Cloning of adipor1 and adipor2 genes in Rana dybowskii and its expression pattern upon infection / 生物工程学报

Adiponectin receptor 1 (AdipoR1) and Adiponectin receptor 2 (AdipoR2) can bind to adiponectin (AdipoQ) secreted by adipose tissue to participate in various physiological functions of the body. In order to explore the role of AdipoR1 and AdipoR2 in amphibians infected by Aeromonas hydrophila (Ah), the genes adipor1 and adipor2 of Rana dybowskii were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by bioinformatics. The tissue expression difference of adipor1 and adipor2 was analyzed by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and an inflammatory model of R. dybowskii infected by Ah was constructed. The histopathological changes were observed by hematoxylin-eosin staining (HE staining); the expression profiles of adipor1 and adipor2 after infection were dynamically detected by qRT-PCR and Western blotting. The results show that AdipoR1 and AdipoR2 are cell membrane proteins with seven transmembrane domains. Phylogenetic tree also shows that AdipoR1 and AdipoR2 cluster with the amphibians in the same branch. qRT-PCR and Western blotting results show that adipor1 and adipor2 were up-regulated at different levels of transcription and translation upon Ah infection, but the response time and level were different. It is speculated that AdipoR1 and AdipoR2 participate in the process of bacterial immune response, providing a basis for further exploring the biological functions of AdipoR1 and AdipoR2 in amphibians.

Cloning and tissue expression analysis of the LepROT gene of Rana dybowskii / 生物工程学报

Leptin receptor overlapping transcript (LepROT) plays multiple roles in the regulation of immune systems. However, very little information is available about the anti-infectious mechanisms of amphibians LepROT. In this study, the cDNA sequence of the Rana dybowskii LepROT gene was determined by using RT-PCR and bioinformatics analysis. Then, the Aeromonas hydrophila (Ah) and lipopolysaccharides (LPS) infected models of R. dybowskii was constructed to obtain histopathological characteristics. Constitutive expression of LepROT mRNA and NF-κB signaling pathway were detected by real-time quantitative PCR. The full-length cDNA of LepROT gene was 396 bp and encoded 131 amino acids. Amino acid sequence analysis revealed LepROT shares 93.74% and 86.39% identity with homologues from other amphibians and mammals respectively, and the LepROT gene was quite conserved among different species. After infection, the relative expression levels of LepROT, NF-κB, IKKα and IKKβ mRNA were all significantly upregulated (P < 0.01), but showed a diverse temporal pattern of up-regulation in different tissues. Therefore, it was proposed that the LepROT gene of R. dybowskii might activate the NF-κB signaling pathway to exert anti-infectious effects, thus providing evidence for further extending the biological function of LepROT.

Histology and structure of the testicles in three species of Atelopus frogs (Anura: Bufonidae) endemic to the Sierra Nevada de Santa Marta, Colombia / Histología y estructura de los testículos en tres especies de ranas Atelopus (Anura: Bufonidae) endémicas de la Sierra Nevada de Santa Marta, Colombia

Abstract Introduction: Testicular histology constitutes one of the least explored aspects in frogs of the genus Atelopus. This taxonomic group shows an alarming population decline; therefore, its reproductive biology is one of the greatest topics of interest for its conservation. Objective: To describe the testicular morphology and the spermatogenetic lineage cells in adult males of Atelopus laetissimus, Atelopus nahumae, and Atelopus carrikeri in the Sierra Nevada de Santa Marta, Colombia. Methods: During June - July 2017 and 2018, sampling was conducted in the localities of San Lorenzo and Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), to collect 15 adult males, 5 per species. Testes samples were fixed in Bouin to be processed by the standard paraffin-embedding technique. Histological sections (3 μm) were stained with Hematoxylin-eosin and Mallory-Heidenhain-Azan-Gomori's. For the description and photographic register of the germ cells, the photonic microscopy technique was used with the differential interference contrast system. Results: The testes are oval organs, compact, light yellow color, and with little vascularization. Externally, they are surrounded by a thin albuginea tunic constituted by regular dense connective tissue. Inside this layer, they are composed of numerous seminiferous tubules of hexagonal contour, in which germ cell cysts are distinguished at different stages of spermatogenesis (spermatogonia I and II, spermatocyte I and II, and early and late spermatids) and spermiogenesis (spermatozoa in fascicles and free spermatozoa). Separating the seminiferous structures is the interstitial tissue in which Leydig cells and blood vessels stand out. Additionally, in the cranial part of the testis, the Bidder's organ was found, formed by two distinguishable regions, the cortex and the medulla. In the cortex, there are previtellogénic oocytes of different sizes surrounded by a monolayer of flat follicular cells. For its part, the medullary region is the connective tissue that nourishes the oocytes and is constituted by blood capillaries. Conclusions: The gonads of the three species analyzed present a cystic cellular organization similar to other anurans, where all stages of spermatogenesis and spermiogenesis were identified, possibly indicating a continuous reproductive activity. Likewise, the Bidder's organ is reported for the first time in the three Atelopus species, which allows suggesting a possible sexual reversion in case of a population decrease of females as a reproductive strategy.

