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Made available in DSpace on 2022-04-28T19:44:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-30 Medical Research Council Australian Research Council Endoparasitoid wasps use complex biochemical arsenals to suppress the normal humoral and cellular immune responses of their hosts in order to transform them into a suitable environment for development of their eggs and larvae. Venom injected during oviposition is a key component of this arsenal, but the functions of individual venom toxins are still poorly understood. Furthermore, there has been little investigation of the potential biotechnological use of these venom toxins, for example for control of agricultural pests. The endoparasitoid Cotesia flavipes (Hymenoptera: Braconidae) is a biocontrol agent reared in biofactories and released extensively in Brazil to control the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae). The objectives of this work were to reveal venom components produced by C. flavipes and explore the function of a major venom peptide, Cf4. Using a combined proteomic/transcriptomic approach, we identified 38 putative venom toxins including both linear and disulfide-rich peptides, hydrolases, protease inhibitors, apolipophorins, lipid-binding proteins, and proteins of the odorant binding families. Because of its high abundance in the venom, we selected Cf4, a 33-residue peptide with three disulfide bonds, for synthesis and further characterization. We found that synthetic Cf4 reduced the capacity of D. saccharalis hemocytes to encapsulate foreign bodies without any effect on phenoloxidase activity, consistent with a role in disruption of the cellular host immune response. Feeding leaves coated with Cf4 to neonate D. saccharalis resulted in increased mortality and significantly reduced feeding compared to caterpillars fed untreated leaves, indicating that Cf4 is a potential candidate for insect pest control through ingestion. This study adds to our knowledge of endoparasitoid wasp venoms composition, host regulation mechanisms and their biotechnological potential for pest management. School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Institute for Molecular Bioscience The University of Queensland Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science The University of Queensland School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Australian Research Council: CE200100012

Made available in DSpace on 2022-05-01T08:44:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-01 Like conventional crops, transgenic plants expressing insecticidal toxins from Bacillus thuringiensis (Bt) are subjected to water deprivation. However, the effects of water deprivation over the insecticidal activity of Bt plants are not well understood. We submitted Bt maize and Bt soybean to water deprivation and evaluated biochemical stress markers and the insecticidal activity of plants against target insects. Bt maize (DAS-Ø15Ø7-1 × MON-89Ø34-3 × MON-ØØ6Ø3-6 × SYN-IR162-4) containing the PowerCore Ultra traits, Bt soybean (DAS-444Ø6-6 × DAS-81419-2) with the Conkesta E3 traits, and commercial non-Bt cultivars were cultivated and exposed to water deprivation in the greenhouse. Leaves were harvested for quantification of hydrogen peroxide, malondialdeyde (MDA), and total phenolics and insecticidal activity. Maize or soybean leaf disks were used to evaluate the insecticidal activity against, respectively, Spodoptera frugiperda (J.E Smith) and Chrysodeixis includens (Walker) neonates. Except for Bt soybean, water deprivation increased hydrogen peroxide and MDA contents in Bt and non-Bt plants. Both biochemical markers of water deficit were observed in lower concentrations in Bt plants than in non-Bt commercial cultivars. Water deprivation did not result in changes of phenolic contents in Bt and non-Bt maize. For Bt or non-Bt soybean, phenolic contents were similar despite plants being exposed or not to water deprivation. Water deprivation did not alter substantially insect survival in non-Bt maize or non-Bt soybean. Despite water deprivation-induced biochemical changes in plants, both Bt plants maintained their insecticidal activity (100% mortality) against the target species. São Paulo State University (UNESP) School of Agricultural and Veterinarian Sciences Corteva Agriscience Alameda Itapecuru, 506, Alphaville - SP Corteva Agriscience, 7000NW 62nd Ave São Paulo State University (UNESP) School of Agricultural and Veterinarian Sciences

Made available in DSpace on 2022-05-01T13:11:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Immature endoparasitoids develop inside their hosts. Consequently, endoparasitoids need to circumvent the action of the immune system and reprogram the physiology of their hosts to develop successfully. For this, parasitoids release host regulation-related molecules into their host’s hemolymph produced by the parasitoid itself or expressed in specific host cells infected by parasitoid symbiotic viruses. In addition to their roles in host regulation, these molecules may also present biotechnological potentials, such as for insecticidal purposes in crop protection. We performed a survey for the biotechnological potential of compounds present in the hemolymph serum with and without circulating cell contents of Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) parasitized by Cotesia flavipes Cameron (Hymenoptera: Braconidae) as insecticides in feeding assays. Hemolymph serum from D. saccharalis larvae at 6 days after the start of the parasitism with and without circulating cell contents were collected and spread over artificial diet or over sugarcane leaves for feeding assays with D. saccharalis neonates. Hemolymph serum with or without circulating cell contents from non-parasitized D. saccharalis larvae at the same age were used as controls. Subsequently, hemolymph serum plus circulating cell contents was fractioned using molecular filters and fractions with different molecular weight range were used for feeding assays with D. saccharalis neonates. Feeding larvae with leaf disks amended with hemolymph serum plus circulating cell contents from parasitized hosts resulted in an increase of ca. 30% in the mortality of D. saccharalis neonates. After fractioning hemolymph serum plus circulating cell contents, feeding assays with the >100 or 3–10 kDa fractions did not result in the previously observed increase in larval mortality, but resulted in a reduction of leaf consumption by neonates. Finally, we selected a parasitism-specific protein band observed in the hemolymph >100 kDa fraction for mass spectrometry identification. This parasitism-specific protein was similar to hymenopteran apolipophorins-I. We conclude that there is an insecticidal potential in the hemolymph serum plus circulating cell contents of D. saccharalis larvae parasitized by C. flavipes and further investigations will be performed to identify, characterize, and uncover the biotechnological application of these molecules. School of Agricultural and Veterinarian Science São Paulo State University (UNESP), 14.884-900, SP University of Franca (UNIFRAN), 14.870-370, SP School of Agricultural and Veterinarian Science São Paulo State University (UNESP), 14.884-900, SP

