Spodoptera frugiperda (J. E. Smith) e Pseudoplusia includens (Walker) são importantes insetos-praga no algodoeiro (Gossypium hirsutum L.) devido às injúrias de desfolha e destruição de estruturas reprodutivas no caso de S. frugiperda. Os eventos de algodão Bt que expressam as proteínas Cry1Ac/Cry2Ab2 (Bollgard® II) e Cry1Ac/Cry1F (WideStrike(TM)) de Bacillus thuringiensis Berliner são ferramentas disponíveis para o controle dessas espécies-praga. A fim de subsidiar o Manejo da Resistência de Insetos (MRI) foram conduzidos estudos em laboratório para avaliar a sobrevivência e desenvolvimento de S. frugiperda e P. includens nos eventos de algodão Cry1Ac/Cry2Ab2 e Cry1Ac/Cry1F. Em bioensaios com discos de folhas, a eficácia de controle de neonatas nos dois eventos de algodão Bt foi superior a 80% para S. frugiperda e de 100% para P. includens. Em bioensaios com brácteas com neonatas de S. frugiperda, a eficácia de controle de ambos os eventos de algodão Bt também foi superior a 80%. As lagartas de S. frugiperda sobreviventes em algodão Bt apresentaram severa inibição de desenvolvimento larval em folhas (> 75%) e brácteas (> 44%). Em bioensaios com simulações de alimentação larval, as quais consistiam em grupos de lagartas alimentadas com o algodão Bt aos 0, 3, 6, 9, 12, 15 e 18 dias após a inoculação (DAI), S. frugiperda e P. includens demonstraram que a suscetibilidade dessas espécies diminuiu com o avançar do desenvolvimento larval. Para S. frugiperda, em todas as simulações de alimentação com o algodão Cry1Ac/Cry2Ab2 houve lagartas que atingiram as fases de pupa e adulto. Por outro lado, quando expostas ao algodão Cry1Ac/Cry1F, somente algumas das lagartas de 5º e 6º ínstares atingiram as fases de pupa e adulto. Para P. includens, somente lagartas no 6º ínstar atingiram as fases de pupa e adulto quando alimentadas com os dois eventos de algodão Bt. Os parâmetros biológicos de S. frugiperda sobreviventes em algodão Cry1Ac/Cry2Ab2 foram afetados negativamente com aumento da duração da fase larval (? 9 dias), baixa viabilidade larval (1,4%) e de insetos que completaram o ciclo biológico (0,9%), aumento no intervalo entre gerações (? 9 dias) e redução da taxa intrínseca de crescimento populacional (? 83%). Os eventos de algodão Cry1Ac/Cry2Ab2 e Cry1Ac/Cry1F são promissores no controle de S. frugiperda e P. includens. No entanto, a atividade inseticida dos dois eventos de algodão Bt em lagartas de S. frugiperda e P. includens diminui com o desenvolvimento larval e essa constatação deve ser considerada em programas de MRI, especialmente na disposição espacial do refúgio. / Spodoptera frugiperda (J. E. Smith) and Pseudoplusia includens (Walker) are important insect pests in cotton (Gossypium hirsutum L.) due to damage on leaves and reproductive structures in the case of S. frugiperda. The events of Bt cotton expressing proteins Cry1Ac/Cry2Ab2 (Bollgard® II) and Cry1Ac/Cry1F (WideStrike(TM)) from Bacillus thuringiensis Berliner are tools available to control these pest species. To develop an Insect Resistance Management (IRM), we performed laboratory studies to evaluate the survival and development of S. frugiperda and P. includens. In fresh leaf discs bioassays, the control efficacy of neonates in both Bt cotton events was greater than 80% mortality for S. frugiperda and 100% for P. includens. In fresh bracts bioassays to neonates of S. frugiperda, the control efficacy of both Bt cotton events was over 80%. The surviving larvae of S. frugiperda in Bt cotton showed severe growth inhibition (weight and instar) in leaves (> 75%) and bracts (> 44%). In simulations feed bioassays with larvae, which consisted of groups of larvae fed on Bt cotton at 0, 3, 6, 9, 12, 15 and 18 days after inoculation (DAI), S. frugiperda and P. includens showed that the susceptibility of species decreases with advancing larval development. For S. frugiperda, in all feed simulations with cotton Cry1Ac/Cry2Ab2 had caterpillars that reached pupae and adult stages. Moreover, when exposed to cotton Cry1Ac/Cry1F, only some caterpillars of 5th and 6th instars reached pupae and adult stages. For P. includens, only some caterpillars of 6th instar reached pupae and adult stages when fed with two events of Bt cotton. The biological parameters of S. frugiperda fed on cotton Cry1Ac/Cry2Ab2 were negatively affected with increasing duration of the larval stage (? 9 days), reduced larval viability (1,4%) and insects that completed the life cycle (0,9%), increased the generation time (? 9 days) and decreased the intrinsic rate of increase (? 83%). The events of cotton Cry1Ac/Cry2Ab2 and Cry1Ac/Cry1F are promising for the control of S. frugiperda and P. includens. However, the insecticidal activity of both events of Bt cotton in larvae of S. frugiperda and P. includens decreases through larval development and this finding should be considered in programs of MRI, especially in the spatial arrangement of the refuge.