Resumen Introducción: La histología testicular constituye uno de los aspectos menos explorados en las ranas del género Atelopus. Este grupo taxonómico ostenta un declive poblacional alarmarte, es por ello, que su biología reproductiva resulta uno de los temas de mayor interés para su conservación. Objetivo: Describir la morfología testicular y las células del linaje espermatogénico en machos adultos de Atelopus laetissimus, Atelopus nahumae y Atelopus carrikeri en la Sierra Nevada de Santa Marta, Colombia. Métodos: Durante Junio - Julio de 2017 y 2018 se realizaron muestreos en las localidades de San Lorenzo y Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), para recolectar 15 machos adultos, 5 por especie. Las muestras de testículo se fijaron en Bouin para ser procesadas mediante la técnica estándar de inclusión en parafina. Las secciones histológicas (3 μm) se tiñeron con Hematoxilina-eosina y Mallory-Heidenhain-Azan-Gomori's. Para la descripción y registro fotográfico de las células germinales, se utilizó la técnica de microscopía fotónica con el sistema de contraste diferencial de interferencia. Resultados: Los testículos son órganos ovalados, compactos, de color amarillo claro y con poca vascularización. Externamente, están rodeados por una delgada túnica albugínea constituida por tejido conectivo denso regular. Al interior de esta capa se componen por numerosos túbulos seminíferos de contorno hexagonal, en los que se distinguen quistes de células germinativas en diferentes etapas de la espermatogénesis (espermatogonia I y II, espermatocito I y II y espermátidas tempranas y tardías) y espermiogénesis (espermatozoides en fascículos y espermatozoides libres). Separando las estructuras seminíferas se halla el tejido intersticial en el que se destacan las células de Leydig y los vasos sanguíneos. Adicionalmente, en la parte craneal del testículo se encontró el órgano de bidder formado por dos regiones diferenciables, la corteza y la medula. En la corteza se aprecian ovocitos previtelogénicos en diferente tamaño rodeados por una monocapa de células foliculares planas. Por su parte, la región medular es el tejido conectivo que nutre los ovocitos y está constituido por capilares sanguíneos. Conclusiones: Las gónadas de las tres especies analizadas presentan una organización celular quística de manera similar con otros anuros, donde se identificó todos los estadios de la espermatogénesis y espermiogénesis indicando posiblemente una actividad reproductiva continua. Así mismo, se reporta por primera vez el órgano de bidder en las tres especies de Atelopus, lo cual permite sugerir una posible reversión sexual en caso de una disminución poblacional de las hembras como una estrategia reproductiva.

Structure prediction and biological activity analysis of dybowskin-1ST antimicrobial peptide in Rana dybowskii / 生物工程学报

The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.

