Host recognition behaviour of two braconid larval parasitoids Cotesia sesamiae and Cotesia flavipes was studied using suitable stemborer hosts [i.e. Busseola fusca for C. sesamiae, and Chilo partellus for C. flavipes] and one non-host [Eldana saccharina]. The wasps displayed similar sequences of behavioural steps when locating their hosts largely depending on their antennae for host recognition and both antennae and tarsi for final host acceptance and oviposition. Tactile and contact chemoreception stimuli from the hosts seem to play a major role in oviposition decision by the parasitoids. In addition, the external morphology and distribution pattern of sensilla present on antennae, tarsi and ovipositor of the parasitoids were examined by scanning electron and optic microscopy after staining with silver nitrate. Three sensillar types were identified on the distal antennomeres: (i) non-porous sensilla trichoidea most probably involved in mechanoreception, (ii) uniporous sensilla chaetica likely to be gustatory and, (iii) multiporous sensilla placodea likely to be olfactory. The tarsi possess a few sensilla chaetica which could be gustatory while the manubrium is likely to be used in detection of vibrations. The distal end of the ovipositor bears numerous multiporous dome-shaped sensilla. Additionally, the ability of the wasps to discriminate between contact cues was studied. When host larvae were washed in distilled water the wasps did not insert their ovipositors. However, ovipositor insertion resumed when washed host or non-host larvae were painted with water extracts of their respective host larvae. The water extracts of the suitable hosts were more attractive to the wasps than those of non-hosts. Similarly, the frass is important in host recognition during short-range examination as those of respective hosts are more intensely antennated than of non-hosts. The parasitoids were able to discriminate the regurgitant of E. saccharina by not antennating the cotton wool ball of this host; while the regurgitant of B. fusca and C. partellus appeared not useful in discriminating between the two species for both parasitoid species. Further analysis suggests the presence of a protein(s) component(s) in the regurgitant possibly responsible for host recognition and oviposition by C. flavipes. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2009.

Submitted by (edna.saturno@ufrpe.br) on 2016-12-01T14:23:37Z No. of bitstreams: 1 Eliana Maria dos Passos.pdf: 2439142 bytes, checksum: 5cc4ce23eb07068031e4913be088cd08 (MD5) / Made available in DSpace on 2016-12-01T14:23:37Z (GMT). No. of bitstreams: 1 Eliana Maria dos Passos.pdf: 2439142 bytes, checksum: 5cc4ce23eb07068031e4913be088cd08 (MD5) Previous issue date: 2013-02-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Lepidopteran larval parasitism by endoparasitoids is associated with immune suppression and manipulation of the host physiology. This study investigated if the immune response in Diatraea flavipennella (Box) is suppressed by Cotesia flavipes (Cam.) and if changes occur in biochemical profile, haemolymph, and fat body, cytochemistry of hemocytes to ensure parasitism. Levels concentration of nitric oxide, phenoloxidase, and protein was determined. Total number of hemocytes per microliter of hemolymph and the average percentage of these cells were verified. Capacity of melanization and encapsulation by injection and ultrastructural analysis of microbeads was evaluated. Presence of apoptotic cells in fat body was investigated using TUNEL technique. Levels of carbohydrate, lipid, and protein in hemolymph and fat body were quantified, as well as the proteic profile of hemolymph was characterized. Histological sections of stained caterpillars were obtained using PAS (Periodic acid/Schiff's) reaction. Marking of acidic polysaccharides, mucin, and lipids was investigated on hemocytes. Results showed reduction in nitric oxide production, phenol oxidase activity, and total number of hemocytes. In the differential count, increase in number of plasmatocytes and reduction of spherulocytes were observed. The encapsulation capacity was affected. Apoptosis in hemocytes and fat body was registered. Reduction of proteins and lipids occurred in hemolymph and protein; reduction of carbohydrate occurred in body fat. Cytochemistry of hemocytes was changed, reducing the acidic polysaccharides in prohemocytes and oenocytoids, mucin in plasmatocytes and granulocytes, and lipids in adipohemocytes, granulocytes, and prohemocytes. It was also observed a drastic body fat degeneration. The proteic profile showed presence of four specific proteins of parasitism in the hemolymph. It is concluded that C. flavipes is effective for controlling D. flavipennella by suppressing the immune, humoral, and cellular response, altering the biochemical profile of hemolymph, fat body, and cytochemistry of hemocytes. / As espécies Diatraea saccharalis (Fabr.) e Diatraea flavipennella (Box) representam um problema para cultura da cana-de-açúcar, sendo o endoparasitoide Cotesia flavipes (Cam.) introduzido em 1974 para o controle destas pragas. Nos últimos 30 anos houve inversão na população destas espécies de brocas no estado de Alagoas e hoje há predominância de D. flavipennella nos canaviais nordestinos, surgindo entre os produtores à idéia de que C. flavipes não controla satisfatoriamente esta praga. Como o parasitismo de larvas de lepidópteros por endoparasitoides esta associada com a supressão da imunidade e manipulação da fisiologia, do hospedeiro, para aumentar a disponibilidade de nutrientes. Neste trabalho investigou se a resposta imune (níveis de óxido nítrico, atividade da fenoloxidase, contagem total e diferencial dos hemócitos, capacidade de encapsulação e apoptose) em D. flavipennella seria suprimida por C. flavipes e se alterações no perfil bioquímico, da hemolinfa e do corpo gorduroso (proteínas, carboidratos e lipídeos), bem como modificações na citoquímica dos hemócitos, ocorreriam para garantir o parasitismo. Os resultados mostraram redução na produção de óxido nítrico, na atividade da fenoloxidase e no número total de hemócitos. Na contagem diferencial, observou-se aumento no número de plasmatócitos e redução de esferulócitos. A capacidade de encapsulação foi afetada. Registrou-se apoptose nos hemócitos e corpo gorduroso. Ocorreu redução de proteínas e lipídeos na hemolinfa e proteína e carboidrato no corpo gorduroso. A citoquímica dos hemócitos foi alterada, tendo reduzido polissacarídeos ácidos nos prohemócitos e oenocitóides, mucina nos plasmatócitos e granulócitos e lipídeos nos adipohemócitos, granulócitos, e prohemócitos. Observou-se também uma drástica degeneração do corpo gorduroso. Já o perfil protéico revelou a presença de quatro proteínas específicas do parasitismo na hemolinfa. Conclui-se, então, que C. flavipes é eficiente no controle de D. flavipennella por suprimir a resposta imunológica, humoral e celular, alterar o perfil bioquímico da hemolinfa e corpo gorduroso e citoquímica dos hemócitos.