Made available in DSpace on 2021-06-25T10:56:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Several secondary metabolites are associated with plant resistance against herbivores. Cassava genotypes present a wide variety of metabolites with insecticidal potential, but little is known about the identity of these compounds. The present work was conducted for the identification of insecticidal molecules present in cassava genotypes. To this purpose, we firstly evaluated the development of the generalist herbivore Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) fed with leaves from six cassava genotypes (IAC-14, IAC-90, IAC-12, IAC-Caapora, Baianinha and MEcu 72). Following bioassays, we measured the levels of two anti-herbivory related groups of compounds (phenolics and steroidal saponins) in all genotypes using colorimetric methods. The metabolic fingerprinting (GC–MS) of three contrasting cassava genotypes selected by their biological interferences over S. frugiperda was performed. The most unsuitable nutritional indices were observed for larvae fed with MEcu 72. Lower reproductive indices were observed for adults in which larval stages were fed with Baianinha or MEcu 72. Steroidal saponin content was similar in all genotypes, but phenolic content was 25% higher in MEcu 72. GC–MS metabolite fingerprinting of MEcu 72, Baianinha and IAC-Caapora resulted in the annotation of 53 metabolites in which 20 presented different relative abundance among the evaluated genotypes. Molecules such as myo-inositol-2-monophosphate, p-coumaric acid, chlorogenic acid, p-hydroxybenzoic acid, octadecadienoic acid and n-hexacosane were more abundant in MEcu 72 or Baianinha than in IAC-Caapora. The possible roles of these molecules in the development of S. frugiperda are discussed. School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Institute of Biosciences São Paulo State University (UNESP) Institute of Chemistry São Paulo State University (UNESP) Departamento de Ciências da Produção Agrícola Setor de Fitossanidade Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Via de Acesso Prof. Paulo Donato Castellane s/n School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Institute of Biosciences São Paulo State University (UNESP) Institute of Chemistry São Paulo State University (UNESP) Departamento de Ciências da Produção Agrícola Setor de Fitossanidade Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Via de Acesso Prof. Paulo Donato Castellane s/n CAPES: Finance Code 001

Made available in DSpace on 2021-06-25T10:28:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The intense spraying of pesticides to control different arthropod pests has resulted in negative side effects for the management of pests. It was previously discovered that exposure to non-acaricidal insecticides alone or in a mixture, results in lower efficiency of the acaricide spirodiclofen used for Brevipalpus spp. control. We investigate here whether the induced expression of detoxification enzymes by non-lethal insecticides may antagonize spirodiclofen toxicity. Brevipalpus californicus mites exposed to the insecticide phosmet alone or in combination with spirodiclofen showed increased activity of P450 monooxygenases (P450s). No antagonistic effects in mite mortality were observed by the combination of phosmet and spirodiclofen. On the other hand, mites exposed to the insecticide imidacloprid alone or in combination with spirodiclofen showed an increase in the activity of P450s, carboxylcholinesterases (CCE), and glutathione-S-transferases (GST). An antagonistic effect on mite mortality was observed when mites were exposed to the LC25 of spirodiclofen combined with the field rate treatment of imidacloprid. The addition of PBO (a P450 monooxygenase inhibitor) to the mixture of spirodiclofen and imidacloprid resulted in a synergistic effect over mite mortality but the addition of DEM (a GST inhibitor) resulted in an antagonist effect. Taken together, this study showed that the combination of imidacloprid with spirodiclofen is antagonistic for the control of B. californicus, and this results from the induction of detoxification enzymes, such as P450s, CCE, and GST. The use of inhibitors highlights the role of these enzymes in the antagonism of the mixture. Department of Agricultural Sciences São Paulo State University (UNESP) School of Agricultural and Veterinarian Sciences, Via de Acesso Prof. Paulo Donato Castellane S/N. Zip code: 14 Laboratory of Agrozoology Department of Crop Protection Faculty of Bioscience Engineering Ghent University, Coupure Links 653 Department of Agricultural Sciences São Paulo State University (UNESP) School of Agricultural and Veterinarian Sciences, Via de Acesso Prof. Paulo Donato Castellane S/N. Zip code: 14 FAPESP: #2019/08384-9 FAPESP: #301492/2018-2

Made available in DSpace on 2019-10-06T17:12:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Biological control using entomopathogens and natural enemies is an ecofriendly method for pest management in agriculture. Biological control agents often can be simultaneously employed and compatibility between agents may improve pest suppression. We investigated the influence of the entomopathogen Bacillus thuringiensis (Bt) on the immune system of the sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) to determine if such changes impact parasitization by Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae). The immune response of surviving D. saccharalis larvae fed with an LC25 dosage of a Bt-based biopesticide (Dipel®) was analyzed (total hemocyte count, hemocyte adhesion, and activities of phenoloxidase and lysozyme). Furthermore, the suitability of surviving Bt-fed larvae as hosts for C. flavipes was assessed by measuring parasitoid attributes such as number and size of teratocytes, weight of pupae, length of adult female tibia and number of emerged adults. Total hemocyte count, but not hemocyte adhesion, total protein content and phenoloxidase activity increased in the hemolymph of non-parasitized Bt-fed larvae (Bt-NP) compared to control larvae (NBt-NP). Lysozyme activity increased only after parasitization without Bt exposure (NBt-P). After parasitization, the immunological parameters activated in Bt-NP larvae decreased to levels at or below those observed in control larvae, showing that C. flavipes can regulate the activated immune response of Bt-fed larvae. The development of C. flavipes was not impaired in Bt-fed larval hosts (Bt-P); no changes were observed for teratocyte size, weight of pupal mass, length of hind tibia and number of adults emerged. São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences

Biological control using entomopathogens and natural enemies is an ecofriendly method for pest management in agriculture. Biological control agents often can be simultaneously employed and compatibility between agents may improve pest suppression. We investigated the influence of the entomopathogen Bacillus thuringiensis (Bt) on the immune system of the sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) to determine if such changes impact parasitization by Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae). The immune response of surviving D. saccharalis larvae fed with an LC

Made available in DSpace on 2018-12-11T17:32:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The giant cicada, Quesada gigas (Olivier) (Hemiptera: Cicadidae), is an important coffee pest and information about the behavior and reproduction of this species, e.g. emergence, senescence and ovarian maturation status, can be valuable to understand giant cicada ecology and to improve the use of a sound trap as a control method. A great number of Q. gigas adult males and females was captured using a sound trap and a protandrous type of emergence possibly associated with chorus centers formation was observed. All giant cicadas collected until 14–15 days after the beginning of male emergence (DAME) had immature ovaries at two different years of evaluation. On the other hand, the majority of cicadas collected from 20 until 48 DAME had mature ovaries with visible chorionated oocytes. Despite the use of the sound trap to collect insects for ecological studies, we believe that next generations of Q. gigas can be reduced by using this sound trap to hinder the formation of giant cicada chorus centers, to reduce male availability to copulate with females and to reduce the number of females to oviposit in coffee plants. School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Departamento de Fitossanidade, Via de Acesso Prof. Paulo Donato Castellane s/n School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) FAPESP: 14/06137-0

Parasitoids exploit host insects for food and other resources; they alter host development and physiology to optimize conditions to favor parasitoid development. Parasitoids influence their hosts by injecting eggs, along with a variety of substances, including venoms, polydnaviruses, ovarian fluids, and other maternal factors, into hosts. These factors induce profound changes in hosts, such as behavior, metabolism, endocrine events, and immune defense. Because endoparasitoids develop and consume tissues from within their hosts, it is reasonable to suggest that internal parasitization would also influence host food consumption and metabolism. We report on the effects of parasitism by Cotesia flavipes on the food consumption and utilization of its host, Diatraea saccharalis. Cotesia flavipes reduces the host food consumption, but parasitized larvae considered a unit with their parasitoid's attained the same final weight as the nonparasitized larvae. Nutritional indices, midgut activities of carbohydrases, and trypsin of parasitized and nonparasitized D. saccharalis were assessed. Parasitized larvae had reduced relative food consumption, metabolic and growth rates, coupled with higher efficiency for conversion of the digested, but not ingested, food into body mass. Parasitism also affected food flux through the gut and protein contents in the midgut of parasitized larvae. The activity of α-amylase and trehalase in parasitized host was enhanced in the first day after parasitism relative to control larvae. Saccharase activity remained unchanged during larval development. Trypsin activity was reduced from the fifth to ninth day after parasitism. We argue on the mechanisms involved in host food processing after parasitism.

