Bufo Melanostictus Research Articles

Application of lhrha and hcg to stimulate spermiation and spawning of the toad, Duttaphrynus melanostictus (Schneider, 1799)

In Vietnam, the population of Duttaphrynus melanostictus in the wild has been declined due to overharvesting for snake farms and consumption and environmental pollution. While Black-lipped Toad in cativity rarely ovulates spontaneuosly. Therefore, it’s necessary to study the assisted reproductive techniques to help preserve them. In this study, we used LHRHa and HCG to stimulate induction of spermiation and of spawning of toad. Male were injected with 2 µg/g LHRHa or 3 IU HCG/g to stimulate sperm release. Female were divided in two groups: the first group was administred a single dose of 500 IU HCG (considered as unpriming group) and the second group was administred first two sequential anovulatory dose of 100 IU HCG, repeat after 72 hours. Forty-eight hours after the second injection, the ovulatory dose of 500 IU HCG was injected (priming group). The results showed that injection of LHRHa and HCG stimulates sperm release graetest in time from 5-7 hours after injection. Five hours after injection of LHRHa or HCG, toad sperm concentration was 2.6 and 1.96 million/ml, sperm motility was 82.5 and 85%. Unprimed females failed to spawn any eggs. Whereas, 60% of primed females spawned in the period from 14-16 hours after the last injection of HCG.

ECOLOGY AND DIVERSITY OF WILDLIFE IN THE ECO-PARK OF THE JAMUNA BRIDGE AND ITS ADJACENT AREA, SIRAJGONJ, BANGLADESH

Animal diversity of an area is a good indicator for understanding a healthy habitat. We conducted a detail study on ecology and the wildlife diversity in the Eco-park of Jamuna bridge and its adjacent area of Sirajgonj district from April 2010 to March 2011. We employed transect line method and interviewed local people to reveal the status and distribution of wild animal and identify their microhabitats. A total of 89 species of wild animals was recorded, of which, 6 (6.74%) species were amphibians, 11 (12.36%) reptiles, 56 (62.93%) birds and 16 (17.94%) mammals. Regarding relative abundance, 18 (20.22%) species of wildlife were very common, 35 (39.33%) common, 28 (31.46%) fairly common, 7 (7.87%) few and only one (1.12%) was occasionally found. In total 25 species of wildlife were identified as threatened category. In amphibians, 3 species were vulnerable nationally. In reptiles, 4 species were vulnerable and one endangered. In birds, 4 species were vulnerable, 6 endangered and one critically endangered (Gallicrex cinerea). In mammals, 3 vulnerable and 3 endangered nationally. Among the amphibians, skipper frog (Euplyctis cyanophlyctis) and toad (Bufo melanostictus) frequently occurred. Among the reptiles, common garden lizard (Calotis versicolor), common skink (Mabuya carinata) and cheekered keel back water snake (Xenochorphis piscator) were frequently occurred. Among the birds, common myna (Acrodotheres tristis), pied myna (A. fuscus), black drongo (Dicrurus macrocercus) and house crow (Corvus splendens) occurred frequently. Among the mammals, shrew (Suncus murinus), flying fox (Pteropus giganteus) and black rat (Rattus rattus) frequently occurred. Our study shows that illegal exploitation of trees, overgrazing of domestic animals, shooting and collection of young animals are the mentionable causes for declining wild animals. We suggest that awareness creation and preparation of proper management action plan in cooperation with related national and international organizations are necessary in order to protect the wildlife resources in the study area. DOI: http://dx.doi.org/10.3126/eco.v20i0.11327Ecoprint: An International Journal of EcologyVol. 20, 2013Page : 27-36

