Para o sucesso das estratégias de manejo de Rhipicephalus (Boophilus) microplus (carrapato bovino) são necessários testes práticos, econômicos e confiáveis que possam detectar a presença de fenótipos resistentes a drogas em suas populações. O fipronil é um acaricida de uso relativamente recente não havendo testes padronizados para o diagnóstico de resistência do carrapato à molécula. No presente trabalho, foram padronizados bioensaios in vitro para esta finalidade: Teste de Imersão de Adultas, Teste de Imersão de Larvas e Teste de Pacote com Larvas. Os testes foram aplicados e, de forma inédita, populações resistentes foram diagnosticadas tanto no Brasil quanto no Uruguai. Ensaios com inibidores enzimáticos não evidenciaram participação importante de enzimas detoxificadoras no mecanismo de resistência. Foi demonstrada reação cruzada entre fipronil e lindano, não verificada para ivermectina. Em algumas situações, foi observado interferência do controle químico de pragas agrícolas no desenvolvimento de resistência dos carrapatos. / For the success of the strategies for the management of Rhipicephalus (Boophilus) microplus (cattle tick), practical, economical and reliable tests are needed to detect the presence of drug-resistant phenotypes in their populations. Fipronil is a relatively new acaricide with no standardized tests for the diagnosis of tick resistance to this molecule. In this study, were standardized in vitro bioassays for this purpose: Adult Immersion Test, Larval Immersion Test and Larval Packet Test. The tests were applied and for the first time, resistant populations were diagnosed in Brazil and Uruguay. Tests with enzymatic inhibitors showed no significant involvement of detoxification enzymes in the mechanism of resistance. Cross-resistance was demonstrated between lindane and fipronil but not with ivermectin. In some situations, it was observed interference of the chemical control of agricultural pests in the development of resistance in ticks.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-17T19:13:24Z No. of bitstreams: 1 felipemartinsferreira.pdf: 5165550 bytes, checksum: dffda206ccbfc77d5aa53ac793323b11 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-18T11:37:40Z (GMT) No. of bitstreams: 1 felipemartinsferreira.pdf: 5165550 bytes, checksum: dffda206ccbfc77d5aa53ac793323b11 (MD5) / Made available in DSpace on 2017-05-18T11:37:40Z (GMT). No. of bitstreams: 1 felipemartinsferreira.pdf: 5165550 bytes, checksum: dffda206ccbfc77d5aa53ac793323b11 (MD5) Previous issue date: 2016-07-20 / Os carrapatos são importantes vetores de doenças em animais e humanos. O carrapato dos bovinos, Rhipicephalus (Boophilus) microplus (Canestrini, 1887) é responsável por sérios prejuízos à pecuária brasileira considerando-se os custos do controle, perda de receita devido a menor produção de leite e carne, danos ao couro, além da transmissão de doenças. O uso de plantas medicinais e de seus derivados com ação carrapaticida é considerado um excelente recurso frente a formação de resistência aos produtos químicos. Diante da necessidade de alternativas mais eficazes e com menores impactos ambientais o objetivo desse estudo foi avaliar a ação carrapaticida in vitro do eugenol, do óleo essencial e do hidrolato de Syzygium aromaticum (cravo-da-índia). Adicionalmente, também foi investigada a atividade carrapaticida do óleo essencial e do eugenol quando incorporados em uma formulação. O óleo essencial e o hidrolato de cravo-da-índia foram obtidos pelo processo de hidrodestilação dos botões florais secos da planta. A caracterização química do óleo essencial de cravo-da-índia por CG/EM revelou a presença dos componentes majoritários eugenol (83,97%), cariofileno (3,04%), humuleno (0,42%) e acetato de eugenol (12,58%). Os carrapatos provenientes de diversas regiões geográficas foram tratados com os compostos em estudo em diferentes etapas de seu ciclo. No teste de pacote de larvas foi avaliada a mortalidade com concentrações de eugenol, óleo essencial e hidrolato que variaram de 