Parasitoides criam condições favoráveis para seu desenvolvimento regulando processos fisiológicos do hospedeiro via a utilização de moléculas produzidas por tecidos maternos ou por tecidos derivados do parasitoide imaturo ou de simbiontes associados (vírus de poliDNA - PDV). Assim, essas moléculas se destacam como uma fonte preciosa para o desenvolvimento de métodos alternativos de controle de pragas. O objetivo desse trabalho foi o de explorar a interação hospedeiro-parasitoide, visando à identificação de moléculas que participem do processo de regulação hospedeira para o desenvolvimento de métodos alternativos de controle de pragas. A busca pela identificação dessas moléculas foi concentrada na interação Diatraea saccharalis (F.) (Lepidoptera: Crambidae) Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) pela avaliação i) do efeito do parasitismo na fisiologia digestiva do hospedeiro e ii) da contribuição da glândula de veneno e do ovário na produção de moléculas bioativas e iii) pela caracterização de transcritos produzidos por teratócitos desse parasitoide. O potencial de aplicação de moléculas derivadas da interação hospedeiro-parasitoide foi verificado pela caracterização da atividade de uma quitinase putativa isolada da associação Heliothis virescens (F.) (Lepidoptera: Noctuidae) Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae), via expressão heteróloga e transformação genética de plantas de fumo. Os resultados demonstraram que o parasitismo afeta a utilização do alimento por lagartas parasitadas, reduzindo as taxas de crescimento (RGR), consumo (RCR) e metabólica (RMR) relativas, enquanto aumenta a eficiência de conversão do digerido (ECD). O parasitismo por C. flavipes também aumentou a permanência do alimento no intestino do hospedeiro e a atividade de -amilase, sacarase e trealase em lagartas de D. saccharalis. Análises do transcriptoma da glândula de veneno e do ovário de C. flavipes indicaram a produção de muitas proteínas desconhecidas. Porém, foi possível identificar proteases, um inibidor de proteinase e uma proteína com ação antimicrobiana, produzidas pela glândula de veneno, e uma proteína de expressão precoce (ep1), como produto do ovário de C. flavipes. Dois transcritos isolados de teratócitos de C. flavipes foram putativamente identificados como serpina e fator de inibição da tradução (CfHTIF). Ambos os transcritos foram detectados em teratócitos após cinco dias do parasitismo, sendo que transcritos do CfHTIF também foram observados em hemócitos de hospedeiros parasitados. CfHTIF apresentou alta homologia a proteínas produzidas por PDVs associados a outras espécies de Cotesia, sugerindo a sua origem viral, sendo este o primeiro relato da utilização de teratócitos para a produção de proteínas virais voltadas à regulação hospedeira. A caracterização da atividade da quitinase de teratócitos de T. nigriceps e a avaliação do seu potencial biotecnológico via transgenia de plantas para o controle de lagartas de H. virescens indicou que, apesar dessa quitinase possuir capacidade de ligação à quitina coloidal e cristalina em ensaios in vitro, a mesma não demonstra qualquer atividade quitinolítica. Avaliações de plantas de fumo transformadas com o Tnchi não indicaram qualquer efeito deletério ao desenvolvimento de lagartas de H. virescens. Apesar da ausência de atividade quitinolítica, essa quitinase apresentou atividade antimicrobiana. A possibilidade de exploração dessa proteína na produção de plantas de interesse econômico é discutida. Parasitoids regulate their hosts physiological processes in order to produce suitable conditions for their own development by employing molecules produced by maternal tissues, the immature parasitoid and derived tissues, and associated simbionts (polidnavirus PDV). These molecules represent, therefore, an untapped resource of new molecules for the development of alternative pest control methods. Our objective was to exploit the hostparasitoid interactions aiming at the identification of molecules involved in host regulation for their use in the development of alternative strategies for pest control. The search for new molecules was concentrated on the interaction Diatraea saccharalis (F.) (Lepidoptera: Crambidae) Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) by assessing i) the effects of parasitization on the host digestive physiology and ii) the contribution of the venom gland and ovary in producing regulatory molecules, and by iii) the characterization of cDNAs isolated from teratocytes of C. flavipes. The potential application of molecules from hostparasitoid associations was verified by characterizing the activity of a putative chitinase isolated from the interaction Heliothis virescens (F.) (Lepidoptera: Noctuidae) Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) using recombinant proteins and geneticallymodified tobacco plants. Data obtained demonstrated parasitized larvae had reduced relative growth (RGR), consumption (RCR) and metabolic (RMR) rates, while an increased efficiency in the conversion of the digested food (ECD) was obtained as compared to unparasitized larvae. Larvae parasitized by C. flavipes retained the food in the gut for a longer period and had higher activities for -amylase, sacarase and trehalase if compared to control larvae. Analysis of the transcriptome of the venom gland and ovary of C. flavipes predicted a large number of unknown proteins. Nevertheless, several proteases, a protease inhibitor and an antimicrobial protein from the venom gland, and an early-expressed protein (ep1), with a putative role in the process of host regulation from the ovaries, were identified. Two cDNAs isolated from teratocytes of C. flavipes were putatively identified as a serpin and an eukaryote translation inhibitory factor (CfHTIF). Both were detected in teratocytes from day 5 of parasitism and onwards, with the CfHTIF being also detected in hemocytes of parasitized hosts. Comparative analysis of the CfHTIF indicated it is highly homologous to host translation inhibitory factors produced by PDVs associated with other species of Cotesia, indicating its possible viral origin and suggesting this to be the first record of teratocytes as a source of PDV-derived proteins for host regulation. The characterization of the activity from the chitinase isolated from teratocytes of T. nigreceps (Tnchi) and the evaluation of its biotechnological potential via plant transgenesis to control larvae of H. virescens indicated this chitinase can bind to colloidal and crystalline chitins in in vitro assays, but it does not display chitinolytic activity. Assays with transformed tobacco plants expressing Tnchi did not result in any effect to H. virescens larval development and survival. Although Tnchi did not have any chitinolytic activity, it did show to have an antimicrobial effect. The possible exploitation of this protein for production of economically-important plants is dicussed.