Expression of MHCⅠ genes in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila / 生物工程学报

The aim of this study was to investigate the expression of MHCⅠ gene in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila (Ah), and to provide evidence for revealing the anti-infective immune response mechanism of amphibians. The experimental animal model of Aeromonas hydrophila infection was first constructed, and the pathological changes were observed by HE staining. The MHCⅠ gene α1+α2 peptide binding region of Rana dybowskii was cloned by RT-PCR and analyzed by bioinformatics. Real-time PCR was used to detect the transcription level of MHCⅠ in different tissues under Ah stress. After Ah infection, the skin, liver and muscle tissues showed signs of cell structure disappearance and texture disorder. The MHCⅠ gene α1+α2 peptide binding region fragment was 494 bp, encoding 164 amino acids, and homology with amphibians. Above 77%, the homology with mammals was as low as 14.96%, indicating that the α1+α2 region of MHC gene was less conserved among different species. The results of real-time PCR show that the liver, spleen and kidney of the experimental group were under Ah stress. The transcript levels of MHCⅠ gene in skin and muscle tissues were higher than those in the control group at 72 h, but the time to peak of each tissue was different (P<0.01), indicating that the response time of MHCⅠ gene in different tissues was different under Ah stress. This study provides a reference for further exploring the immune function of MHC molecules in anti-infection.

Parásitos nematodos de la rana de piscina (Pelophylax lessonae) en la cuenca del Río Volga / Parasitic nematodes of Pool Frog (Pelophylax lessonae) in the Volga Basin

RESUMEN Objetivo. Presentar una revisión moderna de la fauna de nematodos de la rana esculenta Pelophylax lessonae (Camerano, 1882) de las poblaciones de la cuenca del Río Volga. Materiales y métodos. Este trabajo consolida los datos de diferentes trabajos helmintológicos de los últimos 80 años, respaldados por los resultados de nuestras propias investigaciones. Durante el período de 1936 a 2016 diferentes autores examinaron 1460 especímenes de rana esculenta, utilizando el método de autopsia helmintológica completa de 13 regiones de la cuenca del Río Volga. Resultados. En total se registraron 9 especies de nematodos. Nematodo Icosiella neglecta encontrado por primera vez en el huésped estudiado en el territorio de Rusia y la cuenca del Río Volga. Tres especies parecían estar más extendidas: Oswaldocruzia filiformis, Cosmocerca ornata e Icosiella neglecta. Para cada especie de helmintos se incluyó la siguiente información: posición sistemática, áreas de detección, localización, biología, lista de hospederos definitivos, nivel de especificidad del hospedero. Conclusiones. Los nematodos de la rana esculenta, excepto I. neglecta, pertenecen al grupo de helmintos transmitidos por el suelo (geohelmintos) y parasitan en estadios adultos. Algunas especies (O. filiformis, C. ornata, I. neglecta) están muy extendidas en el área de distribución del hospedador. Estos dos últimos son capaces de alcanzar altos índices de invasión y también de ser los parásitos subyacentes de la rana esculenta. Esto sucede debido a la especificidad del ciclo de vida de estas especies de nematodos y a la conexión a largo plazo del anfibio con el agua.

ABSTRACT Objetive. Present a modern review of the nematodes fauna of the pool frog Pelophylax lessonae (Camerano, 1882) from Volga basin populations on the basis of our own research and literature sources analysis. Materials and methods. Present work consolidates data from different helminthological works over the past 80 years, supported by our own research results. During the period from 1936 to 2016 different authors examined 1460 specimens of pool frog, using the method of full helminthological autopsy, from 13 regions of the Volga basin. Results. In total 9 nematodes species were recorded. Nematode Icosiella neglecta found for the first time in the studied host from the territory of Russia and Volga basin. Three species appeared to be more widespread: Oswaldocruzia filiformis, Cosmocerca ornata and Icosiella neglecta. For each helminth species the following information included: systematic position, areas of detection, localization, biology, list of definitive hosts, the level of host-specificity. Conclusions. Nematodes of pool frog, excluding I. neglecta, belong to the group of soil-transmitted helminthes (geohelminth) and parasitize in adult stages. Some species (O. filiformis, C. ornata, I. neglecta) are widespread in the host range. The latter two are able to reach high invasion indices and also to be the background parasites of the pool frog. It happens due to these nematode species life cycle specificity and the long-term connection of the amphibian with water.