0,1 à 2,0%. O índice de produção de ovos, a eclosão larval e a eficácia carrapaticida foram avaliados através do teste de imersão de fêmeas ingurgitadas, em que foram utilizadas concentrações de eugenol e óleo essencial de 1,25 a 10%. O eugenol e o óleo essencial promoveram 100% de mortalidade em larvas a partir das concentrações de 0,25 e 0,5%, respectivamente. O hidrolato não apresentou atividade carrapaticida nas concentrações testadas. No teste de imersão de fêmeas o eugenol e o óleo essencial apresentaram resultado de eficácia superior a 95% a partir da concentração de 5,0%, inibindo completamente a eclosão larval na concentração de 10%. O uso do eugenol, substância purificada (99-100%) não proporcionou superioridade significativa nas eficácias de tratamento em relação ao óleo essencial de cravo, o que reforça o potencial do uso do óleo essencial de cravo como carrapaticida, uma vez que o mesmo é uma alternativa de fácil acesso, baixo custo e com alto rendimento no processo extrativo. A formulação desenvolvida não promoveu melhores eficácias de tratamento quando comparada a solução etanólica, o que sugere que novas pesquisas deverão ser realizadas sob a perspectiva de testar o óleo essencial de cravo em outras formulações que possam melhorar sua atividade. Além disso, testes in vivo são recomendados para validação da eficiência do ativo em condições de campo. / Ticks are important vectors of diseases in animals and humans. The cattle tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) has caused serious harm to livestock raising in Brazil, considering the costs of controlling it, revenue loss as a result of a smaller production of milk and meat, and leather damage, in addition to transmitting diseases. The use of medicinal plants and their derivatives with acaricide action is considered an alternative against the formation of chemicals resistance. Due to the need for more efficient alternatives with less environmental impact, this study aims to evaluate the acaricide action in vitro of eugenol, essential oil and hydrolate of Syzygium aromaticum (clove). Additionally, it has also investigated the acaricide activity of the essential oil and eugenol when incorporated into a formulation. The extraction of the essential oil and hydrolate was performed by steam distillation of dried flower buds of the plant. The chemical characterization of clove essential oil by GC/MS revealed the presence of the major components eugenol (83.97%), caryophyllene (3.04%), humulene (0.42%), and eugenol acetate (12.58%). The ticks from different geographical areas were treated with the test compounds in different stages of the tick cycle. In the larval packet test, mortality was assessed at concentrations of eugenol, essential oil and hydrolate ranging from 0.1 to 2.0%. The eggs production levels, the larvae hatching, and their efficiency on ticks were assessed using the adult immersion test, in which eugenol and essential oil were used at concentrations of 1.25 to 10%. The eugenol and the essential oil caused 100% mortality of larvae, starting at 0.25 and 0.5%, respectively. The test with hydrolate showed no activity at the concentrations tested. In the adult immersion test, the eugenol and the essential oil showed results greater than 95% efficacy at concentrations of 5.0%, completely inhibiting the larvae hatching at 10%. The use of eugenol, purified substance (99-100%), gave no significant superiority in efficacy of treatment with the clove essential oil, which strengthens the potential use of clove essential as an acaricide, being it a low cost and accessible alternative, with high yield in the extraction process. The formulation developed did not promote better efficacies of treatment when compared to ethanol solutions, which suggests that further research should be conducted from the perspective of testing the essential oil of cloves in other formulations that can improve their activity. Furthermore, in vivo tests are recommended for an active efficiency in field conditions validation.