A magnitude do problema de Diatraea saccharalis (Fabricius) tem aumentado nos últimos anos, principalmente nas culturas de cana-de-açúcar e milho no Brasil. Tradicionalmente, D. saccharalis tem sido controlado com agentes de controle biológico, principalmente na cultura da cana-de-açúcar. No entanto, a intervenção química tem sido necessária para o controle de D. saccharalis em ambas as culturas. Visando estabelecer estratégias proativas para o manejo da resistência de D. saccharalis a inseticidas e integrar as táticas de controle químico e biológico, objetivou-se nesse trabalho avaliar a toxicidade de alguns inseticidas (triflumuron, lambdacyhalothrin e fipronil) a D. saccharalis e ao parasitóide Cotesia flavipes (Cameron). A caracterização das linhas-básicas de suscetibilidade de D. saccharalis a esses inseticidas foi realizada com bioensaio de ingestão, mediante contaminação da superfície da dieta artificial com o inseticida, utilizando-se lagartas de 2º instar. Baseado nas curvas de concentração-mortalidade da população suscetível de referência, foram definidas as concentrações diagnósticas de 320 µg de triflumuron /mL de água [I.A.(ppm)], 32 µg de fipronil /mL de água, e 3,2 µg de lambdacyhalothrin/ mL de água, correspondentes à CL99, para o monitoramento da resistência Foram verificadas diferenças significativas na suscetibilidade a inseticidas em populações de D. saccharalis coletadas em diferentes regiões produtoras de milho no Brasil e uma no Paraguai. Para triflumuron, as sobrevivências variaram de 0,85 e 34,2%; para fipronil de 0,21 e 2,71%; e para lambda-cyhalothrin de 0,21 a 7,9%. A toxicidade desses inseticidas sobre C. flavipes foi avaliada mediante determinações de efeitos letal e subletal desses inseticidas sobre adultos do parasitóide. Inicialmente foram avaliadas as concentrações recomendadas de 0,08 mg de triflumuron/L de água e 0,075 mg de lambda-cyhalothrin/L de água, além de 10 e 50% da concentração recomendada, mediante bioensaio de contato residual em discos de folha de canade- açúcar de resíduos com 0, 24 e 48 h de idade. A maior toxicidade foi observada com lambdacyhalothrin (mortalidade de até 100%). Triflumuron causou mortalidade de mortalidade de no máximo 32%. Observou-se também uma redução da longevidade de C. flavipes em todas as concentrações e idade de resíduos para ambos inseticidas. Posteriormente, para avaliar os efeitos indiretos de inseticidas sobre C. flavipes, lagartas sobreviventes de D. saccharalis a CL25 de triflumuron, lambda-cyhalothrin e fipronil foram expostas a C. flavipes para parasitismo. A capacidade de parasitismo foi também avaliada para a geração F1 do parasitóide. Nenhum dos inseticidas afetou a emergência de adultos, a mortalidade de larvas e pupas, e a razão sexual do parasitóide. Por outro lado, lambda-cyhalothrin e fipronil afetaram negativamente a taxa instantânea de crescimento de C. flavipes. Portanto, as estratégias de uso destes inseticidas para o controle de D. saccharalis devem ser implementadas com cautela para preservar a vida útil desses importantes inseticidas em programas de manejo integrado de pragas. / The magnitude of Diatraea saccharalis (Fabricius) problem has increased in the past years, mainly in sugarcane and maize crops in Brazil. Traditionally, D. saccharalis has been controlled with biological control agents. However, chemical intervention has been necessary to control D. saccharalis in both crops. For establishing a proactive insecticide resistance management of D. saccharalis and integrating chemical and biological control tactics, the major objective of this work was to evaluate the toxicity of some insecticides (triflumuron, lambdacyhalothrin e fipronil) to D. saccharalis and to the parasitoid Cotesia flavipes (Cameron). The baseline susceptibility of D. saccharalis to these insecticides was characterized with diet surface treatment bioassay by using 2nd instar larvae. Based on concentration-mortality response of the susceptible reference population, diagnostic concentrations of 320 µg of triflumuron/mL of water [A.I. (ppm)], 32 µg of fipronil/mL of water, and 3.2 µg de lambda-cyhalothrin/mL of water, corresponding to LC99, were defined for monitoring resistance. Significant differences in susceptibilities to insecticides were found in populations of D. saccharalis collected from major maize-production regions and one population from Paraguay. For triflumuron, the survivorship varied from 0.8 to 34.2%; for fipronil, from 0.2 to 2.7%; and for lambda-cyhalothrin, from 0.2 to 7.9%. The toxicity of these insecticides to C. flavipes was evaluated by determining the lethal and sublethal effects of the insecticides to the adults of this parasitoid. Initially, commercial rates of 0.08 mg triflumuron/ L of water and 0.075 mg lambda-cyhalothrin / L of water, and 10% and 50% of commercial rates, were evaluated with residual contact bioassay by using sugarcane leaf disks of 0, 24 and 48 h-old residues. The highest toxicity was detected to lambda-cyhalothrin (mortality up to 100%). Triflumuron caused mortality up to 32%. Reduction in the longevity of C. flavipes was detected in all concentrations and residue ages of both insecticides. Then, to evaluate the indirect effect of the insecticides, D. saccharalis larvae that survived at LC25 of triflumuron, lambda-cyhalothrin and fipronil were exposed to C. flavipes. The parasitism capacity of F1 generation of the parasitoid was also evaluated. None of the insecticide affected the adult emergence, pupal and larval mortality, and sexual ratio of the parasitoid. On the other hand, lambda-cyhalothrin e fipronil negatively affect the instantaneous rate of growth of C. flavipes. Therefore, the strategies of the use of these insecticides for controlling D. saccharalis should be carefully designed to preserve their lifetime in integrated pest management programs.