Using ecological barriers for the conservation of frogs along roads

Due to the fragmentation of biological habitats by road construction, a large population of frogs are killed by cars every year when they cross the roads to aquatic areas for spawning. Ecological barriers can guide frogs to the ecological corridor and cross the road without casualties. This study investigated the effectiveness of using ecological barriers in the conservation of native frogs, while barring exotic amphibian, Polypedates megacephalus. The height and slope adjustment of ecological barriers were also proposed for references. Samples of Bufo melanostictus, Rana limnocharis, Rana guentheri, and Polypedates megacephalus were collected to record their weights, body lengths, jump lengths, jump heights, barrier heights, and angles. According to the results on the barrier effect of the barriers, P. megacephalus and R. guentheri can jump at a distance of 30–40cm from the barriers. R. limnocharis and B. melanostictus can jump at a distance of 5–15cm from the barriers. Regarding the barriers angle for preventing crossing, R. limnocharis and P. megacephalus have no ability to cling at a 65° angle; R. guentheri has no ability to cling at a 90° angle, due to its huge body and lack of suckers. B. melanostictus has no ability to cling at a 25° angle, due to its strong toes and small body. The findings can serve as a reference for ecological conservation on amphibians and inhibition of the P. megacephalus population.

Global distribution of Genus duttaphrynus

29 Duttaphrynus species are reported from all over the world. This genus was previously assigned to Bufo melanostictus group. Frost et al. suggested that Duttaphrynus is only distantly related to other Asiatic bufonids and consequently moved these species in 2006 to a separate genus. Duttaphrynus is a genus of true toads. It is endemic to southwestern and southern China and throughout southern Asia. Out of them, 19 species are reported only from India. Duttaphrynus melanostictus is the most widely distributed species.This melanostictus group is distinguished by presence of orbital cranial ridge, vocal sac when present having surrounding muscles with melanophore.

Solar powered artificial floating island for landscape ecology and water quality improvement

This study uses solar artificial floating islands (SAFI) for water purification and biological conservation. The site of experiment is set up on a lake shore on a university campus, where the eutrophic contents of lake and sewage from the student dormitory are used for result assessment. The study demonstrates that the SAFI is able to reduce the EC of the eutrophic contents by 30% and enhance dissolved oxygen (DO) by 2.8 times. The SAFI is also able to reduce electric conductivity of dormitory sewage by 34% and increase dissolved oxygen by 982 times. After the improvement, the oxidation–reduction property is above +100mV and the oxidation activities in samples are vigorous. The habitation of Ischnura senegalensis, Leucauge magnifica Yaginuma, and Duttaphrynus melanostictus can be observed in the SAFI enhanced water area, while the area without the influence of the SAFI lacks dissolved oxygen and water plants, which results in the common Culicidae, Hirudo nipponica Whitman, and Chironomida in rotten water. This research shows that the SAFI has determinant influence on the ecology and water quality improvement.

Effect of exposure of ultraviolet B radiation on two anuran species, Polypedates cruciger and Duttaphrynus melanostictus under empirical conditions

Increase in ultraviolet (UV)-B radiation has been identified as a suspect in amphibian deformities and decline. Eggs of two amphibian species, Polypedates cruciger and Duttaphrynus melanostictus were exposed to UV-B radiation (312 nm) to receive erythemal dose (0.08 relative response) of the action spectra under laboratory conditions. Biologically estimated doses were calculated to match 50% (high), 25% (medium) and 10% (low) of the average daily irradiance given for the Asian region (17.92 × 10-3 Wm-2). Hatching success of the eggs and survival, growth of tadpoles and development of malformations were recorded until metamorphosis. Although the exposure had no effect on the hatching success of the eggs of the two species, the survival of tadpoles of both species was significantly reduced compared to the tadpoles coming from unexposed eggs in the control. Tadpoles of both species experienced growth retardations due to UV-B exposure. Exposed individuals were smaller at metamorphosis and they took longer to metamorphose. In addition, malformations such as ulcers, edemas, abnormally pigmented skin were common among the exposed tadpoles. Species-specific differences in the growth were observed between D. melanostictus and P. cruciger but not in the other measured parameters. This study shows that the two anuran species are highly sensitive to levels of UV-B radiation that is present in their natural environment and suffered both lethal and sub-lethal effects.DOI: http://dx.doi.org/10.4038/cjsbs.v42i2.6608 Ceylon Journal of Science (Bio. Sci.) 42(2): 45-53, 2013

