Orientadores: Ana Maria Lima de Azeredo Espin, Ana Carolina Martins Junqueira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T08:05:59Z (GMT). No. of bitstreams: 1 Paulo_DanielFernando_M.pdf: 4089154 bytes, checksum: f604296d2e31ee6a5dd7e3aa8f517f69 (MD5) Previous issue date: 2014 / Resumo: MicroRNAs (miRNAs) são pequenos RNAs não codantes que agem como moduladores pós-transcricionais da expressão gênica em todos os eucariotos investigados até o momento. Em animais, a complementariedade imperfeita de bases entre o miRNA e o sítio alvo do RNA mensageiro (mRNA) inibi sua tradução, tornando-os genes chaves no controle da expressão gênica. A identificação de miRNAs pode fornecer uma melhor compreensão de diversos processos biológicos e evolutivos das diferentes espécies. A família Calliphoridae é um grupo que compreende dípteros causadores de miíases, incluindo as espécies Cochliomyia hominivorax (mosca da bicheira) e Cochliomyia macellaria (mosca varejeira). A mosca da bicheira é uma das principais pragas na região Neotropical. Na fase larval, esta espécie causa infestações e alimenta-se de tecidos vivos de vertebrados de sangue quente, acarretando severas perdas na indústria pecuária. Diferentemente, a mosca varejeira, apresenta um hábito saprófago, se alimentando e reproduzindo em carcaças e tecidos em decomposição, ressaltando sua importância para a entomologia forense e para a saúde pública. Por serem filogeneticamente próximas e possuírem diferentes hábitos alimentares e reprodutivos, estas espécies representam modelos para estudos sobre as bases moleculares do parasitismo em Calliphoridae. Para identificar e caracterizar os miRNAs destas duas espécies, o transcriptoma de pequenos RNAs de adultos (macho e fêmea) e larva (terceiro instar) foram sequenciados em plataforma de nova geração MiSeq-Illumina. Os 6.2 milhões de reads gerados foram mapeados contra o genoma de Drosophila melanogaster e o banco de dados miRBase. Foram identificado 84 miRNAs evolutivamente conservados, dos quais 80 foram encontrados em C. hominivorax e 78 em C. macellaria. Também foi investigada a presença dos precursores em forma de grampo (pre-miRNAs) nos dados genômicos e transcriptômicos disponíveis para estas espécies. Foram preditos 10 pre-miRNAs conservados e outros 5 que não apresentaram similaridade com nenhum miRNA já descrito para outras espécies de animais. A caracterização evolutiva dos miRNAs identificados mostrou que essas sequências são altamente conservadas desde Nephrozoa (641 MA), na base de bilatéria, até Brachycera (195 MA). Substituições nucleotídicas observadas foram enviesadas na região 3¿-final com raras mutações na região seed. Análises preliminares de expressão revelaram 79 miRNAs diferentemente expressos entre as espécies e os estágios de desenvolvimento investigados. Os resultados deste trabalho irão contribuir para uma melhor compreensão sobre os hábitos de parasitismo nas espécies C. hominivorax e C. macellaria, com perspectivas para estudos evolutivos e funcionais na família e no controle de insetos-praga / Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional modulators of gene expression in all eukaryotes investigated so far. The imperfect complementarity between miRNA and the target site of messenger RNA (mRNA) inhibits their translation in animals, being key genes for the control of expression in cells. The identification of miRNAs can provide a better understanding of biological processes and evolution of traits in different species. The family Calliphoridae is a group of myiasis-causing flies with different feeding habits, which includes the species Cochliomyia hominivorax (screwworm fly) and Cochliomyia macellaria (secondary screwworm). The screwworm fly is one of the major pests in the Neotropical region. Their larvae infest and feed on live tissues of warm-blooded vertebrates, resulting in severe losses for livestock industry. Differently, the close-related secondary screwworm shows a saprophagous habit, feeding and breeding on carcasses and dead tissues, being crucial for forensic entomology and public health. Because of their close evolutionary relationship and contrasting feeding habits, they represent worthy models to study the molecular basis of parasitism and feeding specialization in the family Calliphoridae. To characterize the miRNAs from both species, the small-RNA transcriptomes of adults (male and female) and larvae (third instar) were sequenced using Illumina-MiSeq next generation sequencing platform. The 6.2 million reads generated were mapped against the Drosophila melanogaster genome and screened in miRBase. We identified 84 evolutionary conserved miRNAs which 80 was founded in C. hominivorax and 78 in C. macellaria. We also investigated the presence of hairpin precursors (pre-miRNAs) in the available genomic and transcriptomic data of these species, and predicted 10 conserved pre-miRNAs and others 5 that show no similarity with previously described animal miRNAs. The evolutionary characterization of identified miRNAs showed that their sequences were highly