O incremento aos programas de bioenergia em nosso país, aponta para uma provável duplicação da área plantada de cana-de-açúcar, num futuro bastante próximo. Neste contexto, a sustentabilidade da cultura deve ser preservada, evitando-se que sejam colocados em risco os avanços obtidos nas últimas cinco décadas no Controle Biológico de suas pragas. Para tanto, são necessárias pesquisas que atendam às necessidade das diversas etapas de um programa de Controlo Biológico. No presente estudo, avaliou-se o uso de temperaturas diferentes para criar cada fase do desenvolvimento de Diatraea saccharalis (Fabricius, 1794), com o objetivo de fornecer informações importantes para sua criação massal. Avaliou-se também, o raio de ação de uma armadilha contendo fêmeas virgens, a dinâmica populacional de D. saccharalis e o seu desenvolvimento em plantas de cana-de-açúcar, visando desenvolver um modelo de previsão da praga, com vistas ao seu controle biológico pelos parasitoides T. galloi e C. flavipes. As combinações de temperaturas 28°C-25°C-25°C, 28°C-28°C-28°C, 30°C-25°C-25°C e 30°C-22°C- 22°C para criar lagartas, pupas e adultos, respectivamente, destacaram-se das demais por promover rápido desenvolvimento aliado a altas taxas reprodutivas e podem assim, serem utilizadas para a criação massal de Diatraea saccharalis (Fabricius, 1794) e de seu parasitoide Cotesia flavipes (Cameron, 1891). O aumento de densidade de criação de 1 lagarta por tubo para 250 lagartas por frasco, não afetou o desenvolvimento e reprodução de D. saccharalis. Foi possível prever o momento de liberação de C. flavipes em cana-de-açúcar com base em modelos de exigências térmicas, assim como, orientar as liberações de T. galloi ao definir o período de ocorrência dos ovos de D. saccharalis. Os parâmetros climáticos determinaram uma faixa térmica de atuação para as armadilhas contendo fêmeas virgens (temperatura média acima de 24°C), faixa na qual foram observadas altas correlações entre a captura de machos e amostragem de lagartas, realizada 21 dias após a captura dos machos de D. saccharalis. De acordo com a dinâmica populacional de D. saccharalis, foram identificadas quatro gerações ao longo do ciclo da cultura. Para a utilização do parasitoide de ovos T. galloi deve-se usar uma armadilha contendo 4 fêmeas virgens de D. saccharalis para amostrar 12,5 ha, área determinada em função do raio de ação de 200 metros. / The increase of bioenergy programs in our country indicates a probable duplication of the planted area with sugar cane in a near future. In this context, the sustainability of the culture must be preserved, avoiding the risk of loose the progress achieved in the last five decades in the Biological Control of its pests. For this, it is necessary researches that attend the needs of the various steps of a biological control program. In this study, was evaluated the use of different temperatures to rear each phase of the development of Diatrea saccharalis (Fabricius, 1794), aimed to provide important informations for its mass rearing. It was also evaluated the radius of action of a trap containing virgin females, the population dynamics of D. saccharalis and its development in sugar cane, aimed the development of a prediction model of the occurence of D. saccharalis in sugar cane, suporting its biological control by the parasitoids T. galloi and C. flavipes. The temperatures combination 28°C-25°C-25°C, 28°C-28°C-28°C, 30°C-25°C-25°C, and 30°C-22°C- 22°C used to rear caterpillar, pupae and adults stood out from the others by promoting fast development combined with high reproductives rates and can be used for mass rearing of Diatraea saccharalis (Fabricius, 1794) and its parasitoids Cotesia flavipes (Cameron, 1891). The rearing of caterpillars individually or in groups of 250 did not interfere in the development and reproduction of D. saccharalis. It was possible to predict the release time of C. flavipes in sugar cane based in thermal requirements models and advise the releases of T. galloi deffining the period of occurrence of D. saccharalis eggs. The climatic parameters determined a temperature range of action for the pheromone traps with the virgin females (average temperatures above 24°C), range at which high correlations were observed between the males capture and caterpillar sampling performed 21 days after the capture of the D. saccharalis males. According to the population dynamics of D. saccharalis, four generations have been identified during the crop cycle. For the use of the egg parasitoid T. galloi, it should be used a trap containing four virgin femeales of D. saccharalis to sample 12.5 ha, area determined by the action radius of 200 meters.