Genetic and Morphologic Identification of Spirometra ranarum in Myanmar

In the present study, we identified a Spirometra species of Myanmar origin (plerocercoid) by molecular analysis using mitochondrial cox1 and nad1 genes, as well as by morphological observations of an adult tapeworm. Spargana specimens were collected from a paddy-field in Taik Kyi Township Tarkwa Village, Yangon, Myanmar in December 2017. A total of 5 spargana were obtained from 20 frogs Hoplobatrachus rugulosus; syn: Rana rugulosa (Wiegmann, 1834) or R. tigrina (Steindachner, 1867). The plerocercoids were used for experimental infection of a dog. After 4 weeks of infection, an adult tapeworm was recovered from the intestine of the dog. Morphologically, the distinct features of Spirometra sp. (Myanmar origin) relative to S. erinaceieuropaei and S. decipiens include a uterine morphology comprising posterior uterine coils that larger than the terminal uterine ball and coiling of the uteri diagonally (swirling) rather than spirally. The cox1 sequences (1,566 bp) of the Myanmar-origin Spirometra species showed 97.9% similarity to a reference sequence of S. decipiens (GenBank no. KJ599679) and 90.5% similarity to a reference sequence of S. erinaceieuropaei (GenBank no. KJ599680). Phylogenetic tree topologies were identical and presented high confidence level of values for the 3 major branches of the 3 Spirometra species in cox1 and nad1 genes. These results indicated that Myanmar-origin Spirometra species coincided with those of S. ranarum and may be considered as a valid species.

Influence of the number of feeding environments on the performance of bullfrog tadpoles / Influência do número de ambientes de alimentação no desempenho de girinos de rã-touro

Feeding is one of the major factors that influences the growth and development of animals, justifying thus the present experiment that analyzed the effect of the number of feeding environments on the performance of bullfrog tadpoles through the growth (weight and length) and performance (food remains, average feed intake, average weight gain, and feed conversion). The treatments consisted of subunits of two, four, and eight feeding environments in the experimental units at disposal for the tadpole population, and one without subdivisions, comprising a single environment, during 60 experimental days. The design was completely randomized, with four treatments and three replications. The values were tested using an analysis of variance and Duncan test (α = 0.05). The growth in weight and length and the indices of animal performance were influenced by the number of feeding environments, with the best results found for the treatments with four and eight environments, and the worst, for the treatment with a single environment. The increased number of feeding environments may be an alternative to improve the performance of bullfrog tadpoles.

Alimentação íntegra a um conjunto de fatores que influenciam o crescimento e o desenvolvimento dos animais. Com isso, foi realizado o experimento com o objetivo de verificar pelo crescimento (peso e comprimento) e o desempenho (sobras diárias de ração, consumo médio de ração, ganho de peso médio e conversão alimentar) dos girinos de rã-touro a influência do número de ambientes de alimentação no desempenho de girinos de rã-touro. Os tratamentos foram subdivisões em dois, quatro e oito ambientes de alimentação das unidades experimentais em relação à população de girinos e um sem subdivisões com único ambiente, durante 60 dias experimentais. O delineamento foi inteiramente casualizado, com quatro tratamentos e três repetições. Os valores encontrados foram submetidos à análise de variância e ao teste de Duncan (α = 0,05). O crescimento em peso e comprimento e os índices de desempenho zootécnicos foram influenciados pelo número de ambientes de alimentação, sendo que os melhores resultados foram para os tratamentos com quatro e oito ambientes e o piores para com único ambiente. O aumento do número de ambientes de alimentação pode ser uma alternativa para melhorar o desempenho dos girinos de rã-touro.

Liver histopathological changes in breeding bullfrogs / Alterações histopatológicas do fígado de reprodutores de rã-touro

In order to complement the histopathological study of juveniles and tadpoles of the bullfrog, Lithobathes catesbeianus, fed commercial diet used by frog farms in Rio de Janeiro, containing 40% crude protein, we performed necropsy and histopathology of the liver to establish a relationship between the quality of crude protein in the diet and animal health. For this, it was used twenty breeding male bullfrogs, with average weight 591.30 g (± 91.90 g) and length 165.02 mm (± 14.22 mm), and ten females with average weight and length of 629.80 g (± 134.47 g) and 169.32 mm (± 21.82 mm). The liver histopathology showed hyperemia, high number of elanomacrophages and cytoplasmic rarefaction, probably due to protein deficiency and fatty liver degeneration and presence of inflammatory processes. These lesions indicate a degenerative nutritional process. These findings suggest that the animals were fed with proteins of low biological value, indicating poor quality of feed, undermining the sanity. The impairment of liver function by these injuries will lead to reduced availability of precursors of sex hormones, since the liver is important in the metabolism of the same, and reproductive performance of these animals may be impaired.