conserved since the Nephrozoa ancestor (641 MYA) in the basis of Bilaterian clade, until Brachycera ancestor (195 MYA), with nucleotide substitutions biased to 3¿-end portion of the miRNAs with rare substitutions in the seed region. The preliminary expression profile revealed 79 differentially expressed miRNAs between species, gender and life stages, given by hierarchical clustering and statistically significant change fold analysis. The results presented here will provide new information about the genetic background of parasitic habits in C. hominivorax and C. macellaria, with prospects to functional and evolutionary studies in Calliphoridae and pest control / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Made available in DSpace on 2016-04-28T20:15:36Z (GMT). No. of bitstreams: 1 2006-Carlos Cesar Mascarenhas da Silva de Oliveira.pdf: 2632878 bytes, checksum: d43615ce0a787516f24b8ef5193ca4a6 (MD5) Previous issue date: 2006-03-14 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The objective of this study was to identify the bacterial microbiota in calf navels myiasis caused by Cochliomyia hominivorax larvae. Two places were choosed to colect the material: the Nhumirim ranch belonging to Embrapa Pantanal, Corumb?-MS and the Santo Ant?nio ranch, Miguel Pereira-RJ. There were collected 62 cotton swabs from calf navels naturally caused by C. hominivorax larvae. After that, the cotton swabs were sent to the Veterinary Bacteriology Laboratory at UFRRJ inside carriage test tubes with agar. The primary isolation used bovine blood agar and brain heart infusion (BHI) agar. After the incubation period, the isolates were evaluated by means of Gram staining, catalase testing and 30% KOH. After stained, the isolates were evaluated by their stain morphologic and color characteristics. Species of Staphylococcus spp. were streaked in selectives agar to Staphylococcus spp. for the species identication and was used coagulase test, nitrate reduction test, VP, urease production and sugar fermentation. To Streptococcus spp. was used selective agar to Streptococcus spp. species througout inoculation in metilene blue milk, esculine test and hipurate test. The specimens of the family Enterobacteriaceae used THI test, motility in tube test, indol production test, gelatine production, urease production, citrate and malonate degradation. A total of 89 colonies were isolated; 14 genera and five families were identified. The most frequent family was Enterobacteriaceae and the genera more frequent was Bacillus and Micrococcus. Pathogenic species were isolated, but no secondary infection ocurred in the calves; some antibacterial or bacteriostatic substances may be produced by larvae of C. hominivorax or bacterial microbiota of the myiasis of the navel. / O objetivo deste trabalho foi identificar a microbiota bacteriana de mi?ases umbilicais de bezerros rec?m-nascidos. Dois locais foram escolhidos para coleta de material: a Fazenda Nhumirim pertencente ? Embrapa Pantanal-MS e a Fazenda Santo Ant?nio, no munic?pio de Miguel Pereira-RJ. Amostras do exsudato de les?es por larvas de Cochliomyia hominivorax foram coletados com swabs em 62 bezerros, acondicionadas em tubos de ensaio contendo meio de transporte e encaminhadas ao Laborat?rio de Bacteriologia Veterin?ria da UFRRJ. No isolamento prim?rio foi utilizado ?gar sangue ou ?gar infus?o de c?rebro e cora??o. As col?nias isoladas foram submetidas ? colora??o pelo m?todo de Gram, teste da catalase e hidr?xido de pot?ssio a 3%. De acordo com as caracter?sticas morfotintoriais e de crescimento, os isolados foram processados para melhor identifica??o. Para a identifica??o das esp?cies de Staphylococcus spp.; as col?nias isoladas foram semeadas em meios seletivos, para observa??o dos aspectos fenot?picos caracter?sticos do g?nero. A identifica??o foi efetuada por meio do procedimento padr?o: prova da coagulase livre e ligada, provas de redu??o de nitratos, VP, produ??o de urease e fermenta??o de a??cares. A identifica??o dos isolados de Streptococcus spp foi efetuada atrav?s do repique em meio seletivo e, posteriormente, atrav?s da inocula??o em leite adicionado com azul de metileno, e das provas de hidr?lise da esculina e do hipurato.Para as amostras suspeitas de enterobact?rias, foi utilizado o meio MacConkey no isolamento prim?rio e as seguintes provas de identifica??o: comportamento em ?gar tr?plice a??car-ferro, motilidade em tubo, produ??o do indol, produ??o de ?cidos a partir da glicose, fermenta??o de a??cares, redu??o do nitrato, produ??o de gelatinase, produ??o de urease, degrada??o do citrato e do malonato. Das amostras coletadas, foram isoladas 89 col?nias, representando 14 g?neros diferentes, de cinco fam?lias . A fam?lia mais freq?ente foi a Bacillaceae; sendo os g?neros mais prevalentes o Bacillus e o Micrococcus. Esp?cies patog?nicas foram isoladas, mas infec??o bacteriana n?o foi percebida nos bezerros do experimento; indicando que alguma subst?ncia bactericida ou bacteriost?tica seja produzida pelas larvas ou por alguma bact?ria da microbiota umbilical.

