Ctenocephalides felis is a major pest of companion animals worldwide. This project aimed to generate novel genetic resources for C. felis and develop tools to aid drug-target identification and validation. Sample handling methods were assessed and candidate reference genes validated, to ensure quality of RNA samples and reliable gene expression normalisation. Piercing C. felis samples prior to storage in RNAlater ensured RNA integrity was maintained over time. Glyceraldehyde 3-phosphate dehydrogenase , 60S ribosomal protein L19 and elongation factor-1α were demonstrated as stable reference genes across all comparisons tested. A C. felis transcriptome encompassing multiple developmental stages, sexes and tissues was sequenced and de novo assemblies produced with two assemblers, Trinity and Oases. Each assembly contained >100000 contigs. Annotation of the assemblies generated functional insight, such as top BLAST hits, GO annotations and signal peptide predictions. The Trinity assembly was deemed the highest quality and searched for genes of interest, involved in development. Expression analysis of selected transcripts across stadia gave insight into developmental processes, and demonstrated the utility of the transcriptome. This study was the first to demonstrate that C. felis can mount an RNAi response upon exposure to dsRNA. Knockdown of glutathione S-transferase σ (GSTσ), was demonstrated in adult C. felis: ≈80 % knockdown following microinjection of dsGSTσ; ≈64 % knockdown after soaking in dsGSTσ; ≈96 % knockdown after continuous feeding on dsGSTσ. RNAi machinery was identified in C. felis. siRNAi pathway components, Dicer 2 and Argonaute 2, were upregulated following dsRNA exposure. Dicer 2 was knocked-down by soaking in dsDicer2, although results of an “RNAi of RNAi” experiment were inconclusive. Transcripts encoding machinery putatively involved in dsRNA uptake and breakdown were also identified. Through these studies, this project has generated novel insights into C. felis biology and opened up new avenues for research.

Made available in DSpace on 2016-04-28T20:16:22Z (GMT). No. of bitstreams: 1 2007-Thais Ribeiro Correia.pdf: 769614 bytes, checksum: dc53e7ee3e823c6268372508d266efaa (MD5) Previous issue date: 2007-02-28 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / The purpose of the present study was to evaluate the activity of the neonicotinoid dinotefuran on the cat flea Ctenocephalides felis felis. Fleas used in controlled and in vitro tests were obtained from a colony maintained at Laboratory of Development of Parasiticide Products (LDPP), and the Beagle dogs used for the controlled tests were kept at the same place. Five concentrations of dinotefuran, 0.2085, 0.417, 0.834, 1.668 and 3.336%, were evaluated for in vitro activity on eggs and adults of C. felis felis. Dinotefuran did show low activity on flea eggs, but showed efficacy on adult fleas. Two formulations of dinotefuran were titulated. For spray formulation, 0.834% was selected and for strip-on formulation, 30% was selected. Later, the efficacy of two strip-on formulations, one containing 30% dinotefuran and other with 30% dinotefuran plus 2.575% pyriproxyfen, were evaluated in the control of fleas on dogs. Dogs were infested with 100 adult fleas and evaluated after 48 hours. The efficacy was to 99.5 and 100% for day 1 declining to 76.1 and 72.2% on day 35, respectively. Two spray formulations, one with 0.834% dinotefuran and other with 0.834% dinotefuran plus 0.148% pyriproxyfen, were tested for the control of fleas on dogs. Dogs were infested and evaluated after 48 hours. The efficacy was 100% on day 1 for both formulations, reducing for 66.6% on day 28 and 59.4% on day 35, respectively. Four formulations previously described were tested on naturally infested housed kept-dogs from Serop?dica City, RJ. Animals were evaluated weekly. The efficacies were 95.2 on day 7 and 91.4% on day 56 for the spray formulation with dinotefuran; and 100% on day 7 and 88.1% on day 56 for the spray association. For dinotefuran strip-on