Made available in DSpace on 2014-08-20T14:38:00Z (GMT). No. of bitstreams: 1 dissertacao_anibal_torres.pdf: 438835 bytes, checksum: d43cf21b0af9852e79231f87a87e06ea (MD5) Previous issue date: 2010-02-22 / The equine theileriosis, caused by Theileria equi, is an endemic disease of south of Rio Grande do Sul Brazil. There are signs that tick Rhipicephalus boophilus microplus still transmit that disease and in few time, some studies had experimental demonstrated it. The purpose of the first study of this paper was to demonstrate the tick infestation and the role of it in the transmission of the disease. In equines with contact directly with cattle, the serum incidence of the disease was 81,8% and in this group, 31,8% of this horses had tick infestation. In the equines that didn t have contact with cattle, the incidence of the disease was 12% and no tick was found in this horses. The most efficient drug in the treatment of the disease is imidocarb dipropionate. Many studies had demonstrated different doses and protocols of treatment of the disease and the toxicity of it was obvious. No study had show the sterile of T. equi with this drug, but it s a way to treat the acute disease and to control the chronic theileriosis. The acute form of the disease is seemed with fever, icteric and death. The chronic kind of it is seemed with covered coat, hyporexia, low performance in athlete horses and loss of weight. The goal of the second study of this paper was to show the toxicity and the metabolic effects of imidocarb dipropionate drug into two protocols of treatment ( two groups, 2 and 4mg/kg) in serum positive horses to theileriosis. It was seemed through hepatic and renal profile that 2mg/kg of the drug have low toxicity. Horses infected with T. equi keep with the disease for the rest of your lives, and the drug control with imidocarb dipropionate is necessary. The goal of the third study of this paper was to demonstrated, through immunologic test and clinic examination that 2mg/kg of imidocarb dipropionate monthly can control the chronic disease even in stress situations. One group received monthly, through 6 mouths, 2mg/kg of imidocarb dipropionate and the other group, once, in the beginning of it, 4mg/kg. The conclusion of this paper is that the incidence of equine theileriosis have a directly relation with the catle contact because it cause the tick Rhipicephalus Boophilus mircoplus infestation. Mensal dosis with 2mg/kg of imidocarb dipropionate is efficient in the disease control and it had demonstrated that it is not toxic for equine. / A theileriose equina, causada pelo hemoprotozoário Theileria equi, é uma doença endêmica na região da campanha do Rio Grande do Sul. Há indícios de que o carrapato Rhipicephalus Boophilus microplus transmita também a theileriose equina e, há pouco tempo, estudos demonstraram experimentalmente esta transmissão. O objetivo do primeiro trabalho foi demonstrar a infestação de carrapatos Rhipicephalus Boophilus microplus e o papel importante deste parasita na transmissão de theileriose equina. Em cavalos com contato direto com bovinos, a incidência sorológica da doença foi de 81,8%, sendo que em 31,8% destes animais se encontrou carrapatos Rhipicephalus Boophilus microplus. Nos equinos sem contato com bovinos, a incidência sorológica foi de 12% e não se encontrou carrapatos. Diversos estudos têm demonstrado a eficiência no tratamento da doença com dipropionato de imidocarb em diferentes doses, porém a toxicidade deste fármaco se manifesta em alguns animais. A forma aguda é caracterizada por febre, icterícia e morte. A forma crônica é descrita por pêlo arrepiado, hiporexia, queda no desempenho em animais atletas e perda de peso. O objetivo do segundo trabalho foi demonstrar a toxicidade e os efeitos metabólicos do dipropionato de imidocarb em duas doses terapêuticas (2 e 4mg/kg). Foi sugerido, através do perfil hepático e renal, após a administração da droga, que 2mg/kg deste fármaco tem uma toxicidade leve e temporária no período de metabolização da droga. Uma vez infectados com a T. equi, os equinos permanecem a vida toda positivos para a doença. Com isso o controle da enfermidade depende da utilização do dipropionato de imidocarb. O objetivo do terceiro trabalho foi demonstrar, através do teste de imunofluorescência indireta e exame clínico, que doses mensais de dipropionato de imidocarb á 2 mg/kg são eficazes para se manter a parasitemia e a clínica da doença controladas, mesmo que os animais sejam submetidos á situações de estresse. Um grupo recebeu mensalmente, por seis meses, 2mg/kg de dipropionato de imidocarb e o outro grupo, 4mg/kg apenas uma vez no primeiro mês. A conclusão desta dissertação é que a incidência da theileriose equina tem relação direta com a convivência com bovinos por facilitar a infestação destes com o carrapato Rhipicephalus Boophilus microplus; e que, doses mensais de dipropionato de imidocarb, à 2mg/kg, são eficazes no controle da parasitemia da doença mesmo em situações de estresse e não se demonstram tòxicas para os equinos.