DETERMINATION OF EIGHT BUFADIENOLIDES IN THE SKIN OF BUFO BUFO GARGARIZANS CANTOR AND BUFO MELANOSTICTUS SCHNEIDER USING HPLC COUPLED WITH TRIPLE QUADRUPOLE MASS SPECTROMETRY

Chan Pi (the toad skin) is an effective Chinese medicine in the treatment of boils, pyogenic infections, and cancer in clinical settings. However, the inhomogeneity of its quality is often observed due to confusing sources, namely Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. In the present paper, a simultaneous determination method of eight bufadienolides in two species of Chan Pi was established using HPLC coupled with triple quadrupole mass spectrometry (HPLC-QqQ MS). Eight bufadienolides in 7 batches of B. bufo gargarizans skin and 8 batches of B. melanostictus skin were analyzed with an acceptable linear regression relationship (r ≥ 0.9990), precisions (RSD, 1.97–4.38%), repeatability (RSD, 2.65–4.70%), stability (RSD, 2.39–4.51%), and recovery (96.39–103.82%). Additionally, cluster analysis was used for the successful discrimination of these two species of toad skin by the ratio of these eight bufadienolides. The present study greatly facilitates clarification of the difference between these two species of Chan Pi, which improves the quality control of this traditional Chinese medicine.

Invasive apple snails (Pomacea canaliculata) are predators of amphibians in South China

Invasive species are one of the most serious threats to amphibian populations. We investigated the effects of two invasive (Pomacea canaliculata and Physella acuta) and one native (Radix sp.) snail species on five species of wetland-breeding frogs in Hong Kong. We quantified embryonic survivorship and determined whether particular attributes of amphibian egg masses influenced consumption by snails. P. canaliculata preyed on four of the species, consuming >90 % of eggs of Microhyla fissipes and Fejervarya limnocharis, nearly 70 % of eggs of Kaloula pulchra, approximately 40 % of eggs of Duttaphrynus melanostictus, but no eggs of Polypedates megacephalus. P. acuta and Radix sp. consumed only the eggs of K. pulchra, but those eggs were probably non-viable. This study shows that P. canaliculata, which occurs at high densities in tropical East Asia, may be an important predator of amphibian eggs. Future research should evaluate their effects on amphibian populations, community structure, and food web dynamics.

A Comparative Study Of Serum Proteins In Some Vertebrate Representatives

Serum is the liquid part of blood, which can be analysed through protein electrophoresis (PEP) technique to determine the levels of different types of proteins in a blood sample. In present investigation a comparative electrophoretic study of serum protein of Gallus gallus domesticus, Hemidactylus frenatus, Schizothorax plagiostomus, Duttaphrynus melanostictus was carried out with serum fraction of man. There was no albumin like large fraction observed in Duttaphrynus metanostictus, Hemidactylus frenatus and Schizothorax plagiostomus. The number of serum protein fractions and their separation pattern differed in all these vertebrate representatives analysed. There were 20 fractions in man, 21 in Gallus gallus domesticus, 14 in Duttaphrynus melanostictus, 11 in Hemidactylus frenatus and 16 in Schizothorax plagiostomus.

