Made available in DSpace on 2016-04-28T20:15:36Z (GMT). No. of bitstreams: 1 2009 - Tatiane Kawamura de Almeida.pdf: 416296 bytes, checksum: 10f392469ed0d8f6721023bb52beb9dd (MD5) Previous issue date: 2009-03-05 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Amblyomma parvum is a tick of wide geographical distribution which parasite wild and domestic animals. In Brazil, the literature records only data on their geographical distribution and hosts. There is no record on the action of different temperatures in Brazilian populations of this species. The objective of this work was to evaluate the effects of five different temperatures on the free living stages on the life cycle of A. parvum. The experiment was conducted under 18, 21, 24, 27 and 32?1?C and relative humidity of 80?10%. For the eggs stage, 15 animals were used for infestation in the back after previous trichotomy. The females that dropped were randomly detach at different temperatures. The eggs were packed in glass jars after the fifth day of posture and observed daily for analysis of the average pre-hatching, average hatching and mean percentage of hatching. For others stages, three rabbits were used for each temperature, totaling 15 animals for each stage. The engorged ticks that dropped were collected daily. In the laboratory, they were cleaned, weighed and observed daily. The average of pre-ecdysis of the larvae and nymphs varied inversely proportional to the temperature. The average percentage of ecdysis for the larvae was statistically similar for the five treatments. The average moulting period had the same change that the average of preecdysis period for larvae and nymphs, where longer periods corresponds to lower temperatures. The average period of larval survival was similar statistically for the treatments 18, 21 and 24?1?C and was lower in the temperatures of 27 and 32?1?C. The average ecdysis period of the nymphs were statistically different only in the treatments of 18 and 32?1?C and was much longer in the temperature of 18?1?C (19.1 days). Similar to larvae, the average percentage of ecdysis for the nymphs was statistically similar to the five treatments. The lowest average period of survival of nymphs, with average of 117.5 days, was observed at 32 ?C. The average of pre-oviposition and oviposition periods were similar. Both were inversely proportional to the increase of temperature, however, only had significant difference in the three lower temperatures. Regarding the average weight of oviposition, the heavier weight was found at 27?1?C (average of 107?48.3 mg). The average production of eggs index and the average nutritional efficiency index had the highest average at 27?1?C. The average of pre-hatching period differed significantly in five treatments, and was inversely proportional to the increase of temperature. The average hatching period and the average percentage of hatching were longer and lower in the temperature of 18?1?C. The temperature of 18?C can be used for delaying the life cycle, however, it may be deleterious to the ticks. / Amblyomma parvum ? uma esp?cie de carrapato de ampla distribui??o geogr?fica, capaz de parasitar animais silvestres e dom?sticos. No Brasil, a literatura registra apenas dados sobre sua distribui??o geogr?fica e hospedeiros. N?o existe nenhum registro sobre a a??o de diferentes temperaturas em popula??es brasileiras desta esp?cie. O objetivo deste trabalho foi avaliar os efeitos de cinco diferentes temperaturas sobre os diversos processos das fases de vida livre do ciclo biol?gico de A. parvum. Todas as fases de vida livre foram avaliadas nas temperaturas de 18, 21, 24, 27, 32?1?C e umidade relativa de 80?10%. Para a etapa dos ovos foram utilizados 15 animais para infesta??o no dorso ap?s pr?via tricotomia. As f?meas que se desprenderam foram distribu?das aleatoriamente nas diferentes temperaturas. Os ovos foram acondicionados em frascos de vidro ap?s o quinto dia de postura e observados diariamente para an?lise do per?odo m?dio de pr?-eclos?o, per?odo m?dio de eclos?o e percentual m?dio de eclos?o. Para os demais est?gios, foi realizada uma infesta??o com tr?s coelhos para cada temperatura, totalizando 15 animais para cada est?gio. Os carrapatos ingurgitados que se desprendiam eram coletados diariamente. No laborat?rio, estes foram limpos, pesados e acondicionados nas cinco temperaturas, para a observa??o cont?nua dos exemplares. Os per?odos m?dios de pr?-ecdise das larvas e das ninfas variaram de forma inversamente proporcional ? temperatura. Em rela??o ao percentual m?dio de ecdise das larvas, os tratamentos foram estatisticamente semelhantes. O per?odo m?dio de muda sofreu a mesma varia??o que o per?odo m?dio de pr?-ecdise de larvas e ninfas, onde per?odos mais prolongados correspondem a temperaturas mais baixas. O percentual m?dio de sobreviv?ncia das larvas foi estatisticamente semelhante nos tratamentos a 18, 21 e 24?1?C e diminuiu consideravelmente nas temperaturas de 27 e 32?1?C. Os per?odos m?dios de ecdise das ninfas foram diferentes estatisticamente somente nos tratamentos a 18 e 32?1?C, sendo muito mais longo na temperatura de 18?1?C (19,1 dias). ? semelhan?a do ocorrido com as larvas, o percentual m?dio de ecdise das ninfas foi estatisticamente semelhante para os cinco tratamentos. O menor per?odo m?dio de sobreviv?ncia das ninfas, com m?dia de 117,5 dias, foi observado a 32?C. Os per?odos m?dios de pr?-postura e de postura foram semelhantes. Ambos foram inversamente proporcionais ao aumento de temperatura, no entanto, somente houve diferen?a significativa nas tr?s temperaturas mais baixas. Com rela??o ao peso m?dio da postura, o maior peso foi encontrado a 27?1?C (m?dia de 107?48.3 mg). O ?ndice m?dio de produ??o de ovos e o ?ndice m?dio de efici?ncia nutricional apresentaram as maiores m?dias a 27?1?C. O per?odo m?dio de pr?