Parasitoids exploit host insects for food and other resources; they alter host development and physiology to optimize conditions to favor parasitoid development. Parasitoids influence their hosts by injecting eggs, along with a variety of substances, including venoms, polydnaviruses, ovarian fluids, and other maternal factors, into hosts. These factors induce profound changes in hosts, such as behavior, metabolism, endocrine events, and immune defense. Because endoparasitoids develop and consume tissues from within their hosts, it is reasonable to suggest that internal parasitization would also influence host food consumption and metabolism. We report on the effects of parasitism by Cotesia flavipes on the food consumption and utilization of its host, Diatraea saccharalis. Cotesia flavipes reduces the host food consumption, but parasitized larvae considered a unit with their parasitoid's attained the same final weight as the nonparasitized larvae. Nutritional indices, midgut activities of carbohydrases, and trypsin of parasitized and nonparasitized D. saccharalis were assessed. Parasitized larvae had reduced relative food consumption, metabolic and growth rates, coupled with higher efficiency for conversion of the digested, but not ingested, food into body mass. Parasitism also affected food flux through the gut and protein contents in the midgut of parasitized larvae. The activity of α-amylase and trehalase in parasitized host was enhanced in the first day after parasitism relative to control larvae. Saccharase activity remained unchanged during larval development. Trypsin activity was reduced from the fifth to ninth day after parasitism. We argue on the mechanisms involved in host food processing after parasitism.

Os insetos possuem diferentes enzimas digestivas que catalisam as reações de hidrólise do alimento consumido e essas se diferenciam entre os insetos de acordo com suas dietas e estado fisiológico. Foram avaliadas as atividades de algumas enzimas digestivas da praga do cafeeiro - Leucoptera coffeella (Guérin-Mèneville & Perrottet, 1842) (Lepidoptera: Lyonetiidae) - popularmente conhecida como bicho-mineiro do cafeeiro, para o entendimento de seu processo digestivo. Lagartas do bicho-mineiro do cafeeiro foram coletadas em campo e em casa-de-vegetação. O extrato enzimático utilizado foi obtido pela maceração das lagartas em água (4ºC). Determinaram-se os pH's ótimos e as atividades das enzimas α e β-glicosidases, α-amilase, aminopeptidase, fosfatase alcalina, sacarase, trealase e tripsina, incubando o extrato enzimático do bicho-mineiro do cafeeiro com substratos específicos. A análise dos resultados sugere que o processo digestivo e o ambiente intestinal do bicho-mineiro do cafeeiro sejam similares com o dos demais lepidópteros encontrados na literatura. Insects are fitted with different digestive enzymes that catalyses the food hydrolysis. Those enzymes differ from one insect to another according to their diets and physiological status. In this work, one intended to verify the activities of some digestive enzymes of the coffee leaf miner - Leucoptera coffeella (Guérin-Mèneville & Perrottet, 1842) (Lepidoptera: Lyonetiidae) - as a pre-requisite to understand its digest process, since this insect is a major plague in coffee production systems. Coffee leaf miner caterpillars were collected in fields and in greenhouse. The enzyme extract utilized in determining the enzyme activities was obtained through grinding the caterpillars in cold water. The optimum pH and the activities of the enzymes α and β-glucosidases, α-amylase, aminopeptidase, alkaline phosphatase, saccharase, trehalase and trypsin were measured by incubating the enzyme extract with specific substrates. The analysis of the optimum pH's indicated that the digestive process and intestinal environment of this insect are similar to another lepidopterans consulted in the literature.

A soja (Glycine max (L.) Merrill) com o evento DAS-81419-2 (ConkestaTM technology) que expressa as proteínas de Bacillus thuringiensis (Bt) Cry1F e Cry1Ac protege a cultura de danos de alimentação de lepidópteros-praga primários e secundários. Experimentos de campo foram conduzidos de 2011 à 2016 na região sul e central do Brasil para caracterizar a eficácia da soja DAS-81419-2 infestada com Anticarsia gemmatalis (Lepidoptera: Erebidae), Chrysodeixis includens (Lepidoptera: Noctuidae), Chloridea virescens (Lepidoptera: Noctuidae), Spodoptera cosmioides (Lepidoptera: Noctuidae), Elasmopalpus lignosellus (Lepidoptera: Pyralidae), Agrotis ipsilon (Lepidoptera: Noctuidae) e Helicoverpa armigera (Lepidoptera: Noctuidae) durante os estágios vegetativo (V4) e reprodutivo (R2 e/ou R4) da cultura. A eficácia da soja DAS-81419-2 foi comparada com a variedade isogênica não-Bt manejada com ou sem inseticidas foliares comerciais para controle de lepidópteros. DAS-81419-2 reduziu significantemente o dano de alimentação causado por A. gemmatalis, C. includens, C. virescens, S. cosmioides e H. armigera durante os estágios vegetativo e reprodutivo, bem como, a mortalidade de plântulas causada por E. lignosellus. Entretanto, DAS-81419-2 apresentou moderada atividade em A. ipsilon. Além disto, um estudo de campo de três anos avaliou os potenciais efeitos adversos de DAS-81419-2 por meio da inspeção da diversidade e abundância de artrópodes não-alvos comparado com a isolinha não-Bt com ou sem aplicações convencionais de inseticidas. Estes estudos foram conduzidos em três regiões produtoras de soja onde os artrópodes não-alvos da parte aérea e da superfície do solo foram monitorados. Em geral, a abundância e diversidade total não foram afetadas signifativamente pela soja Bt. Desta maneira, DAS-81419-2 mostrou eficácia consistente ao longo dos anos, localidades e diferente estágios da cultura contra lepidópteros-pragas primários e secundários de importância econômica da cultura da soja e não afetou adversamente os artrópodes não-alvos que ocorrem nos campos de soja. Soybean (Glycine max (L.) Merrill) event DAS-81419-2 (ConkestaTM technology) expresses Bacillus thuringiensis (Bt) Cry1F and Cry1Ac proteins to provide protection against primary and secondary lepidopteran pest feeding damage. Field experiments were conducted from 2011 to 2016 in southern and central Brazil to characterize the efficacy of DAS-81419-2 soybean infested with Anticarsia gemmatalis (Lepidoptera: Erebidae), Chrysodeixis includens (Lepidoptera: Noctuidae), Chloridea virescens (Lepidoptera: Noctuidae), Spodoptera cosmioides (Lepidoptera: Noctuidae), Elasmopalpus lignosellus (Lepidoptera: Pyralidae), Agrotis ipsilon (Lepidoptera: Noctuidae), and Helicoverpa armigera (Lepidoptera: Noctuidae) during vegetative (V4) and reproductive (R2 and/or R4) crop developmental stages. The efficacy of DAS-81419-2 was compared to a non-Bt isogenic variety managed with or without applications of commercial foliar insecticides for lepidopteran control. DAS-81419-2 significantly reduced the feeding damage caused by A. gemmatalis, C. includens, C. virescens, S. cosmioides, and H. armigera during both vegetative and reproductive crop growth stages as well as the seedling stage plant mortality caused by E. lignosellus. However, DAS-81419-2 showed moderate activity on A. ipsilon. In addition, a 3-yr in-field study evaluated the potential adverse effects of DAS-81419-2 by examining the diversity and abundance of non-target arthropods (NTAs) compared to the non-Bt near isoline with and without conventional insecticide applications. These studies were conducted in three Brazilian soybean producing regions where both soil surface and aerial NTA were monitored. Overall, the total abundance and diversity were not significantly affected by Bt soybean. Therefore, DAS-81419-2 showed consistent efficacy across years, locations, and crop stages against the most economically important primary and secondary lepidopteran pests of soybean and did not adversely affect non-target arthropods occurring in soybean field.