Cold stress-induced lipid peroxidation and non-enzymatic antioxidant defense in tissues of the common Indian toad, Bufo melanostictus

The aim of this study was to investigate the effect of exposure to short-term cold stress (30 min at 0-4 0 C) on lipid peroxidation and the non-enzymatic antioxidant defense system in liver, kidney and brain tissues of an ectothermic animal, the common Indian toad, Bufo melanostictus. Cold stress resulted in a significant increase in lipid peroxidation in terms of TBARS formed in the liver, kidney and brain tissues, indicating increased generation of reactive oxygen species (ROS). Elevated levels of GSH measured in the liver, kidney and brain tissues of cold-stressed animals were probably due to an increased requirement for antioxidant defenses to meet the elevated peroxidative challenge. The observed decrease in ascorbic acid content may be due to its increased consumption and/or decreased synthesis following cold stress. Similarly, increased uric acid content in kidney and brain tissue following cold exposure and its decrease in liver tissue indicates that the effect of cold stress on the antioxidant system is both tissue-specific and dependent on the extent of cold exposure. Cold stress results in increased ROS production causing lipid peroxidation and compensatory alterations in the non- enzymatic antioxidant status in different tissues.

On a new species of Aplectana (Nematoda, Cosmocercidae) from Kulti, Burdwan, West Bengal, India.

During routine survey for amphibian nematodes from coalfield areas of West Bengal, India 7 male and 12 female nematodes were recovered from the rectum of one out of three Duttaphrynus melanostictus (Schneider, 1899) examined for helminth infection from Kulti, Burdwan. On examination the recovered nematodes were found to belong to a new species of the genus Aplectana (Nematoda: Cosmocercidae) and the name Aplectana duttaphryni sp. nov. is proposed for them. This species is most similar to those species which possess gubernaculum but differs from all by distribution pattern of caudal papillae. Aplectana duttaphryni sp. nov. represents 49(th) species assigned to the genus, but only the 4(th) species reported from Oriental realm and second from India.

Consequences of physical disturbance by tadpoles and snails on chironomid larvae.

Indirect interactions among community members impact on organisms. The effects of two snails, banded pond snail, Bellamya bengalensis (Lamarck), and Red-rimmed melania, Melanoides tuberculata (Müller), and tadpoles of Asian common toad, Duttaphrynus melanostictus (Schneider), on nonbiting midge larvae, Chironomus striatipennis Kieffer, were observed in experimental microcosm. Decrease in tube number and tube length of midge larvae was observed compared to control condition due to introduction of selected above mentioned organisms. The direct effects of non-predator organisms on the midge larvae are due to physical disturbance that destroys their tubes. This may result in vulnerability of midge larvae to predators in the wild. So the community structure may be altered by indirect effects, where one or more species, through their direct disturbance, indirectly change the abundance of other species.

Comparative light and electron microscopic studies of dorsal skin melanophores of Indian toad, Bufo melanostictus

Indian toad, Bufo melanostictus is an amphibian, which is able to change the body color to adapt to its ambient need. This ability is due to specialized skin pigment cells known as melanophores which are excellent animal-cell models to study most of the physiological phenomenon related to color changes. In the present investigation morpho-anatomic details of dorsal skin melanophores of B. melanostictus were studied by means of light and electron microscope to establish their phylogenetic relevance with other vertebrates. Light microscopic observations revealed that toad skin contains dominantly present black pigment cells, the melanophores in its sub epidermis while dermal melanophores are rare. The ultrastructure of the melanophore is characterized by oval nucleus and numerous pigment granules, the melanosomes which remain scattered in the cytoplasm. Other sub-cellular organelles like mitochondria, well-developed tubular endoplasmic reticulum and Golgi vesicles were also found to be present in the remaining cytoplasmic area. Toad melanophore contains four distinct stages of melanosomes which are similar in development pattern to the mammalian melanocytes. These findings indicate that toad melanophores contain phylogenetically significant information of anatomical similarity with lower as well as higher vertebrates which can help to better understand the inter relationships between vertebrate pigment cells and their role in skin dysfunctions.

Invasive toxic prey may imperil the survival of an iconic giant lizard, the Komodo dragon.