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-11Bitstream added on 2014-06-13T18:56:54Z : No. of bitstreams: 1 amos_caa_me_jabo.pdf: 563456 bytes, checksum: 521b25625aa1bc8ab88bc8830fdcd90b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Três experimentos foram conduzidos, um com infestação artificial (I) e outros dois com infestação natural (II, III). No artificial (Exp. I), os animais foram distribuídos em 9 grupos, com três repetições/tratamento. Cada animal foi infestado com 50 larvas em quatro incisões cutâneas, totalizando 200/animal. As larvas L1 foram infestadas com um intervalo de três a seis horas, de 24 e de 48 horas. Os grupos receberam os seguintes tratamentos: spinosad (50 e 150 ppm) e um controle sem tratamento. Os animais foram examinados com: 15, 30, 60 minutos e 6, 24 e 48 horas. Larvas que abandonavam as lesões foram colhidas e colocadas para pupar em BOD a 25°C e 90% de UR. Os resultados demonstraram que spinosad (50 e 125 ppm) atingiu 100% de eficácia no tratamento contra os três estádios larvários de C. hominivorax. A concentração de 125 ppm alcançou este valor mais rapidamente. Nos estudos com infestações naturais (Exp. II e III), os animais após receberem incisões cutâneas foram liberados a pasto para que ocorressem infestações. Detectada a presença de larvas, o animal era alocado, por sorteio, para um dos grupos experimentais. No experimento II utilizou-se spinosad 50 e 150 ppm e no experimento III 250 e 400 ppm. Como tratamentos positivos, foram utilizados: chlorpyriphos e uma associação de chlorphenvinphos + dichlorvos. Os animais foram examinados diariamente do dia zero (D0) até dia 12 (D12). Ficou demonstrado nos experimentos com infestação natural que spinosad 50 e 150 ppm apresentaram eficácia inferior a 100%, enquanto que a 250 e 400 ppm proporcionaram 100% de eficácia decorridas 24 horas do tratamento, a concentração com 400 ppm alcançou este valor decorridos 12 horas pós tratamento. / Three experiments were conducted, the first one with an artificial infestation and the other two with natural infestation. In the first experiment (artificially infested) the animals were divided into 9 experimental groups, three repetition per treatment. Each animal was inoculated with 200 larvae in four skin incisions, two on the palette and two in the rump, each one containing 50 larvae. The placement of L1 larvae followed a range of 3 to 6 hours, 24 hours and 48 hours. The experimental groups had the following repetitions: spinosad 50 and 125 ppm, and a control group (untreated). After the treatment the animals were examined on the following period: 15, 30, 60 minutes, 6, 24 and 48 hours. Larvae which left the incision were taken for pupation on 25°C and 90% relativity humidity. The results demonstrated spinosad 50 and 125 ppm reached 100% effectiveness on the treatment of artificially caused myiasis by the three larval stages (L1, L2 and L3) of Cochliomyia hominivorax, in which the spinosad 125 ppm achieved it quicker. In the other experiments with natural infestation, after the skin incisions the animals were release on the field for the concurrency of the natural infestation. Once detected the presence of the larvae, the animals were randomly allocated in one of the experimental groups. On the experiment I were used the following concentration: spinosad 50 and 150 ppm on the experiment II spinosad 250 and 400 ppm. On both experiments were used two positives treatments: chlorpyriphos and a association of chlorphenvinphos plus dichlorvos. The animals were daily examined, from day zero (D0) to day 12 (D12).The results of the two experiments with natural infestation indicate that spinosad 50 and 150 ppm were not effective, showing effectiveness lower than 100%. However, spinosad 250 and 400 ppm reached 100% of efficacy were after 24 hours... (Complete abstract click electronic access below)

Orientador: Ana Maria Lima de Azeredo-Espin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T10:06:29Z (GMT). No. of bitstreams: 1 Carvalho_RenatoAssisde_D.pdf: 2025643 bytes, checksum: 24641039150d3a87bfa143c90c98fc64 (MD5) Previous issue date: 2010 / Resumo: A mosca-da-bicheira, Cochliomyia hominivorax, é uma das principais moscas causadoras de miíases da América do Sul, sendo responsável por severos prejuízos à pecuária através da mortalidade e redução da produtividade dos animais infestados. No Brasil, o seu controle tem sido realizado principalmente através da aplicação de inseticidas organofosforados (OF). Porém, o uso indevido e/ou prolongado desses agentes químicos pode provocar seleção de indivíduos resistentes, devido à alteração do sítio alvo ou à desintoxicação metabólica do inseticida antes de atingir o sítio alvo. Nesse sentido, o objetivo desse estudo foi investigar as bases moleculares da resistência a inseticidas OF na mosca da bicheira, através da caracterização da carboxilesterase E3, do sítio alvo acetilcolinesterase (AChE) e da expressão diferencial de enzimas metabólicas (caiboxilesterases, P450 e GST) em indivíduos resistentes. A seqüência predita da enzima carboxilesterase E3 em C. hominivorax apresentou domínios altamente conservados dentre as carboxil/colinesterases que contribuem para o mecanismo catalítico do sítio ativo. Duas mutações, já descritas em outras espécies, foram encontradas, a G137D, associada com resistência principalmente aos dietil-OPs, e a W251S, associada com resistência aos dietil-OPs e piretróides. A freqüência dessas mutações foi investigada em períodos distintos de diferentes populações do Uruguai, indicando uma correlação da freqüência de cada mutação com a classe de inseticida utilizada no controle da espécie. A região codificante da acetilcolinesterase foi sequenciada em C. hominivorax possibilitando a investigação, em populações naturais, de mutações já caracterizadas por conferir resistência a OF em outras espécies (I298V, G401A, F466Y). Apenas 2/135 indivíduos apresentaram uma das mutações (F466Y). Em contraste, a mutação G137D na carboxilesterase E3, que confere resistência metabólica aos inseticidas OF, foi encontrada em mais de 50% dos indivíduos na maioria dessas