Genes de plantas que codificam inibidores de enzimas digestivas de insetos têm sido introduzidos em plantas cultivadas visando o controle de pragas. Os inibidores de proteinases estão presentes nos tecidos vegetais, principalmente nas sementes, e atuam em resposta a ataques por herbívoros e patógenos. Inibidores de serino-proteinases (IPs) dos tipos Bowman-Birk e Kunitz isolados de sementes de soja foram inseridos em variedades de cana-de-açúcar para aumentar a resistência à broca Diatraea saccharalis (Fabr.), principal praga desta cultura. Para utilização de plantas geneticamente modificadas contendo inibidores de proteinases é necessário um conhecimento profundo de sua sustentabilidade e segurança ambiental, determinando a estabilidade da característica inserida e os seus efeitos nos organismos não-alvo. O objetivo desta pesquisa foi avaliar os efeitos diretos e indiretos de inibidores de proteinases de soja em organismos não-alvos: um parasitóide larval, Cotesia flavipes (Cam.) (Hymenoptera: Braconidae); um patógeno, Metarhizium anisopliae (Mestch.) Sorokin (Deuteromycotina: Hyphomycetes); um polinizador, Apis mellifera L. (Hymenoptera: Apidae) e um decompositor, Scheloribates praeincisus (Berlese) (Acari: Oribatida: Scheloribatidae); associados à cultura da cana-de-açúcar. O consumo de Kunitz e BBI não afetou a sobrevivência de S. praeincisus. Por outro lado, a ingestão dos inibidores semi-purificados e purificados do tipo Kunitz diminuiu a duração das fases imaturas de S. praeincisus. A ingestão de folhas de cana GM expressando inibidores de proteinases (Kunitz e BBI) não afetou o tempo de desenvolvimento e a sobrevivência dos imaturos deste oribatídeo quando comparada à ingestão de suas isolinhas. Os inibidores de proteinases semi-purificados e purificados não afetaram a duração dos períodos larval e pupal, o peso e número de pupas e percentual de emergência do parasitóide C. flavipes em D. saccharalis. Por outro lado, a proporção de fêmeas em relação a machos de C. flavipes foi maior no tratamento onde as lagartas foram alimentadas com dieta contendo 0,5% de inibidores semi-purificados comparado-se à testemunha. A proporção fêmea:macho foi significativamente maior também quando os parasitóides foram alimentados com o inibidor do tipo Kunitz em relação ao controle e aos parasitóides alimentados com BBI. A adição de 0,5% (p/v) de inibidores de proteinases semi-purificados e 0,05% (p/v) de inibidores purificados do tipo Kunitz nos meios de cultura MC e BDA resultaram em maiores crescimento vegetativo e produção de conídios de M. anisopliae. Os inibidores purificados do tipo BBI não alteraram a esporulação do fungo. Os resultados dos estudos com A. mellifera não foram conclusivos e novas investigações precisam ser conduzidas para esclarecer os potenciais efeitos de inibidores de proteinases em abelhas. De uma forma geral, observou-se que os inibidores de proteinases (Kunitz e BBI) não afetaram negativamente os organismos não-alvo testados. Por outro lado, a ingestão de inibidor do tipo Kunitz alterou positivamente alguns parâmetros biológicos de C. flavipes, M. anisopliae e S. praeincisus. / Genes of plants expressing insect proteinase inhibitors have been introduced into plants for pest control. Proteases inhibitors are present in plant tissues, mainly in seeds, and act in response to predators and pathogens. The Bowman-Birk type and Kunitz type of serine proteases inhibitors (PI) from soybean seeds are been used to increase resistance of sugarcane to Diatraea saccharalis (Fabr.), the most important pest of this crop. The sustainability and environmental safety of PI crops is still unknown. For these reasons, it is necessary to understand the stability and the non-target effects of this new trait. The objective of this study was to evaluate the direct and indirect effects of soybean PI on the following non-target organisms associated to sugarcane: the larval parasitoid, Cotesia flavipes (Cam.) (Hymenoptera: Braconidae); the entomopathogen, Metarhizium anisopliae (Mestch.) Sorokin (Deuteromycotina: Hyphomycetes); the pollinator Apis mellifera L. (Hymenoptera: Apidae) and the soil mite involved in the process of recycling organic matter, Scheloribates praeincisus (Berlese) (Acari: Oribatida: Scheloribatidae). Kunitz and BBI did not affect S. praeincisus survival. On the other hand, Kunitz semi-purified and purified inhibitor ingestion reduced duration of the immature stages of S. praeincisus. Ingestion of GM senescent leaves did not have an effect on mite immatures development time and survival compared