Made available in DSpace on 2014-08-20T14:38:04Z (GMT). No. of bitstreams: 1 tese_felipe_geraldo_pappen.pdf: 1221798 bytes, checksum: 72f7cef0d47255d268dc994513126704 (MD5) Previous issue date: 2011-09-21 / Practices of Rhipicephalus (Boophilus) microplus control are problematic in many livestock production systems and have worldwide increased. In the south region of Rio Grande do Sul State, Brazil, where beef cattle are breed, dip baths are the main option for acaricide treatments. Thus, the aim of this study consist in: (i) to know the epidemiological aspects of dip baths use for R. (B.) microplus control in south of Brazil, (ii) to evaluate chemical sensibility of R. (B.) microplus in beef farms with cattle dip baths, (iii) to evaluate dip baths structure and maintenance conditions, and (iiii) to evaluate tick control handlings procedures. An epidemiologic inquiry was applied to 110 beef farmers holding plunge cattle dips, which were located in eight tows in the south region. The obtained results showed that dip baths were unable on 27.3% (30/110) of the properties,mainly due to the presence of resistance to most of the acaricide classes (40% - 12/30). Dip baths have been active in 72.7% (80/110) and amitraz is the most used acaricide in 82.5% (66/80) of these farms, followed by associations of pyrethroids and organophosphates in 16.2% (13/80), and nonassociated pyrethroids in 1.3% (1/80) of properties. Suitable number of engorged female ticks to laboratory sensibility test were obtained in 17.5% (14/80) of farms, demonstrating that 42.9% (6/14) of tick populations were sensible to amitraz, while 85.7% (12/14) were susceptible to associations, and none was sensible to pyrethroids. Moreover, in vitro tests revealed that dip baths solutions were only efficient in 35.7% (5/14) of them. Although the dip baths presents a mean age of 35 years, most farmers have no claims of physical infrastructure issues. The main criterion for the decision of the immersion baths application is the visualization of the immature ticks on cattle in 66% (53/80) of the properties and five or more applications per year were performed in 65% (52/80) of them. Moreover, the simultaneous use of other acaricides on injectable forms and pour-on, and the extension of the parasitism period to the winter months suggested that tick control in the region is reason for concern. It was also concluded that pyrethroids have been replaced by amitraz in last years, and nowdays this acaricide is being replaced by associations of pyrethroids with organophosphates, a viable alternative for most of the farms according to the in vitro tests. / O controle do carrapato Rhipicephalus (Boophilus) microplus é problemático em vários sistemas de criação, estando disseminados os casos de resistência por todo o mundo. A situação também é preocupante na região sul do Rio Grande do Sul, que por sua vez utiliza o banheiro de imersão como principal método de aplicação de acaricidas em propriedades de criação de bovinos de corte. Assim, o presente trabalho teve os seguintes objetivos: (i) conhecer os aspectos relacionados à utilização dos banheiros de imersão para o controle do carrapato R. (B.) microplus; (ii) avaliar a sensibilidade de R. (B.) microplus destas propriedades; (iii) avaliar as características e condições de estrutura física dos banheiros de imersão; e (iiii) avaliar itens de manejo relativos ao controle do carrapato. Para tanto, um questionário epidemiológico foi aplicado em 110 propriedades de criação de bovinos de corte com banheiro de imersão, distribuídos em oito municípios da região de estudo. Constatou-se que em 27,3% (30/110) delas, o banheiro carrapaticida encontra-se desativado, principalmente em virtude do surgimento de resistência aos princípios ativos disponíveis (40% - 12/30). Naquelas em que o banheiro de imersão está carregado (72,7% - 80/110), o amitraz é o ingrediente ativo mais utilizado (82,5% - 66/80), seguido das associações de piretróides com organofosforados (16,2% - 13/80) e dos piretróides não associados (1,3% - 1/80). Obteve-se número suficiente de