populações, indicando ser um dos principais mecanismos de resistência nessa espécie. Para a identificação de genes diferencialmente expressos em indivíduos resistentes foi gerado um banco de dados através da caracterização do transcriptoma utilizando uma das técnicas de sequenciamento de nova geração (Roche 454). Ao todo, foram obtidas 548.940 seqüências, resultando em 36.650 "contigs" e 782 "singlets" Após a anotação desses transcritos, foram selecionados genes candidatos pertencentes às famílias de enzimas de detoxificação metabólica (carboxilesterases, monooxigenases P450 e GSTs) para terem sua expressão comparada entre os indivíduos que sobreviveram ao tratamento (CL90) com o inseticida diclorvos (dimetil-OF) e um grupo controle (sem tratamento). Apenas um gene foi diferencialmente expresso, ortólogo ao CYP6GJ, e estudos futuros serão necessários para determinar a associação da expressão dessa P450 e resistência ao inseticida OF. Em ambos os grupos, o gene da AChE não se encontrou alterado. Já o gene da carboxilesterase E3 apresentou a mutação W251S em todos os indivíduos do grupo resistente (n=44), enquanto que no grupo controle (n-40) 13 indivíduos apresentaram a mutação, confirmando a associação dessa mutação com resistência aos dimetil-OP. Assim, a identificação das mutações nesse gene em populações naturais da mosca-da-bicheira pode ser uma ferramenta importante de monitoramento da resistência ao longo da atual distribuição geográfica da espécie, contribuindo para a implementação de estratégias mais efetivas de controle por meio da escolha apropriada dos produtos químicos. / Abstract: The New World Screwworm (NWS) fly Cochliomyia hominivorax is one of most important myiasis-causing flies in the Neotropics. It is responsible for severe losses to the livestock industry through both mortality and loss of productivity of infested animals. In Brazil, it has been controlled by the application of chemical insecticides, mainly the organophosphate (OP) compounds. However, the intensive use of these compounds over many years may select resistant individuals which have the potential to compromise the efficacy of current control strategies. Major mechanisms of insecticide resistance in insects involve either mutation in the target site of the insecticide, or an alteration in the rate of insecticide detoxification. Therefore, the aim of this study was to investigate the molecular basis of resistance to organophosphate insecticides in NWS throughout characterization of the carboxylesterase and acetylcholinesterase genes and expression evaluation of detoxification enzymes. The predicted amino sequence of the E3 gene showed highly conserved domains within carboxyl/cholinesterases involved in the catalytic mechanism of active site. Two mutations previously described in other dipteran species were found, G137D, associated mainly with diethyl-OP hydrolysis, and W251S, associated with dimethyl-OP and pyrethroid hydrolysis. The frequency of these mutations was analyzed in different Uruguayan regions in 2003 and 2009, indicating a correlation between each mutation and the insecticide class used for NWS control. The entire coding sequence of acetylcholinesterase was sequenced allowing surveying of mutations previously known for conferring insecticide resistance (I298V, G401A, F466Y). Only 2/135 individuals from NWS natural populations showed one of these mutations (F466Y). In contrast, G137D mutation in carboxylesterase E3. that also confers resistance to OP insecticides, was found in a high frequency in the same populations, suggesting this is one of the most important resistance mechanisms and that metabolic resistance has been preferentially selected rather altered target site in this species. Differentially expressed genes in resistant NWS individuals were analyzed throughout candidate gene expression evaluation. For this, NWS transcriptome was sampled by deep sequencing of polyadenilated transcripts using 454 sequencing technology, which generated a total of 548,940 sequences resulting in 37,432 unigenes (36,650 contigs and 782 singlets). Following functional annotation, gene expression of candidate genes belonging to detoxification enzyme families (carboxylesterases, monooxygenases P450 and GSTs) were evaluated in NWS resistant individuals surviving bioassays (1X90) with the active ingredient dichlorvos (dimethyl-OP) and from a control group (without treatment). No genes over expressed were found in the resistant group, and the ortholog to CYP6G1 was down-regulated in this group, requiring further studies to determine the association between reduced expression of a P450 gene and OP resistance. In both groups the target site was not altered. In contrast, E3 gene showed W251S mutation in all resistant individuals (44), while 13 individuals showed such mutation in the control group (40). This result corroborates the association between this mutation and the dimethyl-OP resistance in this species. Therefore, identification of mutations in carboxylesterase gene in NWS natural populations can be an important tool in monitoring insecticide resistance. The selection of appropriate chemicals for NWS control may contribute to implement more effective control strategies. / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Cochliomyia macellaria (Fabricius) has great importance in forensics and is commonly utilized in estimating the period of insect activity (PIA) on a corpse due to its rapid colonization time of fresh remains, active oviposition during daylight, and its abundance throughout the southern United States. The purpose of this study is to determine what effects tissue and temperature will have on C. macellaria. The purpose of my secondary studies on attraction to larval resource is to gain insight into the behavior of adult blow flies in order to determine what factors drive colonization of remains. Forensic entomology can use development data of calliphorid flies to estimate their PIA on human remains. C. macellaria has major implications in medico-legal entomology as well as in veterinary entomology and are often found on human remains in