to ingestion of its isolines leaves. The semi-purified and purified proteinases inhibitors did not alter either the duration of larval and pupal stages of C. flavipes on D. saccharalis, or weight and number of pupae and parasitoid emergence. In other hand, the parasitism and proportion of female was higher on the treatment where caterpillars were fed with diet containing 0.5% of semi-purified inhibitors, comparing to control. The ratio female:male was significantly higher also when parasitoids were fed to the Kunitz type inhibitor compared to the control and BBI. The addition of 0.5 % (w/v) of semi- purified proteinase inhibitors and 0.05% (w/v) of Kunitz type purified inhibitors on two culture media (CM and PDA), resulted in increase of vegetative growth and production of conidia. BBI type purified inhibitors did not change the fungus sporulation. The results from the studies with A. mellifera were not conclusive and investigations are needed to clarify the potential impact of proteinase inhibitors on A. mellifera. Overall, proteinase inhibitors (Kunitz and BBI) did not negatively affect the non-target organisms tested. Conversely, ingestion of the Kunitz type of proteinase inhibitors altered positively some biological parameters of C. flavipes, M. anisopliae and S. praeincisus.

Orientador: Arlindo Leal Boiça Junior / Banca: José Carlos Barbosa / Banca: Marcelo Francisco Arantes Pereira / Resumo: A broca da cana-de-açúcar Diatraea saccharalis (Fabricius, 1794), destaca-se como a principal praga da cultura da cana-de-açúcar e dependendo do nível de infestação, pode causar sérios prejuízos financeiros a produtores. O sucesso no seu controle depende de estudos comportamentais das variedades e avaliação de seu parasitismo, principalmente por Cotesia flavipes (Cameron, 1891). Assim o objetivo da presente pesquisa foi verificar a infestação e os danos causados por D. saccharalis e seu parasitismo por C. flavipes em duas idades de corte das plantas em variedades de cana-de-açúcar. O experimento foi conduzido na Usina Laguna, localizada no município de Batayporã no Estado do Mato Grosso do Sul. O delineamento experimental utilizado foi em parcelas subdivididas, com 5 variedades, RB86-7515, SP81-3250, RB85-5453, SP80-1842 e RB82-5211 duas idades de corte, cana planta e cana soca de segundo corte e 4 repetições, dispostas em blocos casualizados, aplicando as variedades nas parcelas e as idades de corte nas subparcelas. Foram avaliadas a porcentagem e intensidade de infestação, porcentagem de danos no colmo, população da broca por hectare e a porcentagem de parasitismo. Pelos resultados obtidos conclui-se que as variedades menos atacadas por D. saccharalis foram SP81-3250 e RB92-5211; a variedade mais atacada por D. saccharalis foi SP80-1842; as liberações de C. flavipes incrementaram o parasitismo na área; o aumento da porcentagem de parasitismo proporcionou redução na população da broca; e o parasitismo independe da idade de corte da cana-de-açúcar / Abstract: In the culture of sugar cane, and all, there are pests that directly affect production. The sugarcane borer Diatraea saccharalis (Fabricius, 1794), stands out as the main and depending on the level of infestation, can cause serious financial losses to producers. The success in controlling this pest depends on behaviour studies of varieties and evaluation of their parasitism, mainly by Cotesia flavipes (Cameron, 1891). Thus the aim of this research was to verify the infestation and the damage caused by D. saccharalis and its parasitism by C. flavipes in two plant ages in varieties of sugar cane. The experiment was conducted at the Laguna Plant, located in Batayporã borough in Mato-Grosso-do-Sul state. The varieties used in the experiment were RB86-7515, SP81-3250, RB85-5453, SP80-1842 and RB82-5211 conducted in plots of 10 080 m2, repeated four times, adopting the split plot design, applying the varieties in the plots and subplots cutting ages. It was evaluated the rate and intensity of infestation, percentage of damage in the stem borer population per hectare and percentage parasitism. By the results it is concluded that varieties less attacked by D. saccharalis were SP81-3250 and RB92-5211, the variety most attacked by D. saccharalis was SP80-1842, an increase of the parasitism decreased the population of the drill, the release of C. flavipes increased the parasitism the area, and the parasitism is independent of the sugar cane cut age / Mestre