teleóginas para a avaliação de sensibilidade em laboratório em 17,5% (14/80) das propriedades, constatando-se que 42,9% (6/14) das populações eram sensíveis ao amitraz, 85,7% (12/14) às associações de piretróides com organofosforados, e, nenhuma aos piretróides não associados. Os testes in vitro também revelaram que as caldas utilizadas eram eficazes em apenas 35,7% (5/14) dos casos. Embora os banheiros de imersão da região possuam, em média, 35 anos de existência, e alguns problemas conservação tenham sido constatados, a maioria dos proprietários alega possuir estrutura física adequada. O critério para a realização dos banhos é a visualização de formas imaturas do carrapato sobre o bovino em 66% (53/80) das propriedades e são realizadas cinco ou mais aplicações em 65% (52/80) delas. Além disso, a utilização simultânea de outros ingredientes ativos nas formas injetável e pour-on, e a extensão do período de parasitismo aos meses de inverno sugerem que a situação do controle do carrapato na região está se agravando. Também conclui-se que os piretróides foram ao longo dos últimos anos substituídos pelo amitraz, que por sua vez vem dando lugar às associações de piretróides com organosfosforados, uma alternativa viável para a maioria das propriedades segundo os testes in vitro.

Orientador: Sergio Daishi Sasaki / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2013

Orientador: Adjair Antonio do Nascimento / Banca: Gilson Pereira de Oliveira / Banca: Júlio Mendes / Resumo: O presente trabalho procurou verificar a possível interferência do pH do diluidor na eficiência de ixodicidas comerciais. Para tanto, utilizou-se amitraz, clorpirifós, cipermetrina, mais um grupo controle, para verificar os possíveis efeitos de soluções tampão com pH 3, 4, 5, 6, 7, 8, 9, 10 e 11, como diluidores dos ixodicidas, sobre as variáveis: peso médio da oviposição (PMO), relação PMOxPT (peso das teleóginas), porcentagem de eclosão (PE), eficiência reprodutiva (ER) e eficácia da calda (EC). Teleóginas de R. (B.) microplus, obtidas de bovinos infestados com cepa de campo, foram submetidas à biocarrapaticidogramas, uma hora e 24 horas após preparo (HAP) das caldas. O pH do diluente pouco influenciou na eficácia do amitraz nos parâmetros avaliados, embora as linhas de tendência dos resultados para ER e EC tenham mostrado menor ação do ixodicida, à medida que o pH do diluidor aumentava, tanto para uma HAP como para 24 HAP. Para o clorpirifós, os resultados obtidos mostraram diferenças significativas no PMO, PMOxPT e PE nos resultados de uma hora. A ER (uma HAP) e EC (24 HAP) foram influenciadas negativamente pelo pH quando este se elevava. As linhas de tendência dos resultados para ER e EC mostraram diminuição da ação à medida que o pH do diluidor aumentava, tanto para uma hora como para 24 horas. A cipermetrina sofreu pouca influência do pH. Entretanto, a linha de tendência dos resultados para EC foi influenciada pela elevação do pH, quando usada 24 HAP. / Abstract: The objective of the present research is to check, with commercial ixodicides, the possible interference of the pH of the thinner. For this amitraz, chlorpyrifos, cypermethrin, plus a control group, was used to verify the possible effects of buffer solutions with pH 3, 4, 5, 6, 7, 8, 9, 10 and 11, as thinners of the ixodicides, on the following: medium weight of egg mass (PMO), relationship PMOxPT (weight of the teleogines), percentage of larvae hatch (PE), estimated reproduction (ER) and % control (EC). Teleogines of R. (B.) microplus, obtained from cattle infested with field's strains have been submitted to the in vitro immersion tests, one hour and 24 hours after the solution has been prepared (HAP). The pH of the thinners made little difference on the amitraz on the parameters assessed and, statistically, did not show any significant differences, although the tendency of the results for ER and EC showed less action of ixodicide, as the pH of the thinner increased, as much for one HAP as for 24 HAP. As for chlorpyrifos, the results obtained, statistically showed significant differences on the PMO, PMOxPT and PE in the results of one hour. The ER (one HAP) and EC (24 HAP), were negatively affected by pH when this was augmented. The tendency of the results for ER and EC showed a diminution from the action, as the pH from the thinner augmented, as much for one HAP as for 24 HAP. The cypermetrin suffered less influence from pH. However, the tendency of the results for EC were affected by the elevation of pH, when used 24 HAP. / Mestre