Os insetos são conhecidos por estabelecer relações de simbiose com bactérias há muito tempo, mas o papel das associações secundárias ainda é pouco conhecido, e por isso há necessidade de avaliações continuadas dos efeitos dessas interações nos insetos hospedeiros. Assim, esse trabalho teve como objetivo a caracterização da diversidade de simbiontes associados ao pulgão Lipaphis pseudobrassicae Davis (Hemiptera: Aphididae), bem como o efeito dessas associações na biologia e utilização hospedeira por esse inseto. Linhagens clonais de L. pseudobrassicae estabelecidas em laboratório foram submetidas a PCRs diagnósticos para a verificação da associação a simbiontes secundários, sendo selecionadas cinco linhagens clonais infectadas exclusivamente por Buchnera (S-) e cinco linhagens clonais infectadas pelo simbionte secundário Spiroplasma (S+). Assim, as linhagens clonais S- e S+ foram utilizadas para condução de estudos de biologia comparada em duas plantas hospedeiras, Brassica oleracea var. capitata (repolho 60 dias) e Brassica oleracea var. acephala (couve manteiga Geórgia), para a verificação do efeito da infecção por Spiroplasma na biologia e utilização hospedeira por L. pseudobrassicae. O delineamento experimental foi em blocos completamente casualizados, sendo isolinhagem e planta hospedeira tratados como fatores, com o fator infecção sendo aninhado ao fator isolinhagem. Os pulgões foram avaliados quanto à sobrevivência (%) e duração da fase ninfal de desenvolvimento (dias), fecundidade diária e total (número de ninfas por fêmea) e longevidade (dias). Para a avaliação da duração da fase ninfal, ninfas neonatas (10) de cada linhagem clonal S- e S+ foram utilizadas na infestação das plantas hospedeiras selecionadas. A mortalidade das ninfas foi inicialmente avaliada 24 horas após a sua transferência para as plantas hospedeiras para se verificar a mortalidade devido à manipulação, tendo a sua sobrevivência avaliadas diariamente para observação da mortalidade e duração da fase ninfal. Cada grupo de dez ninfas de cada linhagem clonal, inoculada em cada planta hospedeira foi considerada uma repetição biológica, sendo utilizadas 12 repetições/tratamento (=120 insetos/tratamento). Para a avaliação do efeito da infecção por Spiroplasma no potencial reprodutivo de L. pseudobrassicae, quando utilizando diferentes hospedeiros, fêmeas recém-emergidas de cada linhagem clonal foram individualizadas em plantas de cada hospedeiro estudado para a observação de sua capacidade reprodutiva e longevidade. Foram utilizadas 25 repetições (1 fêmea = 1 repetição) para cada tratamento (infecção/isolinhagem/planta hospedeira). Os pulgões foram avaliados diariamente para contabilização da fecundidade (número total de ninfas/fêmea), longevidade (dias) e duração da fase reprodutiva do adulto. Os resultados obtidos indicaram que infecções de L. pseudobrassicae por Spiroplasma não influenciaram os atributos de duração da fase ninfal, duração da fase reprodutiva, longevidade e fecundidade quando utilizando couve ou repolho. A sobrevivência ninfal foi afetada pela interação isolinhagem x planta. Nesse caso, linhagens clonais de L. pseudobrassicae, infectadas ou não por Spiroplasma, diferem entre si quando explorando couve como planta hospedeira. Além dessa variável, os parâmetros avaliados pela tabela de vida demonstraram sofrer efeito da isolinhagem (aninhada à infecção), e de algumas interações (isolinhagem x planta, planta x infecção). Assim, concluímos que Spiroplasma interfere apenas nos parâmetros avaliados através da tabela de vida. Insects have been known to establish symbiotic relationships with bacteria for a long time, however the role of secondary associations is still poorly known, therefore there is a need for continued evaluation of the effects of these interactions on host insects. Thus, this study aimed to characterize the diversity of symbionts associated with the aphid Lipaphis pseudobrassicae Davis (Hemiptera: Aphididae), as well as the effect of these associations in the biology and host use by this insect. Laboratory clonal lines of L. pseudobrassicae were submitted to diagnostic PCRs to verify the association to secondary symbionts. Five clonal lines exclusively infected by Buchnera (S-) and five clonal lines infected with the secondary symbiont Spiroplasma (S +) were selected. Thus, clones S- and S + were used to conduct comparative biology studies in two host plants, Brassica oleracea var. capitata (cabbage 60 days) and Brassica oleracea var. acephala (kale butter Georgia), for the verification of the effect of Spiroplasma infection on biology and host utilization by L. pseudobrassicae. The experimental design was completely randomized blocks, with isoline and host plant treated as factors, with the infection factor being nested to the isoline factor. The aphids were evaluated for survival (%) and duration of nymphal phase of development (days), daily and total fecundity (number of nymphs per female) and longevity (days). In order to evaluate the duration of the nymphal phase, neonatal nymphs (10) of each S- and S + clonal lines were used in the infestation of the selected host plants. The mortality of the nymphs was initially evaluated 24 hours after their transfer to the host plants to verify the mortality due to manipulation, and their survival was evaluated daily to observe the mortality and duration of the nymphal phase. Each group of ten nymphs of each clonal line inoculated in each host plant was considered a biological replicate, using 12 replicates / treatment (= 120 insects / treatment). For the evaluation of the effect of Spiroplasma infection on the reproductive potential of L. pseudobrassicae, when using different hosts, newly emerged females of each clonal lineage were individualized in plants of each host studied to observe their reproductive capacity and longevity. Twenty five replicates (1 female = 1 replicate) were used for each treatment (infection / isolation / host plant). The aphids were evaluated daily for fecundity (total number of nymphs / female), longevity (days) and duration of the adult reproductive phase. The results indicated that L. pseudobrassicae infections by Spiroplasma did not influence the attributes of nymphal phase and reproductive phase duration, longevity and fecundity when using kale butter or cabbage. The only variable influenced by the studied factors was nymphal survival, which was affected by the isolation x plant interaction. In this case, clonal lines of L. pseudobrassicae, infected or not by Spiroplasma, differ among them when using cabbage as host plant. In addition to this variable, the parameters evaluated by the life table showed an effect of the isolation (nested to the infection), and of some interactions (isolation x plant, plant x infection). Therefore, we conclude that Spiroplasma interferes only in the evaluated parameters through the life table.