O presente estudo objetivou identificar as interações do Algodão Bt que expressa Cry1Ac (Bollgard®), com o predador Podisus nigrispinus Dallas (Hemiptera: Pentatomidae), no manejo da resistência de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) a lambda-cyhalothrin em duas condições: ausência e presença do inseticida imidacloprid. Foram utilizadas lagartas de S. frugiperda provenientes das seguintes condições: linhagens resistentes (1) e suscetíveis (2) a lambda-cyhalothrin alimentadas de folhas de algodoeiro Bollgard® (DP 404 BG); e linhagens resistentes (3) e suscetíveis (4) a lambda-cyhalothrin alimentadas de folhas de algodoeiro não transgênico (cultivar DP4049). Os resultados dessa pesquisa revelaram que na ausência de imidacloprid, independente do tratamento, o comportamento de predação foi melhor representado pelo tipo III de resposta funcional, pois a taxa de ataque aumentou linearmente em todas as condições estudadas (a= bN). Houve diferenças entre o tempo de manipulação (Th) de fêmeas do predador que receberam lagartas suscetíveis a lambda-cyhalothrin, previamente alimentadas de algodão não transgênico, em relação aos demais tratamentos. Na densidade de 16 lagartas/predador, o número de lagartas predadas por fêmeas de P. nigrispinus foi significativamente superior em lagartas resistentes a lambda-cyhalothrin, alimentadas de algodão Bt ou não Bt, em relação às lagartas suscetíveis alimentadas de algodão não Bt. Além do mais, se constatou que quando foram ofertadas 16 lagartas de S. frugiperda ao predador, o número de indivíduos predados foi significativamente inferior em lagartas suscetíveis a lambda-cyhalothrin que foram alimentadas de algodão não Bt, em relação as que receberam lagartas previamente alimentadas de algodão Bt. Na presença de imidacloprid, constatou-se que o comportamento de predação de P. nigrispinus foi afetado pelo neonicotinóide imidacloprid, sendo a curva assintótica do tipo II, a que melhor descreveu os dados da sua resposta funcional. Na presença de imadacloprid, o tempo de manipulação (Th) de fêmeas do predador não diferiu entre os tratamentos estudados. Todavia, a taxa de ataque foi representada por um decréscimo em função do aumento da densidade de lagartas ofertadas. Independente do tratamento (linhagem de S. frugiperda ou cultivar de algodão), o número de lagartas de S. frugiperda predadas por fêmeas de P. nigrispinus quando na exposição ao imidacloprid, foi significativamente inferior, especialmente na densidade de 16 lagartas/predador. Diante destes resultados, a pesquisa reforça que o custo adaptativo associado à resistência a lambda-cyhalothrin, assim como a cultivar de algodão Bt afetaram a taxa de predação de lagartas de S. frugiperda por fêmeas de P. nigrispinus, somente na maior densidade testada (16 lagartas/predador). O comportamento de predação de lagartas de S. frugiperda por fêmeas de P. nigrispinus foi negativamente afetado pelo inseticida imidacloprid. / This study aimed to identify the interactions of Bt cotton expressing Cry1Ac (Bollgard®), with the predator Podisus nigrispinus Dallas (Hemiptera: Pentatomidae), in resistance management of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) to lambda-cyhalothrin in two conditions: absence and presence of the insecticide imidacloprid. Larvae of S. frugiperda were used from the following conditions: resistant (1) and susceptible (2) strains to lambda-cyhalothrin fed Bollgard® cotton leaves (DP 404 BG); and resistant (3) and susceptible (4) strains to lambda-cyhalothrin fed non-transgenic cotton leaves (cultivar DP4049). The results of this study revealed that in the absence of imidacloprid, independent of treatment, the behavior of predation was best represented by the type III of functional response, because the attack rate increased linearly in all conditions studied (a = bN). There were differences between the handling time (Th) of females of the predator who received larvae of S. frugiperda susceptible to insecticides previously fed non-transgenic cotton in relation to other treatments. The density of 16 larvae/predator, the number of larvae preyed by female of P. nigrispinus was significantly higher in larvae of S. frugiperda resistant to lambda-cyhalothrin, fed on Bt cotton or non-Bt compared to susceptible larvae fed non Bt cotton. Moreover, when we offered 16 larvae of S. frugiperda to the predator, the number of larvae predate were significantly lower in larvae susceptible to lambda-cyhalothrin that were fed non-Bt cotton, compared to larvae that were previously fed on transgenic cotton. In the presence of imidacloprid, the predatory behavior of P. nigrispinus was affected by the neonicotinoid imidacloprid, and the asymptotic curve type II was the one that best described the data of the functional response. In the presence of imadacloprid, handling time (Th) of females of the predator did not differ among treatments. However, the attack rate was represented by a decrease due to the increase of the density of larvae offered. Regardless of the treatment (strain of S. frugiperda or cultivar of cotton), the predation larvae of S. frugiperda by females of P. nigrispinus when exposed to imidacloprid was significantly lower, especially at density of 16 larvae/predator. Given these results, the research reinforces the fitness cost associated to lambda-cyhalothrin resistance as well as Bt cotton affected the rate of predation on larvae of S. frugiperda by females of P. nigrispinus, only at the highest density tested (16 larvae/predator). The behavior of predation on larvae of S. frugiperda by females of P. nigrispinus was affected by insecticide imidacloprid.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES

O contato com as cerdas da lagarta de Premolis semirufa (Pararama) desperta sintomas da inflamação aguda e nos indivíduos poliacidentados pode causar deformidades comuns às sinovites crônicas (pararamose). No presente estudo foi mostrado que o extrato das cerdas da lagarta apresenta intensa atividade proteolítica, sendo capaz de ativar o Sistema Complemento, promover hidrólise de C3, C4 e C5 e a geração de anafilatoxinas. Análises cromatográficas do extrato permitiram o isolamento de uma serinoprotease de 82 kDa capaz de promover tais atividades. Em modelo murino, foi verificado que o extrato é capaz de induzir altos títulos de anticorpos, pronunciada reação inflamatória, ativação de linfócitos T e APCs, bem como produção de citocinas pró-inflamatórias. Os dados obtidos demonstram a existência, no extrato das cerdas da pararama, de várias enzimas que podem atuar em conjunto na geração e desenvolvimento das manifestações clínicas da pararamose. / The contact with the Premolis semirufas caterpillar bristles (Pararama) causes symptoms of the acute inflammation and, in the individuals after multiple accidents, joint deformities common to chronic synovitis (pararamose) can occur. In the current study it was shown that caterpillar bristles extract has intense proteolytic activity, being able to activate the Complement System, induce cleavage of C3, C4 and C5, and the generation of anaphylatoxins. Chromatographic analysis of the extract allowed the isolation of a serine protease with Mr of 82 kDa able to promote such activities. In murine model, it was demonstrated that the extract is able to induce high antibody titers, a pronounced inflammatory reaction, activation of T lymphocytes and APCs, as well as the generation of proinflammatory cytokines. The data obtained demonstrate the existence, in the pararama bristles extract, of numerous enzymes that can act together in the generation and development of clinical manifestations of pararamose.