Texas during the warm months of the year. Furthermore, C.macellaria is a secondary myiasis producer; this means that its larvae may be found infesting living animal or human muscle tissue and potentially could be involved in veterinary forensic cases. This study will help provide better insight on the behavior of C. macellaria in Texas as it relates to tissue type and temperature. In this experiment larvae were reared at three separate temperatures (21°C, 24°C, and 27°C) on either equine or porcine striated muscle tissues. Eggs were inoculated onto each tissue and monitored every hour for hatch. Once hatch occurred, observations were shifted to every twelve hours. Three larvae were sampled during each observation period. Sampled larvae were weighed and length was recorded. Additionally, stage of development was determined. Pupae were collected and time to adult emergence recorded. Furthermore, life-history traits, such as emergence pattern and adult longevity, were recorded. This study is the first in Texas and second in the United States to examine the development of C. macellaria and could provide significant information for cases of myiasis and neglect of both humans and animals. In contrast, rearing flies on beef liver or pork chops, which tends to be a standard procedure often employed may not be applicable to myiasis cases of equines. Data from this study could provide greater insight to developmental differences of forensically important blow flies on striated muscle from different vertebrate species. Since porcine tissue was used in the Florida study, it was also utilized in our study in order to allow a comparison. To date there is only one data set on the development of C. macellaria in the United States. Fly populations in different climates are suspected to have different development rates. This study will compare data from central Texas to data generated in Florida. If variation of development rates is demonstrated for different populations, then forensic entomologists should use data that are more conducive to their given geographic area. My secondary objective first looked at whether larval resource had an attraction effect on the subsequent adult C. macellaria flies. One cohort of larvae was reared on bovine testicles, while another was reared on bovine liver. The subsequent adults were then tested in a dual-choice olfactometer which possessed testicles at one end and liver at the other end. The second set of experiments also involved using a dual-choice olfactometer; however, all larvae were reared on bovine liver and fresh liver was placed at one end of the olfactometer, while fresh liver with conspecific larvae was placed at the end of the other. The purpose of this experiment was to assess whether adult C. macellaria flies were more attracted to the presence of conspecific larvae. These experiments will give us more insignt into adult blow fly behavior and help us to understand what factors drive colonization of remains.

Indiana University-Purdue University Indianapolis (IUPUI) / Development rate is a quantitative trait that displays significant variation within many species, including Cochliomyia macellaria Meigen (Diptera: Calliphoridae). Calliphorids are a family of dipterans known as blow flies and are commonly used in forensic entomology to estimate the minimum postmortem interval (PMIMIN), given some assumptions are made. In order to dissect the genetic underpinnings of development rate variation in this species, artificial selection for fast and slow development with population-based resequencing was used. The objective of this study is to isolate and characterize genomic regions that are correlated to development rate variation in blow flies. The first approach used known regulatory development genes from Drosophila melanogaster Meigen (Diptera: Drosophilidae) and isolated variants that were associated with development time changes in artificially selected fast and slow development C. macellaria strains. Three variants located in Ras and Eip74EF were associated with fast or slow development in selection strains with a significant change in allele frequency. The second approach involved the comparison of pooled artificially selected fast and slow C. macellaria genomes to investigate the genetic basis of development rate variation. When comparing the fast and slow genomes, 699 sequences were identified that contained 7290 variants with consistent changes in allele frequency. The variants indicated that the genomic regions that are associated with development rate were associated with developmental processes, including regulation of RNA polymerase II activity, and transporter activity, such as protein dimerization. Of the 699 sequences, 69 sequences were related to Achaete-scute complex and 14 were related to Cyp12A7. The identification of the genomic regions that associated with development rates from this study provides an important resource for future studies in identifying potential genetic markers to increase the effectiveness of PMIMIN estimates. By using significantly associated variants as a priori candidates for future studies, the data increases the understanding of natural development variation in blow flies.