O incremento aos programas de bioenergia em nosso país, aponta para uma provável duplicação da área plantada de cana-de-açúcar, num futuro bastante próximo. Neste contexto, a sustentabilidade da cultura deve ser preservada, evitando-se que sejam colocados em risco os avanços obtidos nas últimas cinco décadas no Controle Biológico de suas pragas. Para tanto, são necessárias pesquisas que atendam às necessidade das diversas etapas de um programa de Controlo Biológico. No presente estudo, avaliou-se o uso de temperaturas diferentes para criar cada fase do desenvolvimento de Diatraea saccharalis (Fabricius, 1794), com o objetivo de fornecer informações importantes para sua criação massal. Avaliou-se também, o raio de ação de uma armadilha contendo fêmeas virgens, a dinâmica populacional de D. saccharalis e o seu desenvolvimento em plantas de cana-de-açúcar, visando desenvolver um modelo de previsão da praga, com vistas ao seu controle biológico pelos parasitoides T. galloi e C. flavipes. As combinações de temperaturas 28°C-25°C-25°C, 28°C-28°C-28°C, 30°C-25°C-25°C e 30°C-22°C- 22°C para criar lagartas, pupas e adultos, respectivamente, destacaram-se das demais por promover rápido desenvolvimento aliado a altas taxas reprodutivas e podem assim, serem utilizadas para a criação massal de Diatraea saccharalis (Fabricius, 1794) e de seu parasitoide Cotesia flavipes (Cameron, 1891). O aumento de densidade de criação de 1 lagarta por tubo para 250 lagartas por frasco, não afetou o desenvolvimento e reprodução de D. saccharalis. Foi possível prever o momento de liberação de C. flavipes em cana-de-açúcar com base em modelos de exigências térmicas, assim como, orientar as liberações de T. galloi ao definir o período de ocorrência dos ovos de D. saccharalis. Os parâmetros climáticos determinaram uma faixa térmica de atuação para as armadilhas contendo fêmeas virgens (temperatura média acima de 24°C), faixa na qual foram observadas altas correlações entre a captura de machos e amostragem de lagartas, realizada 21 dias após a captura dos machos de D. saccharalis. De acordo com a dinâmica populacional de D. saccharalis, foram identificadas quatro gerações ao longo do ciclo da cultura. Para a utilização do parasitoide de ovos T. galloi deve-se usar uma armadilha contendo 4 fêmeas virgens de D. saccharalis para amostrar 12,5 ha, área determinada em função do raio de ação de 200 metros. / The increase of bioenergy programs in our country indicates a probable duplication of the planted area with sugar cane in a near future. In this context, the sustainability of the culture must be preserved, avoiding the risk of loose the progress achieved in the last five decades in the Biological Control of its pests. For this, it is necessary researches that attend the needs of the various steps of a biological control program. In this study, was evaluated the use of different temperatures to rear each phase of the development of Diatrea saccharalis (Fabricius, 1794), aimed to provide important informations for its mass rearing. It was also evaluated the radius of action of a trap containing virgin females, the population dynamics of D. saccharalis and its development in sugar cane, aimed the development of a prediction model of the occurence of D. saccharalis in sugar cane, suporting its biological control by the parasitoids T. galloi and C. flavipes. The temperatures combination 28°C-25°C-25°C, 28°C-28°C-28°C, 30°C-25°C-25°C, and 30°C-22°C- 22°C used to rear caterpillar, pupae and adults stood out from the others by promoting fast development combined with high reproductives rates and can be used for mass rearing of Diatraea saccharalis (Fabricius, 1794) and its parasitoids Cotesia flavipes (Cameron, 1891). The rearing of caterpillars individually or in groups of 250 did not interfere in the development and reproduction of D. saccharalis. It was possible to predict the release time of C. flavipes in sugar cane based in thermal requirements models and advise the releases of T. galloi deffining the period of occurrence of D. saccharalis eggs. The climatic parameters determined a temperature range of action for the pheromone traps with the virgin females (average temperatures above 24°C), range at which high correlations were observed between the males capture and caterpillar sampling performed 21 days after the capture of the D. saccharalis males. According to the population dynamics of D. saccharalis, four generations have been identified during the crop cycle. For the use of the egg parasitoid T. galloi, it should be used a trap containing four virgin femeales of D. saccharalis to sample 12.5 ha, area determined by the action radius of 200 meters.