Made available in DSpace on 2016-04-28T20:15:27Z (GMT). No. of bitstreams: 1 2007-Isabele da Costa Angelo.pdf: 1629986 bytes, checksum: 663b35706e830a57e185c3e0a68096c9 (MD5) Previous issue date: 2007-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Boophilus microplus is an ectoparasite which causes great economic losses around the world. The exclusive use of acaricides and the inadequate management have conducted the development of resistance in ticks populations, environmental and food contamination by acaricides and their residues. The use of entomopathogenic fungi to arthropods control has shown interesting responses. Several studies have proved the pathogenicity of fungi toward various ticks species. The aim of the present study was to evaluate the effect of Isaria farinosa, I. fumosorosea, Paecilomyces lilacinus and Lecanicillium lecanii toward engorged females, eggs and larvae of B. microplus tick. The isolates were cultured on malt extract medium at 25 ? 1?C and 80% of relative humidity for 15 days. Conidial suspensions were prepared in Tween 80 water solution (0.1% v/v). There were 4 treatment groups according to the following conidial concentrations: 105, 106, 107 and 108 conidia mL-1. The control group was made up of water and Tween 80 only (0.1% v/v). Treatment was based on immersion of the specimen in 1 mL of the conidial suspension. Each treatment group was made up of 10 repetitions. Changes in biology of engorged females, eggs viability and larvae mortality, were observed every 5 days up to the 20th day after treatment. The results have shown that L. lecanii changed biological parameters in engorged females through the reduction in posture period, nutritional rate, eggs production rate, and increase of incubation period. Isaria farinosa has shown reduction in posture period and in nutritional rate. Paecilomyces lilacinus was the unique isolate able to reduce the eclosion period of larvae from infected females. Isaria fumosorosea has reduced the nutritional rate and was the unique isolate that changed significantly in eclosion period when eggs were infected. Conidial concentrations reduced reproduction capacity of engorged females. The isolates of I. fumosorosea and L. lecanii have presented major potential to control B. microplus engorged females. All tested isolates have shown pathogenicity toward unfed larvae of B. microplus after in vitro infection. / Boophilus microplus ? um ectoparasito que causa grandes perdas na pecu?ria mundial. A utiliza??o exclusiva de produtos qu?micos no controle de carrapatos, associada ao manejo inadequado tem conduzido o desenvolvimento de popula??es de carrapatos resistentes, e a contamina??o dos produtos de origem animal e do ambiente pelos seus res?duos. O uso de fungos entomopatog?nicos no controle de artr?podes tem se tornado uma abordagem cada vez mais atrativa. In?meros trabalhos comprovam experimentalmente a patogenicidade dos fungos sobre diversas esp?cies de carrapatos. O objetivo do presente trabalho foi avaliar o efeito in vitro de Isaria farinosa, I. fumosorosea, Paecilomyces lilacinus e Lecanicillium lecanii sobre f?meas ingurgitadas, ovos e larvas de B. microplus. Os fungos foram repicados em meio de cultura extrato de malte, e mantidos em c?mara climatizada sob temperatura de 25?C ? 