Orientadores: Ana Maria Lima de Azeredo Espin, Pablo Fresia Coronel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T09:56:03Z (GMT). No. of bitstreams: 1 Bergamo_LuanaWalravens_M.pdf: 4822096 bytes, checksum: 6731d49afdda09d9b33086d944c30301 (MD5) Previous issue date: 2014 / Resumo: A pecuária é uma importante atividade econômica do Brasil, mas tem sofrido perdas significativas devido ao impacto de parasitas. Neste cenário destaca-se a mosca-da-bicheira, Cochliomyia hominivorax, que é um importante ectoparasita causador de miíase primária e endêmico das Américas. Sua distribuição geográfica sofreu redução a partir da implementação da técnica do inseto estéril (SIT), sendo considerada erradicada nos Estados Unidos e países continentais da América Central. Na América do Sul, o controle desta espécie é realizado através de inseticidas, cujo uso indiscriminado pode acarretar na seleção de indivíduos resistentes. Em estudos anteriores, as mutações denominadas Gly137Asp e Trp251Leu foram observadas no sítio ativo da enzima carboxilesterase E3 e associadas à resistência a inseticidas dietil e dimetil-organofosforados, respectivamente. A caracterização molecular da região desse gene que compreende desde o final do éxon 2 até o final do éxon 4 para C. hominivorax revelou que o íntron I2 apresenta um tamanho maior que em outras espécies de Muscomorpha. A análise da composição nucleotídica e comparações por métodos estatísticos entre modelos de mutação-seleção de sequências do cDNA da carboxilesterase E3 de C. hominivorax e outros Muscomorpha mostraram sinais de seleção restritiva sobre as substituições sinônimas. Porém, o padrão observado não é exclusivo deste gene, sendo observado em outras regiões do transcriptoma. As pressões seletivas que modelaram a evolução do gene E3 do ponto de vista das mutações não-sinônimas foram investigadas a partir de uma estratégia hierárquica, considerando dados interespecíficos e populacionais. Primeiramente, na investigação de resposta à longo prazo, foram utilizadas as sequências dos éxons das espécies C. hominivorax, Cochliomyia macellaria, Chrysomya megacephala e sequências públicas de outros Muscomorpha. Os testes branch-site relaxado e branch-site estrito, em conjunto com o método Bayes Empirical Bayes (BEB), não indicaram sítios sob seleção positiva no ramo de C. hominivorax. A análise pelo método MM01, que leva em conta as propriedades físico-químicas dos aminoácidos, também não detectou nenhum sinal de seleção. Já na investigação com base em dados populacionais, amostras de C. hominivorax de 21 localidades da América do Sul foram sequenciadas para um fragmento do gene E3, previamente caracterizado. Os resultados da AMOVA e Fst-par-a-par indicam que há estruturação entre as localidades quando consideradas as sequências do gene E3 juntamente com sequências dos genes mitocondriais CR, COI e COII. Porém, os resultados da SAMOVA mostraram uma baixa correlação entre os dados genéticos e geográficos, indicando que esta espécie apresenta uma complexa estrutura populacional. Através do DAPC foi possível distinguir três grupos genéticos entre as localidades. Testes de desequilíbrio de ligação foram significativos entre as duas mutações que são relacionadas à resistência a inseticidas organofosforados, indicando uma associação negativa entre elas. Uma análise baseada na detecção de locos outliers recuperou um dos sítios do primeiro códon associado à resistência com sinal de seleção positiva / Abstract: Livestock production is an important economic activity in Brazil, but has been suffering significant losses due to the impact of parasites. The New World screwworm fly (NWS), Cochliomyia hominivorax, is an important ectoparasite and myiasis causing fly endemic from the Americas, which stands out in this scenario. The geographic distribution of NWS has been reduced after the implementation of the sterile insect technique (SIT), being considered eradicated in North and part of Central America. In South America, NWS is controlled by chemical insecticides, which indiscriminate use can cause the selection of resistant individuals. The Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 have been associated to resistance of diethyl and dimethyl organophosphates, respectively. The molecular characterization of this gene region comprising from the end of exon 2 to the end of exon 4 for C. hominivorax revealed that the intron I2 has a larger size than in other Muscomorpha species. The analysis of nucleotide composition and the comparisons between mutation-selection models by statistical methods of cDNA sequences of carboxylesterase E3 from C. hominivorax and other Muscomorpha showed signs of restrictive selection on synonymous substitutions. However, the observed pattern is not exclusive for this gene, being observed in other regions of the transcriptome. The selective pressures that have shaped E3 gene evolution from the point of view of non-synonymous mutations were investigated from a hierarchical strategy, considering interspecific and populational data. At first, in the investigation of long term response, the sequences of exons of C. hominivorax, Cochliomyia macellaria, Chrysomya megacephala and public sequences from other Muscomorpha were used. The relaxed branch-site and strict branch-site tests, together with the Bayes Empirical Bayes (BEB) method, indicated no sites under positive selection in the C. hominivorax branch. The analysis by MM01 method, which takes into account the physicochemical properties of amino acids, also detected no sign of selection. Secondly, in the investigation based on populational data, samples of C. hominivorax from 21 sites in South America were sequenced for a fragment of E3 gene previously characterized. The results of AMOVA and pairwise Fst indicate that there is structure between sites when considering sequences of E3 gene and CR, COI and COII mitochondrial genes. However, SAMOVA results showed a low correlation between genetic and geographic data, indicating that this specie has a complex population structure. Three genetic groups were distinguished between the sites by DAPC. Linkage disequilibrium tests were significant between the two mutations related to organophosphate insecticides resistance, indicating a negative association between them. An analysis based on outlier loci detection recovered sign of positive selection in one of the sites from the first codon associated with resistance / Mestrado / Genetica Animal e Evolução / Mestra em Genética e Biologia Molecular

