, average day temperature of 15§C; and Bufo spinulosus flavolineatus, from 4100 m, average day temperature of 10§C. Electrophoresis of the hemoglobin showed the same component in each of the three subspecies. At 20§C the blood oxygen affinities (P50) showed small differences between Bufo spinulosus limensis and Bufo spinulosus trifolium, whereas the value for Bufo spinulosus flavolineatus was markedly lower. At 10§C, the ambient temperature of Bufo spinulosus flavolineatus, the P50 was extremely low compared with the other two subspecies at their corresponding ambient temperatures.

A lethal cardiotoxic protein isolated from Bidder's organ of common Indian toad, Bufo melanostictus Schneider.

A lethal cardiotoxic (BO-CT; Bidder's organ cardiotoxin) protein was purified from the Bidder's organ of the common Indian toad B. melanostictus by gel filtration on Sephadex G-200. The homogeneity of cardiotoxin was tested by gel electrophoresis. The molecular weight of lethal BO-CT was 62 KDa and was devoid of glycoprotein. LD50 of the BO-CT was 50 micrograms/20 g (i.v.) in male albino mice. On isolated heart and auricle BO-CT initially increased the rate and amplitude of contraction and finally produced irreversible blockade of contraction. BO-CT induced auricular blockade, was not influenced by verapamil, propranolol and atropine. On isolated chick biventer cervicis preparation BO-CT produced irreversible blockade of electrically induced twitch response followed by contracture. This action was not antagonized by 4-aminopyridine and neostigmine. BO-CT induced contracture on chick biventer cervicis was increased by Ca2+, decreased by Na+ and abolished by K+. Cardiotoxic and neuromuscular activity of BO-CT was heat stable and abolished by proteolytic enzyme.

Isolation, purification and partial characterization of a haemolytic protein from Bidder's organ of toad Bufo melanostictus (Schneider).

A haemolytic protein toxin (BO-HT) from Bidder's organ of toad, B. melanostictus, purified by DEAE-cellulose column chromatography was electrophoretically homogeneous and was glycoprotein in nature (PAS-positive). The molecular weight was estimated to be 14.4 kDa by SDS-polyacrylamide gel electrophoresis. The sensitivity of the haemolysin of different RBC ghost cell preparation was in the order: buffalo > goat > ox > guinea pig > mice > human > chick > rabbit > rat. The haemolytic activity was increased with the decrease in RBC concentration and was produced over a wide range of temperature. Maximum haemolytic effect was produced at 2 hr of incubation. The toxin showed maximum activity at 3 and minimum at 10 pH. Divalent cations (Ca2+, Zn2+, Cu2+, Mg2+) showed inhibitory effect on BO-HT induced haemolysis, whereas sucrose, EDTA, cholesterol, 2-mercaptoethanol and oxygen did not alter the haemolytic activity. Haemolytic activity was reduced by proteolytic enzymes (trypsin, protease) and was totally antagonized by the toad serum.

Changes in lipid composition and some biologically important enzyme activities in the microsomal membranes of developing toad ovary in different seasons.

The microsomal membranes isolated by sucrose density gradient centrifugation from developing toad ovary have been found to differ significantly in lipid composition and various enzyme activities in different seasons. All the enzymes studied, viz. Na+, K(+)-ATPase, delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta HSD) and prostaglandin synthetase, exhibited maximum activity during the breeding season (July-September) at all stages of development (a,b,c & d). The activities of Na+, K(+)-ATPase and delta 5-3 beta HSD increased with development while that of prostaglandin synthetase followed the reverse order. The total phospholipid, cholesterol and fatty acid contents also varied with season and development. The increase in Na+, K(+)-ATPase and delta 5-3 beta HSD activities in the microsomal membranes of toad ovary at breeding season is accompanied with concomitant increase in phospholipid and unsaturated fatty acid contents at different stages in this season, thereby suggesting some correlation between them.

Comparative studies on different small intestinal oligosaccharidase activities in some vertebrates and invertebrates like molluscs.

Oligosaccharidase activities of the small intestinal mucosal homogenates were measured in vertebrates viz fish, toad, garden-lizard (calotes), pigeon, rat and some invertebrates viz, molluscsa. Maximum activities of the enzymes Lactase, Sucrase and Maltase were found in the mammalian species rat, whereas much less activities were found in the non-mammalian vertebrates among which toad shows the highest values and garden lizard the lowest. Among the invertebrates Pila globosa shows higher values of all the enzymes than Achatina fulica. The results obtained have been discussed in the lights of phylogeny and diet habits.