Genes de plantas que codificam inibidores de enzimas digestivas de insetos têm sido introduzidos em plantas cultivadas visando o controle de pragas. Os inibidores de proteinases estão presentes nos tecidos vegetais, principalmente nas sementes, e atuam em resposta a ataques por herbívoros e patógenos. Inibidores de serino-proteinases (IPs) dos tipos Bowman-Birk e Kunitz isolados de sementes de soja foram inseridos em variedades de cana-de-açúcar para aumentar a resistência à broca Diatraea saccharalis (Fabr.), principal praga desta cultura. Para utilização de plantas geneticamente modificadas contendo inibidores de proteinases é necessário um conhecimento profundo de sua sustentabilidade e segurança ambiental, determinando a estabilidade da característica inserida e os seus efeitos nos organismos não-alvo. O objetivo desta pesquisa foi avaliar os efeitos diretos e indiretos de inibidores de proteinases de soja em organismos não-alvos: um parasitóide larval, Cotesia flavipes (Cam.) (Hymenoptera: Braconidae); um patógeno, Metarhizium anisopliae (Mestch.) Sorokin (Deuteromycotina: Hyphomycetes); um polinizador, Apis mellifera L. (Hymenoptera: Apidae) e um decompositor, Scheloribates praeincisus (Berlese) (Acari: Oribatida: Scheloribatidae); associados à cultura da cana-de-açúcar. O consumo de Kunitz e BBI não afetou a sobrevivência de S. praeincisus. Por outro lado, a ingestão dos inibidores semi-purificados e purificados do tipo Kunitz diminuiu a duração das fases imaturas de S. praeincisus. A ingestão de folhas de cana GM expressando inibidores de proteinases (Kunitz e BBI) não afetou o tempo de desenvolvimento e a sobrevivência dos imaturos deste oribatídeo quando comparada à ingestão de suas isolinhas. Os inibidores de proteinases semi-purificados e purificados não afetaram a duração dos períodos larval e pupal, o peso e número de pupas e percentual de emergência do parasitóide C. flavipes em D. saccharalis. Por outro lado, a proporção de fêmeas em relação a machos de C. flavipes foi maior no tratamento onde as lagartas foram alimentadas com dieta contendo 0,5% de inibidores semi-purificados comparado-se à testemunha. A proporção fêmea:macho foi significativamente maior também quando os parasitóides foram alimentados com o inibidor do tipo Kunitz em relação ao controle e aos parasitóides alimentados com BBI. A adição de 0,5% (p/v) de inibidores de proteinases semi-purificados e 0,05% (p/v) de inibidores purificados do tipo Kunitz nos meios de cultura MC e BDA resultaram em maiores crescimento vegetativo e produção de conídios de M. anisopliae. Os inibidores purificados do tipo BBI não alteraram a esporulação do fungo. Os resultados dos estudos com A. mellifera não foram conclusivos e novas investigações precisam ser conduzidas para esclarecer os potenciais efeitos de inibidores de proteinases em abelhas. De uma forma geral, observou-se que os inibidores de proteinases (Kunitz e BBI) não afetaram negativamente os organismos não-alvo testados. Por outro lado, a ingestão de inibidor do tipo Kunitz alterou positivamente alguns parâmetros biológicos de C. flavipes, M. anisopliae e S. praeincisus. / Genes of plants expressing insect proteinase inhibitors have been introduced into plants for pest control. Proteases inhibitors are present in plant tissues, mainly in seeds, and act in response to predators and pathogens. The Bowman-Birk type and Kunitz type of serine proteases inhibitors (PI) from soybean seeds are been used to increase resistance of sugarcane to Diatraea saccharalis (Fabr.), the most important pest of this crop. The sustainability and environmental safety of PI crops is still unknown. For these reasons, it is necessary to understand the stability and the non-target effects of this new trait. The objective of this study was to evaluate the direct and indirect effects of soybean PI on the following non-target organisms associated to sugarcane: the larval parasitoid, Cotesia flavipes (Cam.) (Hymenoptera: Braconidae); the entomopathogen, Metarhizium anisopliae (Mestch.) Sorokin (Deuteromycotina: Hyphomycetes); the pollinator Apis mellifera L. (Hymenoptera: Apidae) and the soil mite involved in the process of recycling organic matter, Scheloribates praeincisus (Berlese) (Acari: Oribatida: Scheloribatidae). Kunitz and BBI did not affect S. praeincisus survival. On the other hand, Kunitz semi-purified and purified inhibitor ingestion reduced duration of the immature stages of S. praeincisus. Ingestion of GM senescent leaves did not have an effect on mite immatures development time and survival compared to ingestion of its isolines leaves. The semi-purified and purified proteinases inhibitors did not alter either the duration of larval and pupal stages of C. flavipes on D. saccharalis, or weight and number of pupae and parasitoid emergence. In other hand, the parasitism and proportion of female was higher on the treatment where caterpillars were fed with diet containing 0.5% of semi-purified inhibitors, comparing to control. The ratio female:male was significantly higher also when parasitoids were fed to the Kunitz type inhibitor compared to the control and BBI. The addition of 0.5 % (w/v) of semi- purified proteinase inhibitors and 0.05% (w/v) of Kunitz type purified inhibitors on two culture media (CM and PDA), resulted in increase of vegetative growth and production of conidia. BBI type purified inhibitors did not change the fungus sporulation. The results from the studies with A. mellifera were not conclusive and investigations are needed to clarify the potential impact of proteinase inhibitors on A. mellifera. Overall, proteinase inhibitors (Kunitz and BBI) did not negatively affect the non-target organisms tested. Conversely, ingestion of the Kunitz type of proteinase inhibitors altered positively some biological parameters of C. flavipes, M. anisopliae and S. praeincisus.