Orientador: Ana Maria Lima de Azeredo-Espin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T11:05:22Z (GMT). No. of bitstreams: 1 Silva_NormaMachadoda_D.pdf: 6129934 bytes, checksum: 5d8a07895afd2e470c9fd5caf8ac9fb8 (MD5) Previous issue date: 2009 / Resumo: Cochliomyia hominivorax é considerada uma importante praga por causar perdas econômicas para a pecuária na região Neotropical. Este ectoparasita tem sido controlado principalmente por inseticidas, entretanto, este método normalmente resulta na seleção de indivíduos resistentes. Nesta tese foram investigados mecanismos associados com resistência a inseticidas organofosforados e piretróides. Os resultados apresentados nesta tese estão divididos em dois artigos. No artigo 1 foram investigados dois mecanismos de resistência a piretróides, um conhecido como kdr (knockdown resistance), associado à mutações no canal de sódio, e a mutação W251S no gene da carboxilesterase E3, a qual têm sido associada à hidrólise de piretróides. Foram investigadas populações da atual distribuição geográfica de C. hominivorax através de PCR-RFLP. Para a mutação kdr (L1014F) nenhum indivíduo mutante foi encontrado. Entretanto, a mutação W251S foi encontrada em todas as populações, com as mais altas freqüências nas amostras da Venezuela (100%) e Colômbia (93.75%). Em algumas populações amostradas, apesar da freqüência do alelo mutante ter sido relativamente baixa, o número de indivíduos portadores de pelo menos um alelo mutante foi alto. No bioensaio com cipermetrina (piretróide tipo II), utilizando amostras de Estiva (MG), os resultados analisados através do teste exato de Fisher indicaram que para a mais baixa concentração (p=0.0003) e para uma concentração intermediária (p= 0.024), a presença da mutação W251S está correlacionada com as chances de sobrevivência. Entretanto, na mais alta concentração (p= 0.221) a presença desta mutação não foi correlacionada com a sobrevivência, o que possivelmente indica dificuldade em hidrolisar este tipo de piretróide em concentrações maiores. No Artigo 2, as mutações G137D no gene da carboxilesterase E3, e as mutações I298V, G401A e F466Y no gene da acetilcolinesterase, associadas com resistência a organofosforados, foram investigadas nas mesmas populações do artigo 1, através de PCR-RFLP e sequenciamento direto de PCR. A região codificante completa da acetilcolinesterase também foi caracterizada e proposto um modelo para a estrutura tridimensional desta proteína com base na estrutura da acetilcolinesterase de Drosophila melanogaster. Possíveis alterações na proteína causadas pelas mutações associadas com resistência a organofosforados foram discutidas. No gene da acetilcolinesterase somente 2 de 135 indivíduos analisados apresentaram uma das mutações investigadas, a F466Y, o que pode estar associado ao alto custo no valor adaptativo do indivíduo, causado por algumas mutações neste gene ou inexistência dos demais alelos mutantes nestas populações. Entretanto, para a mutação G137D no gene da E3 foi encontrado um alto número de indivíduos portadores de pelo menos um alelo mutante na maioria das populações brasileiras e no Uruguai. Em Cuba, Venezuela e Colômbia nenhum indivíduo mutante para G137D foi encontrado, o que pode estar relacionado com os inseticidas usados no controle de C. hominivorax nestas regiões ou com a inexistência do alelo mutante nestas localidades. Os resultados de ambos os trabalhos indicam que mutações no gene da carboxilesterase E3 são um dos principais mecanismos de resistência selecionados nesta espécie, dentre os investigados até o momento. Este estudo representa um importante avanço no entendimento da base molecular da resistência a inseticidas em um importante ectoparasita. / Abstract: Cochliomyia hominivorax is an important ectoparasite, causing considerable economic losses to livestock sector in Neotropical region. This ectoparasite has been controlled mainly by applying insecticides, however, this method usually results in the selection of resistant individuals. In this thesis, mechanisms associated with organophosphate and pyrethroid insecticide resistance were investigated. The results presented in this thesis are divided in two articles. In the Article 1 two mechanisms associated with pyrethroid resistance were investigated, the kdr (knockdown resistance), a generic term for mutations in the sodium channel, and a mutation in the residue 251 of the carboxylesterase E3 gene, which have been associated with pyrethroid hydrolysis. Populations of actual geographical distribution of C. hominivorax were investigated through PCR-RFLP. For the kdr mutation, no mutant individuals were found. However, the W251S mutation was found in all populations investigated, with the highest frequencies in the samples from Colombia (100%) and Venezuela (93.75%). In some populations, despite relatively low mutant allele frequency, the number of individuals having at least one mutant allele was high. A bioassay with cypermethrin (pirethroid type II) was also performed, using samples from Estiva (MG). The results of the Fisher's exact test for the lowest concentration (p=0.0003, a= 0.05) and for an intermediate concentration (p= 0.024, a= 0.05) indicated that the presence of W251S mutation are correlated with the survival. However, at the highest concentration, Fisher's exact test (p= 0.221, a= 0.05) indicated that the presence of this mutation was not correlated with survival. These results indicate that the W251S mutation in C. hominivorax probably presents difficulties in hydrolyzing of this kind of pyrethroid, in high concentrations. In the Article 2, the G137D mutation, in the carboxylesterase E3 gene, and the I298V, G401A and F466Y mutations in the acetylcholinesterase gene, associated with organophosphate resistance, were investigated. The same populations of article 1 were analyzed using PCR-RFLP and PCR direct sequencing. The complete coding region of acetylcholineserase also was characterized and a putative model for the three-dimensional structure of this protein was proposed, based on solved structure of Drosophila melanogaster. Possible alterations in the protein, caused by mutations associated with organophosphate resistance, were discussed. For the acetylcholinesterase gene, only 2 of 135 individuals analyzed presented one of the mutations investigated, the F466Y. This fact may be related with the fitness cost of some mutations in this gene, or the inexistence of mutant alleles in these populations. For the G137D mutation, a high number of individuals having at least one mutant allele were found in most of the Brazilian populations and in Uruguay. In