Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2012-08-13T05:08:16Z No. of bitstreams: 1 ademir_j_martins_junior_ioc_bp_0028_2009.pdf: 3828128 bytes, checksum: 594e5415edb29a09e864f5395428b97c (MD5) / Made available in DSpace on 2012-08-13T05:08:16Z (GMT). No. of bitstreams: 1 ademir_j_martins_junior_ioc_bp_0028_2009.pdf: 3828128 bytes, checksum: 594e5415edb29a09e864f5395428b97c (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / No Brasil o controle das formas aladas do vetor de dengue, o mosquito Aedes aegypti, é feito com inseticidas da classe dos piretróides. Porém, apesar da recente utilização deste composto em escala nacional, várias populações do vetor já estão resistentes. O canal de sódio regulado por voltagem, no sistema nervoso do inseto, é a molécula-alvo de piretróides. Investigamos, em populações brasileiras de Ae. aegypti, a diversidade molecular em uma região deste gene (AaNaV) com o objetivo de identificar potenciais alterações relacionadas à resistência. Clonagem e sequenciamento da região genômica entre os exons 20 e 21 do AaNaV de indivíduos de cinco localidades distintas, confirmaram polimorfismo no íntron e em dois sítios do exon 20 que geram substituições sinônimas. De acordo com estas características, as sequências foram agrupadas em tipos A ou B. Observamos ainda mutações causando substituições de aminoácidos: Ile  Met no sítio 1011 e Val  Ile no sítio 1016, somente em sequências do tipo A. Indivíduos da cepa Rockefeller, referência de susceptibilidade, apresentaram apenas sequências do tipo B e os alelos selvagens nas duas posições, 1011 ou 1016. Tipagem molecular por PCR alelo-específica em indivíduos de 15 localidades revelou que a mutação Ile1011Met está disseminada por todo país, diferentemente da Val1016Ile, concentrada nas regiões mais ao centro. Nas cinco localidades onde os indivíduos avaliados foram divididos em resistentes e susceptíveis, o alelo mutante 1016Ile esteve significativamente mais presente nos resistentes, principalmente quando em homozigose, indicando o caráter recessivo da mutação para a resistência. Surpreendentemente, uma série de observações sugeriu a ocorrência de polimorfismo envolvendo uma duplicação gênica, de forma que o haplótipo duplicado estaria constituído de uma sequência do tipo B ligada à outra do tipo A com a mutação 1011Met. Corroborou esta hipótese a tipagem do sítio 1011 na prole de cruzamentos com genótipos determinados. Finalmente, comparação entre linhagens mantidas na presença ou na ausência de pressão de seleção com piretróide, em laboratório, sugeriu efeitos pleiotrópicos negativos da resistência em aspectos do desenvolvimento e da reprodução destes mosquitos / In Brazil, control of adults of the dengue vector, the mosquito Aedes aegypti, is performed with pyrethroid insecticides. However, despite the recent implementation of this compound in national scale, several populations of this vector are already resistant. The voltage gated sodium channel is the pyrethroid target site, in the insect nervous system. We investigated the molecular diversity of a particular region of this gene (AaNaV) in Ae. aegypti Brazilian populations in order to identify potential substitutions implicated with insecticide resistance. Cloning and sequencing of the genome region between the AaNaV exons 20 and 21 in individuals from five distinct localities, confirmed polymorphism in the intron and in two positions of exon 20, these latter related to synonymous substitutions. According to these characteristics sequences were grouped into types A or B. Two additional predictive substitutions were noted: Ile Met and Val  Ile, respectively, in 1011 and 1016 sites, both only in type A sequences. Individuals from the Rockefeller insecticide susceptible reference strain exhibited only type B sequences and the wild alleles on both 1011 and 1016 positions. Allelespecific PCR molecular typing of individuals from 15 localities showed that the Ile1011Met mutation is widespread throughout Brazil, whereas Val1016Ile is more proeminent toward the middle of the country. In five localities typing was performed separately in susceptible or resistant individuals; the mutant 1016Ile allele was significantly more present in the resistant ones, especially in homozygozity, suggesting the recessive character of this resistant mutation. Surprisingly, a series of observations suggested the occurrence of a gene duplication consisting of both type B and A sequences this last one with the 1011Met mutation. Typing of the 1011 site in the offspring of couples with specific genotypes corroborated this hypothesis. Finally, comparisons among lineages selected or not with pyrethroid suggested negative pleiotropic effects of resistance on development and reproduction aspects.