Eldana saccharina Walker (Lepidoptera: Pyralidae) is an indigenous insect widely distributed throughout Sub-Saharan Africa that is a major pest of sugarcane in southern Africa. Studies have shown that populations from West Africa have distinct behavioural differences compared to populations from eastern and southern Africa. In addition, the parasitoid guilds attacking populations in these regions are markedly different. This marked behavioural and parasitoid guild variation evoked a hypothesis of genetic diversification. To evaluate this hypothesis a project on the phylogeography of E. saccharina was initiated. The project was planned to include sampling of as many regions as possible in its known range in Africa, to obtain specimens of E. saccharina for genetic analysis. When these surveys were initiated in Ethiopia, it was found that there was no published literature available on the occurrence of stem borers in Ethiopian sugarcane. It was thus clear that no stalk borer/parasitoid surveys had been completed in either sugarcane or any large grass and sedge indigenous hosts in Ethiopia. The study was thus expanded beyond the investigation of only the genetic diversity of E. saccharina, to include area-wide surveys to determine ecological aspects of the borer complex in suspected host plants, including sugarcane, in Ethiopia. In this way the host plant range and distribution of E. saccharina and other sugarcane borers in Ethiopia in particular could be determined, samples for a larger phylogeography project could be collected, and the insect's impact on sugarcane could be assessed. Quantified area-wide surveys of the sugarcane estates and small-scale farmer fields of Ethiopia were conducted between December 2003 and February 2004. The surveys verified the presence of four lepidopteran stem borer species on Ethiopian sugarcane. These were Chilo partellus Swinhoe (Lepidoptera: Crambidae), Sesamia calamistis Hampson (Lepidoptera: Noctuidae), Busseola fusca Fuller (Lepidoptera: Noctuidae) and Busseola phaia Bowden (Lepidoptera: Noctuidae). The surveys indicated that Busseola species are the major and most widely distributed sugarcane stem borers in sugarcane farms of Ethiopia. Over 70% of the peasant sugarcane fields visited were infested by these borers, with the highest levels of infestation (35% and 50%) being in the northern and western part of the country, respectively. Busseola was also the predominant stem borer of sugarcane in two of the three estates (Wonji and Finchawa). Chilo partellus and S.calamistis were recovered in very low numbers at all the commercial estates and from peasant farms in the western part of Ethiopia. However, C. partellus was the predominant sugarcane stem borer in lowland areas of northern, southern and eastern parts of the country. Eldana saccharina was recovered from large sedges in waterways of Metehara and Wonji sugar estates in the central part of the country, and sedges growing around lakes in northern and southern Ethiopia, but not from sugarcane anywhere in Ethiopia. The phylogeographic study conducted on E. saccharina populations from eleven countries of Africa clearly showed the population structure of the insect within the continent. Five hundred and two base pairs of the mitochondrial DNA (mtDNA), corresponding to the Cytochrome Oxidase subunit I (COl) region, were sequenced to clarify phylogenetic relationships between geographically distant populations from eastern, northern, southern and western Africa. Results revealed that E. saccharina is separated into four major populations corresponding to their geographical location, i.e. West African, Rift Valley and two southern African populations. Sequence divergence between the four populations ranged from 1% to 4.98%. The molecular data are congruent with an isolation by distance pattern except for some of the specimens from eastern and southern Africa where geographically close populations are genetically distant from each other. Geographical features such as the Rift Valley and large water bodies in the continent seem to have a considerable impact on the genetic diversity in E. saccharina. Identification of field-collected stem borer specimens was done using classical taxonomic techniques, except for Busseola spp. where DNA barcoding was used. As field-collected larval material of Busseola died before reaching the adult stage, identification of species using adult morphology was not possible. 'Sequence divergence in the COl gene was used as a tool to identify the species of Busseola attacking Ethiopian sugarcane. Partial COl sequences from Ethiopian specimens were compared with sequences of already identified noctuid species from the East African region. Results of the sequence analysis indicated that the Busseola species complex in Ethiopian sugarcane comprised B. fusca and B. phaia. Sequence divergences between Ethiopian Busseola species was as high as 5.0 %, whereas divergences within species were less than 1% in both species identified. Several larval parasitoids, bacterial and fungal diseases of stem boring caterpillars were also recorded in Ethiopian sugarcane. Amongst these was Cotesia flavipes Cameron (Hymenoptera: Braconidae). This exotic parasitoid has been introduced into several African countries for the control of C. partellus in maize and sorghum, but had never been released in Ethiopia. To investigate the origin of C. flavipes in Ethiopian sugarcane, molecular analyses were conducted on Ethiopian specimens from sugarcane and specimens of C. flavipes from different countries of Africa released from the Kenyan laboratory colony, again using COl sequences. Results of the analysis revealed that the C. flavipes population that had established in sugarcane fields of Ethiopia was similar to the south east Asian populations released against C. partellus in maize in other parts of Africa, and different from other populations of this species, providing evidence that the Ethiopian C. flavipes is likely to be a descendant of the original Pakistani population that was released in different parts of Africa. The study reveals the importance of lepidopteran stem borers in sugarcane production in Ethiopia and highlights the role of molecular methods in species identification and determining phylogenetic relationships. More importantly, this study establishes the continental phylogeographical pattern of the indigenous moth, E. saccharina. The impact of geological events, geographic barriers and cropping systems on the evolution, distribution and abundance of stem borers are discussed. Future areas of research for understanding more about the phylogeographic relationships of E. saccharina and management of stem borers are discussed. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.