Although invasive species constitute a major threat to global biodiversity, the introduction of toxic prey into naïve predator faunas may become particularly destructive. An example of such an introduction was the deliberate release of the highly toxic cane toad (Bufo marinus) to the toad-free Australian continent in 1935. Naïve large Australian varanid lizards have recently been shown to suffer a massive increase in mortality (> 95%) when attempting to feed on this toxic amphibian. The high susceptibility of Australian varanids to toad toxin is caused by minor mutations in the sodiumpotassium- ATPase enzyme. In the present study we show that Komodo dragons (Varanus komodoensis) have similar mutations within this enzyme as observed in Australian varanids demonstrating that dragons are extremely susceptible to toad toxin. During the last decade the black-spined toad (Bufo melanostictus) has been able to invade areas close to the five toad-free islands constituting the habitat of Komodo dragons. An invasion of highly toxic black-spined toads into dragon habitats may therefore cause similar dramatic increase in dragon mortality as recorded in Australian varanids imperiling the long-term survival of this giant and iconic lizard.

Cardiotonic activity of Parotoid gland secretion of common Indian Toad Bufo melanostictus on isolated heart of Frog

The present investigation has been undertaken to study the cardiotonic activity of parotoid gland secretion of common Indian Toad Bufo melanostictus on isolated perfused frog heart (Rana tigrina). Cardiotonic activity of parotoid gland secretion of B. melanostictus in normal as well as when induced with paraoxon (an organophosphate) was studied. For the evaluation of cardiotonic activity, Syme’s technique is being employed, digoxin and propranolol were used as standard drug and β-blocker respectively to characterize the effects on the receptors. The isolated perfused frog heart and hypodynamic frog heart showed dose dependent positive ionotrophic effects. The parotoid gland secretion exihibited cardiac stimulant activities. The propranolol was not able to block the effects of paraoxon induction on toad secretion. Thus, the present investigation reports that the parotoid gland secretion increased the force of contraction, heart beat and cardiac output in perfused frog’s heart, whereas, there was no change on hypodynamic heart, indicating that there may be existence of two components, one with β–receptor stimulating activity and other acting directly on the heart (independent of β1-adrenoreceptors).

Genetic divergences of South and Southeast Asian frogs: a case study of several taxa based on 16S ribosomal RNA gene data with notes on the generic name Fejervarya

To elucidate the genetic divergences of several Asian frog taxa, the mitochondrial 16S rRNA gene (16S) sequences of 81 populations across 6 Asian countries were analyzed. In total, 109 haplotypes were found, and the concept of a 3% difference in 16S sequence corresponding to species threshold was applied to define candidate amphibian species, for which corroborating evidence, such as morphology, ecological characteristics, and/or nuclear gene data, is required. Polypedates leucomystax, Hylarana chalconota, and Hylarana sp. from Chantaburi, Maelippet Siberut, and Langkawi Island, respectively, correspond to 3 candidate species and H. erythraea from Malaysia/Thailand represents a possible candidate species. Hylarana cf. nicobariensis from Muara Siberut, Amolops larutensis from Gombak, and Microhyla okinavensis from Ishigaki Island showed divergences from the topotypic specimen, all suggesting a relevant candidate species. Microhyla heymonsi from Malaysia and M. ornata from 2 regions (Mudigere and Talapu) in India did not fit any congeneric species based on available 16S data, suggesting 3 possible candidate species in total. Two lineages of Duttaphrynus melanostictus from Malaysia denote 2 possible candidate species. Consequently, this study indicates the occurrence of 6 candidate and 6 possible candidate species, and argues that the generic allocation of the Fejervarya-Minervarya-Zakerana complex needs to be studied in detail.

