ABSTRACT
Objective:To identify 24 <italic>Rana</italic> species such as <italic>Rana dybowskii</italic> by mitochondrial cytochrome C oxidase subunit I （<italic>CO</italic>Ⅰ） gene-based DNA barcoding and build the neighbour-joining （NJ） tree for hierarchical cluster analysis， so as to provide a basis for the identification and classification of <italic>Rana</italic> species as well as the discovery of new species. Method:<italic>R. dybowskii</italic>， <italic>R. chensinensis</italic>， <italic>R. amurensis</italic>， <italic>R. culaiensi</italic>s， and <italic>R. huanrenesis</italic>， ten for each species， were collected for DNA extraction and polymerase chain reaction （PCR） amplification<italic> </italic>and sequencing. A total of 50 <italic>CO</italic>Ⅰ gene sequences were obtained. Then 163 <italic>CO</italic>Ⅰ gene sequences for 24 species of <italic>Rana</italic> and one <italic>CO</italic>Ⅰ gene sequence for <italic>Pelophylax</italic>，<italic> Odorrana</italic>， <italic>Nidirana</italic>， <italic>Hylarana</italic>， and <italic>Amolops</italic> were harvested from GenBank. After sequence alignment by MEGA X， the parsimony-informative sites of <italic>CO</italic>Ⅰ gene sequences were analyzed and the intraspecific and interspecific genetic distances were calculated， followed by the built of NJ tree and hierarchical cluster analysis. Result:The <italic>CO</italic>Ⅰ gene sequences of 24<italic> Rana</italic> species including <italic>R. dybowskii</italic> were 554 bp in length and there were 210 parsimony-informative sites in total. The intraspecific genetic distance of each species was smaller than 2%. Except that the interspecific genetic distance between <italic>R. sangzhiensis</italic> and <italic>R. zhengi</italic> was 0.004， the genetic distances between the other species ranged from 0.024 to 0.228. <italic>R. sangzhiensis</italic> and <italic>R. zhengi</italic> were clustered into one branch and some <italic>R. dybowskii</italic> and <italic>R. uenoi</italic> into one branch. There were two separate branches for <italic>R. chensinensis</italic> and the other species were all clustered independently. Conclusion:<italic>CO</italic>Ⅰ-based DNA barcoding enabled the identification of 24 species of <italic>Rana</italic> including <italic>R.dybowskii</italic>. The findings supported that <italic>R. sangzhiensis</italic>， <italic>R. zhengi</italic>， <italic>R. coreana</italic>， and <italic>R. kunyuensis</italic> were the same species. One branch of <italic>R. chensinensis </italic>might be one of the four undownloaded species in Ranidae or a new species. The results have demonstrated that <italic>CO</italic>Ⅰ-based DNA barcoding allows not only the identification of 24 species of Rana including <italic>R. dybowskii </italic>but also the classification of ranidae species and the discovery of new species or subspecies.
ABSTRACT
Ultradiluição (UHD) é o efeito de uma solução, diluída acima do número de Avogrado, que na dependência da sua dinamização (diluição com sucussão) induz um efeito celular supressivo ou estimulante, com conseqüente obtenção de uma curva dose-efeito oscilatória. Por outro lado, a 3,3,5 Triiodo-L-Tironina (T3) é o hormônio mais importante na indução e manutenção das mudanças metamórficas dos girinos, nelas incluídas a absorção da cauda. O presente estudo, cego e randomizado, tem como objetivo comprovar que o T3 5.10-24M (10ª cH) altera a apoptose induzida pelo T3 100 nM na cauda de girinos de Rana catesbeiana, in vitro. Foram distribuídos 60 explantes em três grupos: Grupo A: sem o estímulo do T3 em dose farmacológica e em UHD; Grupo B (teste): sob a ação de T3 100 nM e T3 10ª cH (5.10-24 M); Grupo C (controle): sob a ação do T3 100 nM e etanol 70% sem sucussão. A análise estatística da área dos explantes, no primeiro e ultimo dia do experimento, e do índice apoptótico foi realizado através do teste t Student e foi considerado estatisticamente significante quando p<0,05. Embora sem diferenças significativas na área dos explantes do grupo teste e no grupo controle, um maior e significante índice apoptótico foi identificado nos explantes do grupo teste. Este resultado confirma que o T3 na 10ª cH altera a ação do T3 em dose farmacológica. Futuros experimentos serão realizados, com diferentes dinamizações, com o objetivo da parametrização da curva dose-efeito.
Ultra High Dilution (UHD) is the effect of a solution, beyond the Avogadro limits, that in the dependence of the applied dinamization (dilution with succussion) elicits a suppressive or a stimulant effect on a living cell, with a consequent generation of an oscillatory dose-effect curve. The entire process of anuran amphibian metamorphosis is under thyroid hormones control, included the complete resorption of the tadpole tail. A random and blind study was performed, with the intent to prove that T3 5.10-24 M (10ª cH) modifies the apoptosis induction of T3 100 nM in Rana catesbeiana tadpoles tail tips, in vitro. 60 Explants were distributed in three ways: Group A: without T3 action, at pharmacological and UHD dose; Group B (test): under the action of T3 100 nM and treated with T3 10ª cH (UHD); Group C (control): under the action of T3 100 nM and treated with ethanol 70% unsuccussed. In order to identify significant differences in the area of the remainder explants, at the first and final day of the experiment, and in the apoptotic index we used a student t-test. Although we didnt find statistical difference in macroscopic tadpoles tail tips area from test and control groups, a high and significant (p<0,05) index of apoptosis in histology was found in explants of test group. This data confirms that T3 10 cH modifies the effect of T3 at pharmacological dose. More studies will be necessary, using different dinamizations, to the parameterization of the dose-effect curve proceeding from these experiments.