Em complementação ao estudo histopatológico de girinos e imagos da rã-touro, Lithobates catesbeianus, alimentados com ração comercial utilizada pelos ranários do Rio de Janeiro, contendo 40% de proteína bruta, realizou-se necropsia e histopatologia do fígado de reprodutores, procurando uma relação entre a qualidade de proteína bruta na ração e a sanidade do animal. Foram utilizados 20 reprodutores de rã-touro, dez machos com peso de 591,30 g (± 91,90 g) e comprimento médios de 165,02 mm (± 14,22 mm), e dez fêmeas com peso e comprimento médios de 629,80 g (± 134,47 g) e 169,32 mm (± 21,82 mm). Os resultados da histopatologia mostraram que o fígado dos reprodutores, tanto machos quanto fêmeas apresentavam hiperemia, grande número de melanomacrófagos e rarefação citoplasmática provavelmente por deficiência proteica e quadro de degeneração hepática gordurosa. Tais lesões indicam quadro degenerativo nutricional. Também presente processos inflamatórios focais. Estes achados sugerem uma alimentação com proteínas de baixo valor biológico, indicando má qualidade da ração, comprometendo sua sanidade. Pode-se inferir que o comprometimento das funções hepáticas, irá diminuir a disponibilidade de precursores dos hormônios sexuais, pois o fígado é importante na metabolização dos mesmos, e a performance reprodutiva destes animais poderá ser prejudicada.

Evaluation of the semen characteristics after induced spermiation in the bullfrog Lithobates catesbeianus / Características e avaliação do sêmen de rã-touro Lithobates catesbeianus induzidos à espermiação

Evaluation of semen characteristics after hormonal induction of the bullfrog could provide valuable information on the gametes of this species, which may be useful for projects related to artificial fertilization, animal improvement, and cryopreservation. Bullfrog males were induced to spermiate with buserelin acetate (GnRHa), and their semen was subsequently analyzed. GnRHa (0.4 µg) was administered to the bullfrog males with secondary sexual characteristics such as weight > 200 g, yellow chin, nuptial callus, and amplexus reflex, being the semen collected after 60 min. The semen volume was 5.76 mL, light-colored. The other characteristics of the semen were: vigor of 4.80, motility of 93%, concentration of 14.24 × 106 mL-1, and content of normal spermatozoa of 70%. The volume, color, vigor, motility, sperm concentration, and content of normal spermatozoa were adequate in these bullfrog semen samples. Evaluation of the bullfrog semen samples based on this set of parameters is essential for decision-making about the quality and destination of the semen.

Avaliação de que as características do sêmen, após a indução hormonal da rã-touro, pode fornecer informações valiosas sobre os gametas desta espécie, podendo ser útil para projetos relacionados à fertilização artificial, melhoramento animal, e criopreservação. Machos de rã-touro foram induzidos à espermiação com acetato de buserelina (GnRHa) e o sêmen foi posteriormente analisado. GnRHa (0,4 mg) foi administrado à rã- touro do sexo masculino com características sexuais secundárias, tais como peso superior a 200 g, papo amarelo, calo nupcial, e reflexo amplexo, e seu sêmen foi coletado após 60 min. O sêmen da rã-touro apresentou volume de 5,76 mL, coloração turva, vigor espermático de 4,80; motilidade espermática de 93%, concentração de 14,24 x 106 SPTZ mL-1 e 70% de espermatozoides normais. O volume, cor, vigor, motilidade, concentração espermática e o número de espermatozoides normais das amostras do sêmen de rã-touro são adequados. O conjunto dos parâmetros para avaliação das amostras de sêmen de rã-touro é indispensável para a tomada de decisão sobre a qualidade e destino do mesmo.