Ao longo do processo evolutivo, parasitoides desenvolveram inúmeras estratégias para a colonização de seus hospedeiros, resultando em mecanismos específicos de regulação. Endoparasitoides cenobiontes depositam seus ovos no hospedeiro e manipulam sua fisiologia e suas repostas imunológicas, a fim de tornar o hospedeiro um ambiente seguro e nutricionalmente adequado para o desenvolvimento do imaturo. A associação de parasitoides das famílias Braconidae e Ichneumonidae com vírus da família Polydnaviridae e sua injeção no hospedeiro configuram-se como um dos principais mecanismos de regulação utilizados durante o parasitismo, devido sua ação sobre a expressão de genes do hospedeiro. O conjunto diverso de fatores maternos e larvais apresentados por parasitoides pode apresentar potencial biotecnológico no controle de pragas, que busca por novos nichos de exploração. Apesar disso, a especificidade das interações hospedeiro - parasitoide limita a exploração de eventos que utilizam tais fatores de virulência, baseada na necessidade de táticas que apresentem ação sobre maior número de espécies-praga. Dessa forma, a presente tese teve por objetivo identificar fatores de virulência expressos durante a interação Cotesia flavipes Cameron (Hymenoptera: Braconidae) - Diatraea saccharalis (F.) (Lepidoptera: Crambidae), buscando a identificação de fatores com potencial biotecnológico, e identificar vias metabólicas do hospedeiro alvos de regulação, buscando por genes-alvos de controle. Além disso, essa tese buscou avaliar o potencial biotecnológico de um fator de virulência (CfHTIF) pertencente ao vírus simbionte de C. flavipes e de um fator de virulência larval (TnQuit) expresso por teratócitos associados a Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) contra hospedeiros incomuns. O parasitismo e a injeção de fatores maternos por C. flavipes resultou na regulação de inúmeras vias metabólicas de D. saccharalis, principalmente relacionadas ao controle do sistema endócrino e do sistema imunológico e identificou a expressão de genes virais e larvais associados a C. flavipes. Plantas transformadas com o fator de virulência TnQuit apresentaram efeitos na sobrevivência e no desenvolvimento dos hospedeiros não-permissivos de hábito mastigador, Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae), Spodoptera albula (Walker) (Lepidoptera: Noctuidae) e Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), minador, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), e sugador, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), enquanto que o gene viral CfHTIF interferiu nas eficiências de conversão do alimento ingerido e digerido de S. albula, mas não de S. frugiperda. Parasitoids developed many strategies for colonization of their hosts during the evolution of associations with their hosts, which resulted in the development of specific mechanisms of host regulation. Koinobiont endoparasitoids deposit their eggs into the host and manipulate the host´s physiology and immune responses in order to turn the host a safe and nutritionally suitable environment for parasitoid immature development. The association of parasitoids belonging to Braconidae and Ichneumonidae with virus of the family Polydnaviridae, which are injected in the host, constitutes one of the main mechanisms of regulation used during the parasitization due to viral regulation of the host gene expression. The diverse set of maternal and larval-derived factors produced by parasitoids may have biotechnological potential for pest control. Nevertheless, the specificity of the host - parasitoid interactions are thought to limit the exploration of events that use such virulence factors, as a consequence on the need of successful tactics for a large number of pest species. Thus, we aimed to identify the virulence factors expressed during the interaction Cotesia flavipes Cameron (Hymenoptera: Braconidae) - Diatraea saccharalis (F.) (Lepidoptera: Crambidae) to identify factors with biotechnological potential and to identify targets of regulation that could aid on the development of additional control tactics. In addition, we evaluated the biotechnological potential of a virulence factor (CfHTIF) belonging to the symbiont virus of C. flavipes and a larval virulence factor (TnQuit) expressed by teratocytes associated to Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) against non-host species. Parasitization and pseudoparasitization of D. saccharalis larvae by C. flavipes resulted in the regulation of several metabolic pathways, particularly those related to the endocrine and immune systems. We also characterized the transcriptome of C. flavipes and of associated bracovirus in parasitized and pseudoparasitized hosts. Plants transformed with the TnQuit virulence factor affected survival and development of the non-permissive hosts Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae), Spodoptera albula (Walker) (Lepidoptera: Noctuidae) and Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and the number of developing adults of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). CfHTIF-transgenic plants affected the efficiency of conversion of the digested and ingested food of S. albula larvae, but not of S. frugiperda larvae.