1 e umidade relativa de 80% por quinze dias. Suspens?es conidiais foram preparadas a partir do crescimento do fungo, cujos con?dios foram adicionados a solu??o de ?gua destilada e Tween 0,1%. As concentra??es 105, 106, 107 e 108 con?dios mL-1 formaram os grupos tratamento, juntamente com o grupo controle, constitu?do por ?gua destilada est?ril e Tween 0,1%. O tratamento constituiu-se de um mililitro da concentra??o conidial testada, e cada grupo foi formado por 10 repeti??es. Os par?metros de avalia??o observados para demonstrar o efeito dos fungos sobre o carrapato foram as altera??es biol?gicas de f?meas ingurgitadas, viabilidade de ovos tratados e percentual de mortalidade de larvas, acompanhado a cada cinco dias at? o 20? dia ap?s infec??o. Os resultados mostraram que L. lecanii causou altera??es nos par?metros biol?gicos de f?meas ingurgitadas, diminuindo o per?odo de postura, o ?ndice nutricional, o ?ndice de produ??o de ovos, e aumentando o per?odo de incuba??o. Isaria farinosa mostrou redu??o no per?odo da postura e no ?ndice nutricional de f?meas ingurgitadas. P. lilacinus foi o ?nico fungo capaz de reduzir o per?odo de eclos?o das larvas provenientes da infec??o de f?meas ingurgitadas. I. fumosorosea reduziu o ?ndice nutricional e foi o ?nico isolado que causou altera??o significativa no per?odo de eclos?o de larvas provenientes da infec??o de ovos. Diferentes concentra??es dos entomomopat?genos reduziram o potencial reprodutivo das f?meas ingurgitadas dos grupos tratados. Os isolados de I. fumosorosea e L. lecanii apresentaram maior potencial de controle para f?meas ingurgitadas de B. microplus. Todos os isolados testados mostraram patogenicidade para larvas n?o alimentadas de B. microplus ap?s infec??o in vitro.

Made available in DSpace on 2016-04-28T20:16:21Z (GMT). No. of bitstreams: 1 2007-Everton Kort Kamp Fernandes.pdf: 2844359 bytes, checksum: a56e9f806dc8f1f0e7cc6b4641b4a78c (MD5) Previous issue date: 2007-03-30 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Beauveria bassiana is an entomopathogenic fungus used worldwide as a biological control agent of pest arthropods. The present study aimed at characterizing several isolates of B. bassiana and selects those with major potential to control ticks population and other arthropods under natural conditions. Thus, 53 isolates of B. bassiana and 7 isolates including 5 other Beauveria spp. and 1 Engyodontium albus (=Beauveria alba) were investigated. These isolates were originally from different regions of Brazil and other nations, and different hosts or substrate. Initially, all the isolates were morphologically analyzed and investigated about the potential of conidial production. Afterwards, experiments were conducted to investigate the genetic diversity of Brazilian isolates of B. bassiana, to evaluate the spatial distribution of genotypes, and to understand the genetic relationship among isolates of different species of Beauveria. Thus, all isolates were analyzed by isozymes electrophoresis, AFLP-PCR and sequencing of ITS1 and ITS2 regions. The results have permitted the taxonomic identification of isolates and the selection of those with major potential to conidial production. The results have also showed the detection of high genetic variability among B. bassiana isolates, indicating that this fungus represents a species aggregate. Furthermore, the genetic distance among isolates was directly related to the geographic distances. In addition, the isolates were evaluated to UV-B tolerance, heat tolerance, and cold activity. Heat and cold are two abiotic factors of the environment that restrict the use of entomopathogenic fungi as agents for biological control of insects. In addition, solar radiation, particularly the UV-B wavelength, is a major environmental factor that can negatively affect this entomopathogenic fungus in the field. High variability in UV-B tolerance was detected among the isolates after 2 h of radiation exposure. Conidial relative germination ranged from 0% to almost 80%, and a delayed germination was detected. Moreover, isolates of B. bassiana originating from lower latitudes tended to have lower UV-B tolerance than isolates from higher latitudes. A high variability in thermotolerance was also observed among the isolates after 2 h of heat exposure at 45 ?C, ranging from low, to medium, to high viability. The thermal death point of most resistant isolates was between 44 ?C and 45 ?C after 6 h of