Buserelin acetate-induced spermiation in Leptodactylus ocellatus and evaluation of semen characteristics / Espermiação com acetato de buserelina em Leptodactylus ocellatus e avaliação de características seminais

By understanding the hormonal action behind the induction of spermiation in Leptodactylus ocellatus can help in the collection of semen for artificial insemination and cryopreservation. The experiments were conducted at the Experimental Frog Farm of the Federal University of Viçosa. Seven L. ocellatus males with the following secondary sexual characteristics were selected: black thorns, developed forelimbs, and amplexus reflex. Spermiation was induced in these animals by applying daily 0.4 µg buserelin acetate hormone (GnRHa) for 7 days, being the semen collected 90 min after the application. The volume, color, vigor, motility, sperm concentration, and spermatozoa morphology were subsequently evaluated. Three animals responded to GnRHa induction, enabling the collection of seven samples of semen, and the first sample was collected 97.5h after the first application. The semen of the creole frog presented the following characteristics: an average volume of 0.38 mL, murky color, sperm vigor of 3.71, sperm motility of 77.14%, sperm concentration of 6.60 x 106 SPTZ mL-1, and 69% of normal sperm. GnRHa can induce spermiation in the creole frog. Although the volume of collected semen was low, the color, vigor, motility, concentration, and spermatozoa content showed to be adequate.

O conhecimento da ação de hormônios na indução à espermiação de Leptodactylus ocellatus pode permitir a coleta de sêmen para estudos de fertilização artificial e criopreservação. O experimento foi realizado no Ranário Experimental da Universidade Federal de Viçosa, onde sete machos de L. ocellatus com as características sexuais secundárias: acúleos negros, membros anteriores desenvolvidos e reflexos ao amplexo foram induzidos à espermiação com aplicação do hormônio acetato de buserelina (GnRHa) na dosagem de 0,4 µg, durante sete dias a cada 24h e o sêmen coletado após 90 min. O volume, cor do sêmen, vigor espermático, motilidade espermática, concentração espermática e morfologia dos espermatozoides foram avaliados. Três animais responderam à indução pelo acetato de buserelina permitindo a coleta de sete amostras de sêmen, a primeira após 97,5h da primeira aplicação. O sêmen da rã-manteiga apresentou volume médio de 0,38 mL, coloração turva, vigor espermático de 3,71; motilidade espermática de 77,14%, concentração de 6,60 x 106 SPTZ mL-1 e 69% de espermatozoides normais. O acetato de buserelina pode induzir à espermiação da rã-manteiga. O volume seminal coletado foi baixo, mas a coloração, vigor, motilidade, concentração e número de espermatozoides de L. ocellatus foram adequados.

Balantidium honghuensis n. sp. (Ciliophora: Trichostomatidae) from the Rectum of Rana nigromaculata and R. limnocharis from Honghu Lake, China

A new trichostome ciliate, Balantidium honghuensis n. sp., was isolated from the recta of Rana nigromaculata and R. limnocharis during parasite surveys in Honghu Lake, Hubei Province, central China in summer of 2010 and 2011. Its detailed morphometric characters based on LM and SEM studies were described herein. The organism is oval in shape and thickly ciliated. The vestibulum is "V" shaped and occupies about 1/3 to 2/5 of the body length. The vestibular and nearby regions possess strong peripheral fibers which form a marked axial fiber about the cytopharynx. More than 10 contractile vacuoles are distributed along the periphery of the latter body. Comparisions were made between this new species and B. sinensis Nie, 1935. They were discriminated from each other in terms of general body forms, body size, and vestibulum shapes. Besides, special attention was paid to its high-speed daughter swarmers which we believed to be the infective stage of B. honghuensis. Possible infection routes of anura amphibian balantidia were discussed.

Discovery of Larval Gnathostoma nipponicum in Frogs and Snakes from Jeju-do (Province), Republic of Korea

A survey was performed to find out the intermediate hosts of Gnathostoma nipponicum in Jeju-do (Province), the Republic of Korea. In August 2009 and 2010, a total of 82 tadpoles, 23 black-spotted pond frogs (Rana nigromaculata), 7 tiger keelback snakes (Rhabdophis tigrinus tigrinus), 6 red-tongue viper snakes (Agkistrodon ussuriensis), and 2 cat snakes (Elaphe dione) were collected in Jeju-do and examined by the pepsin-HCl digestion method. Total 5 gnathostome larvae were detected in 3 (50%) of 6 A. ussuriensis, 70 larvae in 3 of 7 (42.9%) R. tigrinus tigrinus, and 2 larvae in 2 of 82 (8.7%) frogs. No gnathostome larvae were detected in tadpoles and cat snakes. The larvae detected were a single species, and 2.17x0.22 mm in average size. They had characteristic head bulbs, muscular esophagus, and 4 cervical sacs. Three rows of hooklets were arranged in the head bulbs, and the number of hooklets in each row was 29, 33, and 36 posteriorly. All these characters were consistent with the advanced third-stage larvae of G. nipponicum. It has been first confirmed in Jeju-do that R. nigromaculata, A. ussuriensis, and R. tigrinus tigrinus play a role for intermediate and/or paratenic hosts for G. nipponicum.