were 98.5% on day 7 and 74% on day 56; and 100% on day 7 and 81.1% on day 56 for strip-on association. Residual activity of treated dog s hair with dinotefuran on eggs, larvae and adults of fleas were evaluated. One dog was treated with 0.834% dinotefuran spray, other dog was treated with 30% dinotefuran strip-on and the third was not treated. Some areas of dog s hair were clipped on days 2, 16, 23, 30, 37 and 44 after treatment. For the evaluation of adulticidal, larvicidal and ovicidal activities were used adults, larvae and eggs from the laboratory colony. Six repetitions were used with 10 specimens of each flea stage per day, placed in assay tubes. In each repetition were added 0.02 g of treated or untreated dog s hair and larval diet for immature stages. The adulticidal activity was evaluated during 24 hours, the larvae 20 days after treatment and the eggs 72 after challenge. The residue on hair of both treated dogs showed efficacy on the larval control for 44 days and on egg control at day 2. Regarding the adults control the spray and strip-on formulations showed efficacy until days 16 and 23, respectively. / O presente trabalho teve como objetivo avaliar a atividade do neonicotin?ide dinotefuran sobre Ctenocephalides felis felis. As pulgas utilizadas nos testes in vitro e nos testes controlados foram da col?nia mantida no Laborat?rio de Desenvolvimento de Produtos Parasiticidas (LDPP) da UFRRJ, e os nos testes controlados foram utilizados c?es Beagle oriundos do canil de experimenta??o mantido nas mesmas depend?ncias. Em um teste in vitro foram avaliadas as efic?cias de cinco concentra??es de dinotefuran, 0,2085; 0,417; 0,834, 1,668 e 3,336%, sobre ovos e adultos de C. felis felis. O dinotefuran apresentou atividade parcial sobre os ovos de C. felis felis, por?m se mostrou eficaz no controle de adultos. Duas formula??es de dinotefuran foram tituladas. Das concentra??es testadas para a formula??o spray (0,417; 0,834 e 1,668%), a selecionada foi a de 0,834%, e para a formula??o strip-on (25, 30 e 35%) foi a de 30%. Posteriormente, em um teste controlado, foram avaliadas as efic?cias de duas formula??es strip-on , uma contendo dinotefuran a 30% e outra o mesmo associado ao piriproxifen a 2,575%, no controle de adultos de C. felis felis em c?es Beagle. Os c?es foram infestados semanalmente com 100 pulgas adultas oriundas da col?nia e avaliados ap?s 48 horas. As efic?cias foram de 99,5 e 100% no dia +1 declinando para 76,1 e 72,2% no dia +35, respectivamente. Duas formula??es spray, uma contendo dinotefuran a 0,834% e outra o mesmo associado ao piriproxifen a 0,148%, tamb?m foram avaliadas. As efic?cias foram de 100% no dia +1 para as duas formula??es, declinando para 66,6% no dia +28 e para 59,4% no dia +35, respectivamente para a formula??o contendo apenas dinotefuran e a associa??o. As quatro formula??es citadas anteriormente foram testadas em n?vel de campo em c?es infestados naturalmente domiciliados no Munic?pio de Serop?dica, RJ. Para a formula??o spray contendo apenas dinotefuran, a efic?cia foi de 95,2% no dia +7 e de 91,4% no dia +56. J? para formula??o associada, a efic?cia foi de 100% no dia +7 e de 88,1% no dia +56. Para a formula??o strip-on contendo apenas o dinotefuran, a efic?cia foi de 98,5% no dia +7 e de 74% no dia +56, e para associa??o a efic?cia foi de 100% no dia +7 e de 81,1% no dia +49. Foram avaliadas as atividades adulticida, larvicida e ovicida do res?duo no p?lo de c?es tratados com as formula??es spray e strip-on . Pequenas ?reas do corpo do c?o foram tricotomizadas nos dias 2, 9, 16, 23, 30, 37 e 44 ap?s o tratamento. Seis repeti??es com 10 exemplares cada, acondicionados em tubos de ensaio, da etapa correspondente foram utilizados por dia de desafio, e acrescidos de 0,02g de p?lo tratado ou n?o. O res?duo no p?lo de c?es tratados com ambas as formula??es de dinotefuran foi eficaz no controle de ovos apenas no dia +2, para larvas por at? 44 dias, e no controle de adultos as formula??es spray e a strip-on foram eficazes por um per?odo de 16 e 23 dias, respectivamente.