<p>Bed bugs (<i>Cimex lectularius</i> L.) are globally important human ectoparasites. Their management necessitates the use of multiple control techniques. Plant-derived essential oils are extracts from aromatic plants that represent one of the alternative control measures for bed bug control, in addition to mechanical options and synthetic pesticides. However, there is limited information available on the efficacy and toxicology of plant essential oils against bed bugs. This project was designed with the aim to provide in-depth information on efficacy, toxicology and mode-of-action of essential oils and their insecticidal constituents in bed bugs. Initially, I evaluated topical and fumigant toxicity of fifteen essential oil components against adult male bed bugs of the Harlan strain (an insecticide susceptible strain). Neurological effects of the six most toxicologically active compounds were also determined. In both topical and fumigant bioassays, carvacrol and thymol were the most active compounds. Spontaneous electrical activity measurements of the bed bug nervous system demonstrated neuroinhibitory effects of carvacrol, thymol and eugenol, whereas linalool and bifenthrin (a pyrethroid class insecticide) produced excitatory effects. Further, I evaluated the efficacy and neurological impacts of a mixture of three neuroinhibitory compounds; carvacrol, eugenol and thymol in 1:1:1 ratio against adult male bed bugs of the Harlan strain. This mixture of monoterpenoids as well as the mixture of synthetic insecticides exhibited a synergistic affect in topical bioassays. In electrophysiology experiments, the monoterpenoid mixture led to higher neuroinhibitory effects, whereas a mixture of synthetic insecticides caused higher neuroexcitatory effects in comparison to single compounds. </p> <p>In the next objective of my dissertation, I compared the efficacy of five plant essential oils (thyme, oregano, clove, geranium and coriander), their major components (thymol, carvacrol, eugenol, geraniol and linalool) and EcoRaider^® (commercial product) between pyrethroid susceptible (Harlan) and field collected (Knoxville) bed bug populations. Initially, I found that the Knoxville strain was 72,893 and 291,626 fold resistant to topically applied deltamethrin (a pyrethroid class insecticide) compared to the susceptible Harlan strain at the LD₂₅ and LD₅₀ lethal dose levels, respectively. Synergist bioassays and detoxification enzyme assays showed that the Knoxville strain possesses significantly higher activity of cytochrome P450 and esterase enzymes. Further, Sanger sequencing revealed the presence of the L925I mutation in the voltage gated sodium channel gene. The Knoxville strain, however, did not show any resistance to plant essential oils, their major components or EcoRaider^® in topical bioassays (resistance ratios of ~ 1). In the final objective, I evaluated the efficacy of binary mixtures of above-mentioned essential oils or their major components or EcoRaider^® with deltamethrin in susceptible and resistant bed bugs. In topical application bioassays, binary mixtures of essential oils or their major components or EcoRaider^® and deltamethrin at the LD₂₅ dose caused a synergistic increase in toxicity in resistant bed bugs. Further, I studied the inhibitory effects of major essential oil components on detoxification enzyme activities (cytochrome P450s, esterases and glutathione transferases). Detoxification enzyme assays conducted using protein extracts from bed bugs pre-treated with essential oil constituents showed that these compounds significantly inhibited cytochrome P450 activity in the resistant strain, but esterase and glutathione transferase activity were unaffected. No inhibition of detoxification enzyme activities was observed in the Harlan strain bed bugs pre-treated with essential oil constituents.</p> <p>In conclusion, my dissertation research has created the foundation for utilization of natural products for bed bug management by (i) describing the efficacy of plant essential oils and their components against bed bugs, (ii) discovering synergistic interactions between essential oil components at the nervous system level, (iii) determining susceptibility of deltamethrin-resistant bed bugs to plant essential oils and their constituents and (iv) identifying synergistic effects of essential oils or their components on toxicity of pyrethroid insecticides and underlying mechanisms of this synergistic interaction. </p> <br>