Arginase isoforms in frog and lizard tissues.

Isoforms of arginase in the liver and kidney tissues of the ureotelic frog (Rana tigerina) and uricotelic lizard (Calotes versicolor) were fractionated by DEAE-cellulose chromatography (pH 8.3). Four molecular forms, designated as A’1, A2, A3 and A4 based on the KCl concentration required for their elution from the ion-exchange column, were detected in lizard liver, while only two forms were found in lizard kidney (A3 and A4) and frog liver and kidney (A2 and A3). No major differences were found in the pH optimum, substrate affinity and molecular weight of the isoenzymes. The isoforms in lizard tissues were either totally unaffected or only partially immunoprecipitated by antibodies raised against rat liver and beef liver arginases, but those in frog tissues were significantly activated by the two antibodies. While the physiological importance of the presence of four isoforms in lizard liver remains enigmatic, different sets of isoenzymes were present in the liver of the two ureotelic vertebrates, rat and frog. Hence, it appeared that a given mode of nitrotelism was not associated with a specific set of isoenzymes. Also, the data were not consistent with the generally held view that a basic isoform of arginase served as a component of the urea cycle in liver and a neutral/slightly acidic form functions in the synthesis of proline, glutamate and polyamines in extra-hepatic tissues. The isoforms appeared to show considerable functional overlap.

Trans-differentiation of iris pigmented epithelial cells of Euphlyctis cyanophlyctis tadpoles into lens in vitro.

Meshed pigmented iris epithelium along with neural retina of tadpoles of the frog Euphlyctis cyanophlyctis were found to undergo dedifferentiation and subsequently transdifferentiate into lens in culture medium. During lag period, depigmentation (dedifferentiation) occurred in many cells. When culture became confluent 3-4 weeks after seeding tiny lens like structures differentiated from foci of cultured pigmented iris epithelium cells. The percentage of lens formation was higher in vitamin A treated cases. The culture system appears to be a suitable for investigating the changes occurred during trans-differentiation of pigmented epithelial cells into lens.

On origin of Oviductus Ranae in Chinese Pharmacopoeia / 中国中药杂志

<p><b>OBJECTIVE</b>To discuss the problem about the origin of Oviduetus Ranae in the Chinese Pharmacopoeia according to historical documents, the researches reported recently and the author research.</p><p><b>METHOD</b>Through comprehensive analysis of the documents and materials reported, the original animal sources of Oviduetus Ranae was discussed in terms of historical records, morphology, karyotype, Ag-Belt and isoenzyme electrophoresis, gene levels and so on.</p><p><b>RESULT AND CONCLUSION</b>The original animal sources of Oviduetus Ranae is Rana dybowskii,its order element is an effective species in China. In order to avoid the problem of species confusion about the origin of Oviduetus Ranae, author suggests that R. dybowskii should be the original animal of Oviduetus Ranae.</p>

Phenotypic and molecular variation in the green and black poison-dart frog Dendrobates auratus (Anura: Dendrobatidae) from Costa Rica / Variación fenotípica y molecular en la rana dardo venenoso verde y negra Dendrobates auratus (Anura: Dendrobatidae) de Costa Rica

The green and black poison-dart frog Dendrobates auratus exhibits high intraspecific variation in hue color and pattern throughout its range, making it a very popular species in the pet trade. We analyzed the correspondence between color variation and molecular variation of D. auratus from Costa Rica using RAPD analysis. Twenty-six random primers were analyzed for variation in 99 individuals from seven populations. Color pattern was scored from digital images of the dorsal and ventral views. In general, frogs from the Caribbean coast had significantly more light coloration than black color but cannot be grouped by population based only on hue pattern. Only 3 RAPD primers were found to be polymorphic, representing a total of 16 loci. Most of the molecular variation encountered here occurs within populations, thus making unclear the degree of population structure and differentiation. Further examination of COI mtDNA sequences from our samples also supports these results. Partial Mantel correlations suggested that the pattern of molecular variation is not congruent with the variation in color pattern in this species, an outcome that is discussed in terms of phenotypic evolution. Rev. Biol. Trop. 57 (Suppl. 1): 313-321. Epub 2009 November 30.