Male size does not correlate with fertilization success in two bufonid toads that show size-assortative mating

Abstract We examined sexual size dimorphism (SSD), mating pattern, fertilization efficiency and female reproductive traits in two bufonid toads (Bufo gargarizans and Duttaphrynus melanostictus) to test the idea that importance of male body size for egg fertilization success depends on the mating pattern. Female-biased SSD was evident only in D. melanostictus. Female B. gar-garizans laid fewer larger eggs nearly three months earlier than did female D. melanostictus. Fertilization efficiencies on average were higher in B. gargarizans (95%) than in D. melanostictus (91%). Though differing in the degree of SSD, body size, breeding season, clutch size, egg size and fertilization efficiency, the two toads were similar in four aspects: (1) both showed size-assortative mating; (2) females did not tradeoff egg size against egg number; (3) male size, clutch size and clutch dry mass were greater in male-larger than in female-larger pairs after accounting for female snout-vent length (SVL); and (4) the ratio of male to female SVL did not affect fertilization efficiency. Our data show that: (1) a female preference for large males is likely not important in terms of egg fertilization success; (2) a male preference for large females is likely important because larger females are more fecund; and (3) size-assortative mating arises from a male preference for large females. Our study demonstrates that male size is not always important for egg fertilization success in anurans that show size-assortative mating.

Study on anti-osteosarcoma activity of ethanol extract of <i>Venenum Bufonis</i> in vitro

Venenum bufonis is the dried white secretion of the auricular and skin glands of Bufo gargarizans Cantor, or Bufo melanostictus Schneider, Bufonidae. It is used in the treatment of deep-rooted carbuncle, boils and swelling; pain in the throat, heart stroke, coma, abdominal pain, vomiting and diarrhea. The objective of this paper is to preliminarily observe the effects of ethanol extract of Venenum bufonis on growth, and proliferation of human osteosarcoma U2OS cell lines, and to provide a theoretical basis for an in-depth study of the clinical application of Venenum bufonis for osteosarcoma inhibition, with its mechanism of action. SRB assay was used to determine the effect of Venenum bufonis ethanol extract on U2OS cell line activity, and to detect its inhibitory dose-effect on osteosarcoma cells. FCM was applied to determine the effect of Venenum bufonis ethanol extract on U2OS cell apoptosis and to perform cell cycle analysis. As results, different Venenum bufonis ethanol extracts showed apparent concentration-effect relationships on U2OS cell lines. FCM analysis showed that it had a U2OS apoptosis promoting effect, which increased with increasing concentration. Cell cycle analysis revealed that the Venenum bufonis ethanol extract mainly arrested U2OS in the G0/G1 phase, preventing the cells from progressing to the S phase. The study concluded that Venenum bufonis ethanol extract has an inhibitory effect on proliferation of osteosarcoma U2OS cells.

Molecular Cloning and Characterization of Cystatin, a Cysteine Protease Inhibitor, fromBufo melanostictus

Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.

Berberine-induced pigment dispersion in Bufo melanostictus melanophores by stimulation of beta-2 adrenergic receptors

Reduced production of melanin by decreased or the absence of melanocytes leads to various hypopigmentation disorders, and the development of melanogenetic agents for photoprotection and hypopigmentation disorders is one of the top priority areas of research. Hence, the present study was carried out to elucidate the ability of berberine, a principal active ingredient present in the roots of the herb Berberis vulgaris to stimulate pigment dispersion in the isolated skin melanophores of the toad Bufo melanostictus. In the present study, mean melanophore size index of the isolated skin melanophores of B. melanostictus was assayed after treating with various concentrations of berberine. A marked melanin dispersion response leading to skin darkening was observed in the isolated melanophores of toad in response to berberine, which was found to be mediated through beta-2 adrenergic receptors. The physiologically significant dose-related melanin dispersion effects of berberine per se were found to be completely abolished by propranolol, which is a specific beta-2 adrenergic receptor blocker. These per se melanin dispersal effects were also found to be markedly potentiated by isoprenaline, which is a specific beta-adrenoceptor agonist. The results indicate that berberine causes a tremendous, dose-dependent, physiologically significant pigment dispersing in the isolated skin melanophores of B. melanostictus.