heat exposure. At cold temperatures most of the isolates grew at 5 ?C. An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that isolates from higher latitudes were more cold-active than isolates from nearer the equator. The present study also evaluated the virulence of the isolates toward Boophilus microplus larvae, and virulence ranged from very low to very high. It was observed that different populations of B. microplus may present different susceptibility to B. bassiana infection. Therefore, the present study has selected isolates of this entomopathogenic fungus with major effective potential to control B. microplus and other pest arthropods under environmental conditions. / Beauveria bassiana ? um fungo entomopatog?nico utilizado em v?rias partes do mundo como agente de controle biol?gico de artr?podes. Baseado nisso, o presente estudo buscou caracterizar diversos isolados de B. bassiana e selecionar aqueles com maior potencial para controlar popula??es de carrapatos e outros artr?podes em condi??es naturais. Neste sentido, 53 isolados de B. bassiana e mais sete isolados incluindo cinco esp?cies de Beauveria spp. e uma de Engyodontium albus (=Beauveria alba) foram estudadas. Esses isolados foram oriundos de v?rias regi?es geogr?ficas brasileiras e estrangeiras, e de diferentes hospedeiros ou substratos. Os isolados foram inicialmente avaliados morfologicamente e quanto ao potencial de produ??o de con?dios. Em seguida, buscou-se investigar a diversidade gen?tica dos isolados brasileiros de B. bassiana, avaliar a distribui??o espacial de gen?tipos, e conhecer a rela??o gen?tica entre os isolados das diferentes esp?cies. Para esta finalidade os isolados foram submetidos as t?cnicas de eletroforese de isoenzimas, AFLP-PCR e seq?enciamento de regi?es ITS1 e ITS2. Os resultados permitiram a confirma??o taxon?mica dos isolados e a sele??o daqueles com maior capacidade de produ??o de con?dios. Permitiram tamb?m detectar grande variabilidade gen?tica entre os isolados de B. bassiana indicando que este fungo representa um agregado de esp?cies. Al?m disso, a dist?ncia gen?tica detectada entre os isolados foi fortemente relacionada a dist?ncia geogr?fica de origem dos isolados. Posteriormente, os isolados tamb?m foram avaliados quanto a toler?ncia ? radia??o UV-B, toler?ncia ao calor e atividade ao frio. Tanto o calor quanto o frio s?o fatores abi?ticos ambientais que restringem o uso de fungos como agentes de biocontrole, enquanto a radia??o solar, particularmente UV-B, ? o maior fator ambiental que pode afetar negativamente a a??o deste entomopat?geno. Neste sentido, foi observada alta variabilidade entre os isolados analisados em rela??o ? toler?ncia a UV-B ap?s duas horas de exposi??o ? radia??o, obtendose percentuais de germina??o de con?dios que variaram entre 0% e aproximadamente 80%. Al?m disso, foi observado atraso na germina??o dos con?dios, e revelado que os isolados originados de baixas latitudes tenderam a apresentar menor toler?nc ia a UV-B do que aqueles provenientes de latitudes mais elevadas. Foi tamb?m encontrada alta variabilidade em termotoler?ncia dos con?dios ap?s duas horas de exposi??o a 45 ?C, variando entre baixa, m?dia e alta viabilidade. Os con?dios mais resistentes n?o toleraram exposi??o entre 44 ?C e 45 ?C por seis horas. Quando submetidos a 5 ?C a maioria dos isolados apresentaram atividade. Os isolados de B. bassiana obtidos de latitudes mais elevadas foram mais ativos ao frio do que aqueles originados pr?ximos ? linha do equador. Al?m disso, os isolados tamb?m foram avaliados quanto ao potencial virulento sobre larvas de Boophilus microplus, e neste sentido foi poss?vel diferenci?-los em baixa, m?dia e alta virul?ncia. Foi detectado tamb?m que diferentes popula??es desta esp?cie de carrapato podem manifestar diferentes n?veis de suscetibilidade ? infec??o por B. bassiana. Neste sentido, o presente estudo permitiu selecionar isolados deste entomopat?geno com maior potencial para o controle biol?gico de B. microplus e outros artr?podes em condi??es de campo.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.