Cuba, Venezuela and Colombia no G137D mutant was found, a finding that may be related to the insecticides used for C. hominivorax control in these regions. The results of these two works indicate that mutations in the carboxylesterase E3 gene are one of the main insecticide resistance mechanisms selected in this species, so far investigated. This study represents a significant advance in the understanding of the molecular basis of insecticide resistance in an important livestock ectoparasite. / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Resumo: Três experimentos foram conduzidos, um com infestação artificial (I) e outros dois com infestação natural (II, III). No artificial (Exp. I), os animais foram distribuídos em 9 grupos, com três repetições/tratamento. Cada animal foi infestado com 50 larvas em quatro incisões cutâneas, totalizando 200/animal. As larvas L1 foram infestadas com um intervalo de três a seis horas, de 24 e de 48 horas. Os grupos receberam os seguintes tratamentos: spinosad (50 e 150 ppm) e um controle sem tratamento. Os animais foram examinados com: 15, 30, 60 minutos e 6, 24 e 48 horas. Larvas que abandonavam as lesões foram colhidas e colocadas para pupar em BOD a 25°C e 90% de UR. Os resultados demonstraram que spinosad (50 e 125 ppm) atingiu 100% de eficácia no tratamento contra os três estádios larvários de C. hominivorax. A concentração de 125 ppm alcançou este valor mais rapidamente. Nos estudos com infestações naturais (Exp. II e III), os animais após receberem incisões cutâneas foram liberados a pasto para que ocorressem infestações. Detectada a presença de larvas, o animal era alocado, por sorteio, para um dos grupos experimentais. No experimento II utilizou-se spinosad 50 e 150 ppm e no experimento III 250 e 400 ppm. Como tratamentos positivos, foram utilizados: chlorpyriphos e uma associação de chlorphenvinphos + dichlorvos. Os animais foram examinados diariamente do dia zero (D0) até dia 12 (D12). Ficou demonstrado nos experimentos com infestação natural que spinosad 50 e 150 ppm apresentaram eficácia inferior a 100%, enquanto que a 250 e 400 ppm proporcionaram 100% de eficácia decorridas 24 horas do tratamento, a concentração com 400 ppm alcançou este valor decorridos 12 horas pós tratamento. / Abstract: Three experiments were conducted, the first one with an artificial infestation and the other two with natural infestation. In the first experiment (artificially infested) the animals were divided into 9 experimental groups, three repetition per treatment. Each animal was inoculated with 200 larvae in four skin incisions, two on the palette and two in the rump, each one containing 50 larvae. The placement of L1 larvae followed a range of 3 to 6 hours, 24 hours and 48 hours. The experimental groups had the following repetitions: spinosad 50 and 125 ppm, and a control group (untreated). After the treatment the animals were examined on the following period: 15, 30, 60 minutes, 6, 24 and 48 hours. Larvae which left the incision were taken for pupation on 25°C and 90% relativity humidity. The results demonstrated spinosad 50 and 125 ppm reached 100% effectiveness on the treatment of artificially caused myiasis by the three larval stages (L1, L2 and L3) of Cochliomyia hominivorax, in which the spinosad 125 ppm achieved it quicker. In the other experiments with natural infestation, after the skin incisions the animals were release on the field for the concurrency of the natural infestation. Once detected the presence of the larvae, the animals were randomly allocated in one of the experimental groups. On the experiment I were used the following concentration: spinosad 50 and 150 ppm on the experiment II spinosad 250 and 400 ppm. On both experiments were used two positives treatments: chlorpyriphos and a association of chlorphenvinphos plus dichlorvos. The animals were daily examined, from day zero (D0) to day 12 (D12).The results of the two experiments with natural infestation indicate that spinosad 50 and 150 ppm were not effective, showing effectiveness lower than 100%. However, spinosad 250 and 400 ppm reached 100% of efficacy were after 24 hours... (Complete abstract click electronic access below) / Orientador: Alvimar José da Costa / Coorientador: Gilson Pereira Oliveira / Banca: Luciano Melo de Souza / Banca: Giane Serafim da Silva / Mestre

This investigation explored the environmental and physiological factors affecting adult blow fly (Diptera: Calliphoridae) arrival and attendance at pig (Sus scrofa domesticus L.) carcasses in Brazos Co, TX in the summer and winter, and validated a new technique for estimating the pre-colonization interval. It also examined how the offspring of said blow flies compensate for adverse developmental conditions such as starvation or the presence of older competitors by determining the function of minimum viable weight, critical weight, and the terminal growth period in Cochliomyia macellaria (F.) (Diptera: Calliphoridae). Adult blow fly carcass attendance is poorly explained by temperature, wind speed, ambient light intensity, or body size for either winter or summer-active species. Time of day explained approximately 10% in carcass size variation for all four of the most common species. For summer flies, the degree of ovarian development changed significantly from 96%/98% fully developed on day 1 postmortem to 7%/2% fully developed on day 2 postmortem for C. macellaria and Chrysomya rufifacies (Macquart) iv respectively. Using the binomial distribution, the minimum postmortem interval was correctly estimated for 4/6 validation tests. Minimum viable weight for C. macellaria was found to be ~ 0.02 g, and was stable under conditions of starvation and simulated competition. Under starvation conditions, time to pupariation was not altered, whereas under simulated competition, growth rate was increased and terminal growth period shortened. Starved flies under simulated competition entered the pupal state ~12 h faster than starved flies without competition, but required ~12 longer to complete development. These effects should be considered when estimating post-colonization intervals.