Insetos representam a maioria dos seres vivos na Terra. O sucesso biológico desses organismos está, em parte, relacionado às associações com microrganismos que influenciam aspectos de sua bioecologia, incluindo as relações com o primeiro e terceiro níveis tróficos. Assim como os insetos, plantas também apresentam associações com microrganismos que podem interferir na sua nutrição e relação com patógenos e herbívoros. Esse trabalho buscou analisar a capacidade de simbiontes associados a insetos de atuar como endofíticos de planta e influenciar, assim, o potencial biótico da planta e a sua capacidade de defesa contra a herbivoria, utilizando o modelo milho - Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae). Quatro simbiontes de insetos foram selecionados. Três deles (IIL-Sfm05, IILSfc-sus01 e IIL-Sfb05) associados ao trato digestivo de S. frugiperda e outro (IIL-ASP45) de Acromyrmex coronatus (IIL-ASP45). Adicionalmente, um endofítico (IILzm-Idp03) isolado do próprio milho foi adicionado como controle positivo. Os ensaios foram conduzidos após cultivo de sementes inoculadas com os diferentes microrganismos via bio-priming, aplicação no solo e aplicação foliar. Dos cinco isolados testados, três (IILSfc-sus01, IIL-Sfb05 e IILzm-Idp03) induziram maior crescimento em plantas de milho; porém, a forma de disponibilização dos microrganismos influenciou a resposta da planta. O isolado IILzm-Idp03 induziu o maior crescimento da planta pela sua disponibilização via bio-priming; IILzm-Idp03 e IIL-Sfb05 via inoculação do solo; e o isolado IILSfc-sus01 via inoculação foliar. Testes biológicos com S. frugiperda em plantas de milho inoculadas ou não com os microrganismos selecionados indicaram que dois deles (isolados IIL-Sfb05 e IIL-ASP45) resultaram em relações patogênicas, chegando à induzir a completa mortalidade de insetos alimentados em tecidos foliares de plantas inoculadas com esses microrganismos. Testes de preferência alimentar demonstraram que as lagartas não foram influenciadas pela alimentação com folhas de plantas inoculadas com as bactérias via bio-priming. Estudos comparativos dos índices nutricionais de plantas tratadas com as diferentes bactérias, indicaram que aquelas tratadas com IIL-ASP45 resultaram no menor crescimento e desenvolvimento larval de S. frugiperda, corroborado pelo menor consumo e índices nutricionais alcançados nessa fonte de alimento. A alimentação em plantas tratadas com IIL-Sfb05 causou 100% de mortalidade larval, não sendo possível a avaliação dos índices nutricionais. Os resultados desse trabalho demonstram o potencial de manipulação da planta hospedeira por bactérias associadas ao herbívoro, bem como o potencial biocida de microrganismos associados a insetos. Insects represent most of the living things on Earth. The biological success of these organisms is in part related to associations with microorganisms that influence aspects of their bioecology, including relationships with the first and third trophic levels. Like insects, plants also have associations with microorganisms that may interfere in their nutrition and interactions to pathogens and herbivores. This work sought to assess the potential of symbionts associated with insects to act as plant endophytes and thus to influence plant growth and its utilization by the herbivory using the maize - Spodoptera frugiperda (JE Smith, 1797) (Lepidoptera: Noctuidae) model system. Four insect symbionts were selected. Three of them (IIL-Sfm05, IILSfc-sus01 and IIL-Sfb05) associated with the digestive tract of S. frugiperda and another (IIL-ASP45) with the cuticle of Acromyrmex coronatus (IIL-ASP45). Additionally, an endophyte (IILzm-Idp03) from maize was added as a positive control. Experiments were conducted after inoculation of the different microorganisms via bio-priming, soil application and foliar application. Of the five isolates tested, three (IILSfc-sus01, IIL-Sfb05 and IILzm-Idp03) induced higher growth in maize plants; however, the inoculation of the seeds with the microorganisms influenced the plant response. The isolate IILzm-Idp03 induced the highest plant growth when inoculated via bio-priming; IILzm-Idp03 and IIL-Sfb05 via soil inoculation; and IILSfc-sus01 via foliar inoculation. Biological tests with S. frugiperda on maize plants inoculated or not with the selected microorganisms indicated that two of them (IIL-Sfb05 and IIL-ASP45 isolates) were highly pathogenic, inducing very high larval mortality. Our analysis indicated no changes in larval feeding preference. Comparative analysis of larval food consumption and utilization indicated that plants inoculated with IIL-ASP45 resulted in the lowest growth and larval development of S. frugiperda, corroborated by the lower consumption and nutritional indexes reached in this food source. Feeding in plants inoculated with IIL-Sfb05 caused 100% larval mortality, and it was not possible to evaluate the nutritional indexes. Our data demonstrate the potential for manipulation of the host plant by bacteria associated with the herbivore, as well as the biocidal potential of insect - associated microorganisms.

A associação de diferentes seres vivos favoreceu a exploração de novos hábitats e outras fontes de alimento, contribuindo nos processos de adaptação e de diversificação biológica. Essa associação, em que duas espécies passam a viver juntas, é denominada simbiose, e as associações de simbiose podem resultar em relações benéficas (mutualismo), neutras (comensalismo) ou prejudiciais (parasitismo) aos organismos envolvidos. Insetos estabelecem associações de simbiose diversas com outros organismos, como leveduras, vírus, protistas e bactérias. Dentre esses organismos, as bactérias são as mais comumente associadas a insetos. Elas podem se localizar no interior do corpo do hospedeiro, tanto no espaço extracelular ou endocelular (endossimbionte), assim como associadas externamente ao corpo do hospedeiro (ectossimbionte). Endossimbiontes são os que mais diretamente influenciam a fisiologia e o metabolismo do hospedeiro, tornando-se, em alguns casos, essenciais à sobrevivência de seu hospedeiro. Exemplo disso são as bactérias endossimbiontes associadas a pulgões, Buchnera aphidicola, que são responsáveis pela síntese de aminoácidos essenciais e vitaminas para complementar os recursos nutricionais explorados pelos pulgões, seiva de plantas, que normalmente apresentam baixo valor nutricional. Mas vários outros simbiontes, mesmo que não estabelecendo essa obrigatoriedade na relação com o hospedeiro, acabam influenciando inúmeros aspectos da bioecologia de seus pulgões hospedeiros, como, por exemplo, a capacidade de defesa de seus hospedeiros a inimigos naturais. Spiroplasma são bactérias que influenciam o sistema de reprodução de borboletas, moscas-das-frutas, joaninhas e pulgões, além de influenciar a resposta de seus hospedeiros ao ataque de inimigos naturais, como parasitoides e nematoides. Porém, pouco se sabe sobre os efeitos desse simbionte no metabolismo de seus hospedeiros. Assim, este trabalho teve por objetivo avaliar o efeito de infecções por Spiroplasma no metaboloma de seu hospedeiro Aphis citricidus (Kirkaldy) (Hemiptera: Aphididae). As análises foram conduzidas utilizando-se de isolinhagens irmãs de A. citricidus, infectadas ou não por Spiroplasma. Fêmeas adultas de A. citricidus, infectadas ou não por Spiroplasma, foram coletadas (25 mg/amostra), submetidas aos processos de extração com solução de clorofórmio, metanol e água (1:3:1) e derivatização com n-metil-n-(trimetilsilil)trifluoroacetamida e trimetilclorosilano, para posterior análise por cromatografia gasosa acoplada à espectrometria de massas (GCMS-TOF) para a detectação de metabólitos. Após tratamento e análise dos dados obtidos, foi observado que 40 metabólitos foram afetados significativamente em pulgões infectados por Spiroplasma. Dentre os metabólitos de A. citricidus alterados pela infecção por Spiroplasma, merecem destaque os hidrocarbonetos cuticulares, compostos nitrogenados e ácidos graxos. Os efeitos metabólicos da interação Aphis citricidus - Spiroplasma revelou informações importantes sobre a influência do endossimbionte na composição química de seus hospedeiros. The association between different living beings favored the exploitation of new habitats and food sources, contributing to adaptation and biological diversity. This association in which two species live together is named symbiosis, and associations of symbiosis can result in beneficial (mutualism), neutral (comensalism) or prejudicial (parasitism) interactions. Insects established a diverse range of associations with other organisms, such as yeasts, virus, protists and bacteria. Bacteria are by far the most commonly associates of insects. Bacteria can be located within the host body, either at the extra or intracellular space (endosymbionts), as well as on the body surface (ectosymbionts). Endosymbionts are the ones that most influence the host physiology and metabolism, and they may even become essential for the host survival. An example of such dependency is the aphid symbiont Buchnera aphidicola that synthesis essential amino acids and vitamins to complement the natural diet of host, which are of a low nutritional value. But many other symbionts that are not essential do the host can affect a number of bioecological aspects of their host aphids, as the host immune capacity to natural enemies. Spiroplasma are bacteria that can influence the reproduction system of butterflies, fruit flies, ladybugs and aphids, and interfere with the host immune response to parasitoids and nematodes. However, little is known of the effects of secondary symbionts on their host\'s metabolism. Thus, this work is aimed to detect the effects of Spiroplasmainfection on the metabolomics of the apid host Aphis citricidus (Kirkaldy) (Hemiptera: Aphididae). Analysis were conducted using sister isolines A. citricidus, infected or not with Spiroplasma. Infected and uninfected adult females were collected from lab colonied (25 mg/samples), subjected to metabolite extraction in chloroform, methanol and water (1:3:1) and derivatization with n-methyl-n-(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane, followed by metabolite analysis in a gas chromatography coupled with a mass spectrometer (GCMS-TOF). After filtering and data analysis, 40 metabolites were observed in different intensities in between infected and uninfected aphids. The aphid\'s metabolites influenced by infection with Spiroplasma indicated the regulation of the metabolism of fatty acids, hydrocarbons and nitrogen compounds. The metabolic effects of the interaction Aphis citricus - Spiroplasma revealed interesting information on the host metabolism regulation by a secondary symbiont and on the role these changes may play on the associations of the host with other organisms.