-eclos?o diferiu significativamente nos cinco tratamentos, sendo inversamente proporcional ao aumento da temperatura. O per?odo m?dio de eclos?o e o percentual m?dio de eclos?o foram mais longos e menores na temperatura de 18?1?C. A temperatura de 18?C pode ser utilizada quando se pretende retardar o desenvolvimento de uma gera??o, por?m, pode apresentar um efeito delet?rio.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-11T12:09:07Z No. of bitstreams: 1 2016 - Joao Bosco Varela.pdf: 1890246 bytes, checksum: 359c15e2f52f2ea873d394cbbfa5479c (MD5) / Made available in DSpace on 2017-01-11T12:09:07Z (GMT). No. of bitstreams: 1 2016 - Joao Bosco Varela.pdf: 1890246 bytes, checksum: 359c15e2f52f2ea873d394cbbfa5479c (MD5) Previous issue date: 2016-02-24 / The study of ticks and tick-borne disease is increasingly dependent upon the use of molecular biological techniques that are employed in pathogen detection and for the accurate identification of ticks, particularly immature stages. The successful application of molecular methods, principally the polymerase chain reaction (PCR), can only be achieved if the DNA present in the tick was effectively preserved and could be extracted efficiently. The current study compared three fixatives (RNAlater, zinc salts (ZN) and isopropanol) for the ability to preserve the nuclear and mitochondrial (mt) DNA of larvae and nymphs of Amblyomma parvum and larvae of Amblyomma scultptum. DNA was extracted from ticks at times between 72h to 12 months post fixation using a phenol-chloroform procedure and examined using PCR assays for nuclear (internal transcribed spacer 2; ITS2) and mitochondrial (12S rDNA, subunit 1 of cytochrome c oxidase (COI) and D-loop) sequences. The efficiency of amplification was analyzed quantitatively (number of samples which produced amplicon) and qualitatively (relative intensity of bands observed on agarose gels). It was observed that the ITS2 sequence could be amplified in the majority (93,39%, n= 283/303) of the samples, in each of the three fixatives, although qualitative differences were observed, particularly with A. sculptum preserved in ZN. In contrast, fixation in isopropanol effectively abolished the ability to amplify the mitochondrial marker sequences of A. parvum and also resulted in inferior amplification (qualitative), of the D-loop target with A. sculptum. Those effects were observed in samples fixed for as little as 72h. The detrimental effects of isopropanol were also observed in samples extracted using an alkaline lysis method (Hotshot). Samples of A. parvum larvae preserved in RNAlater for 30 months showed an amplification efficacy of 100% in the ITS2 and COI assays, irrespective of the extraction method. Mitochondrial sequences are a central component of the majority of molecular studies of ticks. The findings of this study indicate that isopropanol should be avoided as a fixative for immature stages of ticks. Instead, the use of RNAlater is recommended in order to permit the consistent recovery of amplifiable mtDNA / O estudo de carrapatos e doen?as transmitidas por eles ? cada vez mais dependente da utiliza??o de t?cnicas de biologia molecular que s?o empregadas na detec??o de pat?genos, e a acurada identifica??o desses artr?podes, em particular os est?gios imaturos. A aplica??o bem-sucedida dos m?todos moleculares, principalmente, a rea??o em cadeia da polimerase (PCR), s? pode ser alcan?ada se o DNA presente no carrapato foi eficazmente preservado e extra?do de forma eficiente. O estudo comparou tr?s fixadores (RNAlater, sais de zinco (ZN) e isopropanol) avaliando a sua capacidade de preservar DNA mitocondrial (mtDNA) e nuclear de larvas e ninfas de Amblyomma parvum e larvas de Amblyomma scultptum. O DNA foi extra?do dos carrapatos, em tempos entre 72h e 12 meses ap?s a fixa??o por meio da t?cnica de fenol-clorof?rmio e lise alcalina (?Hot Shot?) e examinadas usando ensaios de PCR para sequ?ncias nucleares (espa?ador interno transcrito 2; ITS2) e mitocondriais (12S rDNA, subunidade 1 do citocromo c oxidase (COI) e D-loop). A efici?ncia de amplifica??o foi analisada quantitativamente (n?mero de amostras que produziram ?amplicon?) e qualitativamente (intensidade relativa das bandas observadas em g?is de agarose). Foi observado que a sequ?ncia ITS2 foi amplificada na maioria (93,39%, n= 283/303) das amostras em cada um dos tr?s fixadores, embora tenham sido observadas diferen?as qualitativas, particularmente com A. sculptum preservado em ZN. Em contraste, a fixa??o em isopropanol afetou negativamente a capacidade de amplificar as sequ?ncias dos marcadores mitocondriais de A. parvum e tamb?m resultou na amplifica??o inferior (qualitativa), do alvo D-loop com A. sculptum. Esses efeitos foram observados em amostras fixadas por apenas 72 horas. Os efeitos prejudiciais de isopropanol tamb?m foram observados igualmente em amostras extra?das usando um m?todo de lise alcalina (?HotShot?). As amostras de larvas de A. parvum preservadas em RNAlater durante 30 meses, mostrou uma efic?cia de amplifica??o de 100% nos ensaios para ITS2 e COI, independentemente do m?todo de extra??o usado. Sequ?ncias mitocondriais s?o um componente central da maioria das pesquisas moleculares com carrapatos. Os resultados deste estudo indicam que isopropanol deve ser evitado como um fixador para fases imaturas de carrapatos. Em vez disso, o uso de RNAlater ? recomendado, a fim de permitir a recupera??o consistente de mtDNA amplific?ve