La rana venenosa Dendrobates auratus posee una gran variación intraespecífica en tonos y patrones de coloración a lo largo de toda su distribución, lo que la hace una especie muy reconocible entre las especies de dendrobátidos. Analizamos la correspondencia entre variación de coloración y variación molecular de D. auratus de Costa Rica empleando análisis de RAPDs. La variación resultante en veintiséis "primers" aleatorios fue analizada en 93 individuos de siete localidades en Costa Rica. El patrón de coloración fue evaluado de imágenes digitales del dorso y vientre para los mismos individuos. En general, las ranas provenientes de localidades en la costa Caribe tienen significativamente una coloración más clara, con menos proporción de color negro que las de localidades en la vertiente Pacífica, pero no pueden ser agrupadas por localidad basadas simplemente en el patrón de coloración. Solamente tres RAPD "primers" fueron encontrados polimórficos, representando un total de 16 loci. Mucha de la variación molecular encontrada habita dentro de poblaciones, lo que hace difícil determinar el grado de estructura poblacional y diferenciación. La reexaminación posterior de secuencias del gen mitocondrial CO1 también apoya estos resultados. Correlaciones parciales de matrices (test de Mantel) sugieren que el patrón de variación molecular no es congruente con la variación en el patrón de coloración en esta especie, un resultado que es discutido en términos de evolución fenotípica.

Effect of Emblica officinalis (Gaertn) on lens regeneration in the frog, Rana cyanophlyctis (Schneider).

Emblica officinalis (Amla) accelerated cell proliferation and dedifferentiation of pigmented epithelial cells of dorsal iris and consequently induced lens regeneration in R. cyanophlyctis. Further it enhanced the percentage of lens regeneration not only in young tadpoles but also is adult frogs. Lens regeneration ability declined with the age of animals in both control as well as treated groups.

Cloning of cDNAs encoding skin antimicrobial peptide precursors from Chinese brown frogs, Rana chensinensis and determination of antimicrobial, anticancer and hemolysis activity / 生物工程学报

Amphibian skin antimicrobial peptides exhibit a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacterium and cytotoxic activity responsible for inhibiting the growth of cancer cells. In this present study, six cDNAs encoding antimicrobial peptide precursors were cloned from the skin of Chinese brown frog, Rana chensinensis by RT-PCR and 3'-RACE procedure and identified as preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, preprobrevinin-1CEa, preprobrevinin-1CEb, and preprochensinin-1, respectively. The nucleotide sequences of cDNA encoding 59-65 amino acid composed of 289-315 bp. Preprotemporin-1CEa, preprotemporin-1CEb and preprotemporin-1CEc are members of temporin family, which usually are short, hydrophobic, and C-terminally alpha-amidated antimicrobial peptides. Preprobrevinin-1CEa and preprobrevinin-1CEb were identified as the members of the brevinin-1 family of antimicrobial peptides since both peptides contain "RANA box" that it's responsible for forming Cys-bridged cyclic heptapeptides at the C-terminal region of peptide. The nucleotide acid sequence and the deduced amino acid Sequence of preprochensinin-1 were not found to be identity with any known amphibian skin defensive peptides, so, preprochensinin-1 was identified as a novel peptide precursor. Four of bioactive peptides: temporin-1CEa, temporin-1CEb, brevinin-1CEa and chensinin-1 were synthesized to investigate their antimicrobial, anticancer and haemolysis activities. The results showed that all of the synthesized antimicrobial peptides in this study inhibited the growth of the Gram-positive bacterium, and exhibited the anticancer activity against the growth of MCF-7 cells and HeLa cells. Analysis of the R. chensinensis bioactive peptides and their gene expression will be beneficial for preservation of this species.