Parasitoides interferem no sistema imunológico de seus hospedeiros, influenciando a expressão de genes relacionados à resposta celular e humoral, podendo interferir na relação hospedeiro - microbiota intestinal. Além disso, parasitoides induzem alterações fisiológicas no hospedeiro que alteram o consumo e a utilização de alimento, e que podem influenciar a microbiata intestinal do mesmo. Alterações nessa microbiota poderiam afetar as relações e contribuições ao hospedeiro e, consequentemente, influenciar o desenvolvimento do próprio parasitoide. O objetivo deste trabalho foi o de verificar o efeito do parasitismo por Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) na estrutura e no potencial funcional de contribuição da microbiota intestinal de Diatraea saccharalis (F.) (Lepidoptera: Crambidae), hospedeiro permissivo, e de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), hospedeiro não-permissivo. Além disso, buscou-se verificar se as secreções utilizadas pelo parasitoide (veneno, fluidos do cálice e virus simbionte) na regulação hospedeira estariam associadas à manipulação da microbiota intestinal do hospedeiro. O efeito do parasitismo na microbiota intestinal associada às porções antero-mediana e posterior do intestino dos hospedeiros estudados foi avaliado na fase inicial (1 DAP - dia após o parasitismo), intermediária (5 DAP) e final (9 DAP) do desenvolvimento larval do parasitoide. A avaliação foi feita por meio da comparação da diversidade e abundância de bactérias associadas ao trato intestinal de D. saccharalis e S. frugiperda parasitadas ou não por C. flavipes. A caracterização das bactérias foi feita via análise metagenômica em plataforma Illumina MiSeq utilizando a região V4 do gene ribossomal 16S. O pacote de softwares QIIME foi utilizado para a atribuição taxonômica das mesmas e o potencial funcional foi inferido por meio do software PICRUSt. O parasitismo afetou a abundância e diversidade de unidades taxonômicas operacionais (UTOs) da microbiota intestinal da porção antero-mediana e posterior de ambos hospedeiros. As alterações observadas para as duas regiões intestinais investigadas não seguiram o mesmo padrão ao longo do desenvolvimento do parasitoide. As análises realizadas também demonstraram que as alterações da microbiota induzidas pelo parasitismo refletiram em alterações significativas no potencial funcional de contribuição da microbiota associada ao trato digestivo de D. saccharalis e S. frugiperda. As análises da microbiota de lagartas pseudo-parasitadas demonstraram que as secreções maternas injetadas pela fêmea do parasitoide no momento do parasitismo estão envolvidas, pelo menos parcialmente, com os processos que levam às modificações na diversidade e abundância da microbita intestinal hospedeira, assim como de seu potencial de contribuição funcional. Esses resultados indicam que outros fatores/alterações produzidos em condições normais de parasitismo, seja pela influência de secreções de teratócitos e das próprias larvas do parasitoide em desenvolvimento também estão envolvidos na manipulação da microbiota hospedeira. Várias das alterações observadas no potencial de contribuição da microbiota intestinal do hospedeiro podem refletir sua qualidade nutricional e, consequentemente, favorecer sua exploração pelo parasitoide. Assim, o processo de regulação hospedeira por parasitoides se estende ao conjunto de organismos associados que compõem o holobionte representado pela lagarta hospedeira. Parasitoids interfere with the immune system of their hosts by influencing the expression of genes related to cellular and humoral responses, which may interfere with the host - gut microbiota relationship. Furthermore, parasitoids induce physiological changes in the host, modifying food consumption and utilization, influencing then the host gut microbiota. These changes can affect the relationship and contributions of the gut microbiota to the host and therefore influence parasitoid development. This study aimed to evaluate the effect of parasitism by Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) in the structure and potential functional contribution of the gut microbiota of the permissive host Diatraea saccharalis (F.) (Lepidoptera: Crambidae) and the non-permissive host Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) . In addition, the participation of the secretions female parasitoids (venon, calyx fluid and symbiotic virus) use in host regulation in the manipulation of the host gut microbiota was also investigated. The effects of host parasitization on the microbiota associated with the anterior (foregut-midgut) and posterior (hindgut) portions of host gut were evaluated at the early (1 DAP - day after parasitism), intermediate (5 DAP) and final (9 DAP) stages of parasitoid larval development. The diversity and abundance of the gut microbiota of D. saccharalis and S. frugiperda was compared in between parasitized and non-parasitized larvae by C. flavipes. The gut microbiota was characterized by sequencing the V4 region of the 16S ribosomal gene using the Illumina MiSeq platform. The software package QIIME was used for taxonomic attribution and the PICRUSt software was used to infer the potential funcional contribution of the gut microbiota. Host parasitization affected the abundance and diversity of operational taxonomic units (OTUs) of the two gut regions investigated (foregut-midgut and hindgut) in both hosts. The changes observed for both gut regions did not follow the same pattern throughout parasitoid development. Changes in the gut microbiota induced by parasitization reflected in significant changes in the potential of the functional contribution of the gut microbiota associated with D. saccharalis and S. frugiperda. Analyses of pseudo-parasitized larvae demonstrated that the maternal secretions female parasitoids inject when ovipositing are involved, at least partially, with the processes that lead to changes in the abundance, diversity and potential functional contribution of the host gut microbita. These results indicate that other factors / changes produced during normal parasitization, such as secretions from teratocytes and/or the developing parasitoid larvae can also be involved in the manipulation of host gut microbiota. Several of the changes observed in the potential contribution of the host gut microbiota may reflect its nutritional quality and therefore favor host exploitation by parasitoids. Thus, the process of host regulation by parasitoids also involves the regulation of the gut-associated bacteria, which altogether comprise the holobionte represented by the host larvae.