L’imagerie par spectrométrie de masse (MSI) est une technique en pleine expansion ayant déjà été utilisée afin d’évaluer la distribution spatiale d’une grande variété de biomolécules. La plupart des applications découlent généralement du domaine médical et le modèle imagé est souvent mammalien, soit la souris ou le rat. L’optimisation de préparations d’échantillons sur ces modèles a permis d’obtenir de l’information reproductible et de grande qualité dans diverses applications biologiques. Il est cependant important de pouvoir reproduire les différentes préparations sur des échantillons diversifiés, permettant ainsi d’utiliser la technique dans plusieurs domaines de recherches différents. Les travaux de recherches présentés dans ce mémoire décrivent l’optimisation de méthodes de préparations d’échantillons afin d’imager des protéines et des lipides à travers des sections tissulaires minces d’un échantillon animal non mammalien. L’échantillon à l’étude était le Spodoptera exigua, un lépidoptère, qui sous sa forme larvaire est bien connu une chenille ravageuse de plantation. L’optimisation d’une méthode permettant la découpe en section tissulaire mince de ce type d’échantillon sera d’abord présentée. Ensuite, l’optimisation d’une méthode de déposition de matrice par sublimation/recristallisation à des fins d’imagerie de protéines sera décrite. Les résultats obtenus ont permis d’obtenir des données de qualité quant à la distribution de différentes protéines à travers des sections de chenilles. De plus, l’optimisation d’une méthode de préparation d’échantillon par sublimation de la matrice sera décrite. Ainsi, l’évaluation de la distribution spatiale de quatre différentes classes de phospholipides à travers ces sections a été possible.Enfin, il est possible de conclure que la qualité des signaux obtenus pour l’imagerie des protéines et des lipides à partir de sections tissulaires minces de chenille est équivalente à celle normalement observée sur des tissus mammaliens. / Imaging mass spectrometry (IMS) is a technique in full expansion that has been used in a large range of studies to assess the spatial distribution of a wide range of biomolecules. Most of the applications ensuing from IMS studies correlate the molecular expression and the health status of a tissue in the medical biology field, using mammalian models such as mice or rats. Optimization of different sample preparation methods for these models has permitted high quality and reproducible information in numerous biological applications. However, it is important to be able to reproduce this level of sample preparation and the quality of resultant data on a wider range of samples. This will open the technology to several scientific fields. The research work presented in this thesis proposes sample preparation methods to image the distribution of proteins and lipids across a thin tissue section of a non-mammalian model. This model is a moth, the Spodoptera exigua, one of the best-know agricultural pest insects in it’s caterpillar form. Firstly, optimization of sample handling prior to cutting thin tissue sections will be presented. Then, optimization of a matrix deposition method using sublimation/recrystallization in order to image the proteins across the sample will be described. Also, a sublimation method for the detection and imaging of lipids is described. The imaging results showed the distribution of four different classes of phospholipids across thin sections of the caterpillar. Lastly, the quality of imaging data for proteins and phospholipids across caterpillar sections can be compared directly to the expected data quality obtained from mammalian tissue sections.

Mopane (Colophospermum mopane) trees are browsed upon by two key species, namely mopane caterpillars (Imbrasia belina) and African elephants (Loxodonta africana), which each inflict a different type of damage while feeding, namely defoliation (leaf removal) and pruning (branch and/or stem breakage). Damage type can have a significant influence on plant responses, and these induced changes in morphological and chemical characteristics of regrowth can influence the subsequent feeding behaviour by each species. The objective of this study was therefore partly to investigate the differential effect of defoliation by mopane caterpillars and pruning by elephants on mopane trees, and then to investigate whether these two taxonomically different species interact through their shared food resource, by looking specifically at the effect of elephant utilisation of mopane on mopane caterpillar abundance. To determine the comparative effect of each browsing type, mopane trees were subjected to simulated mopane caterpillar or elephant utilisation treatments, at various frequencies and times within the year. Regrowth characteristics were then measured on treatment and control trees, as well as on naturally utilised and unutilised trees. Reproductive investment was also recorded on naturally utilised and unutilised trees. Additionally, the impact of mopane caterpillar defoliation and elephant pruning on plant stress was investigated by measuring the level of fluctuating asymmetry (FA) in leaves. Then, to determine whether there is an interaction between elephants and mopane caterpillars, mopane caterpillar egg mass abundance in areas of high elephant impact was compared to that in areas of low elephant impact. Firstly, however, in areas without elephant damage, those tree characteristics determining host tree preference by ovipositing mopane moths were identified. From this, an understanding of how elephant utilisation may influence mopane caterpillar abundance could therefore be gained. Defoliation and pruning had a significant different effect on mopane regrowth responses. Shoot and leaf length were significantly longer on pruned trees than control trees, for both naturally utilised and simulated elephant treatment trees, while there was no difference in shoot density. Defoliation, however, resulted in shorter shoots and leaves, particularly on naturally defoliated trees, which also had leaves of a higher nutritional value (tannin:protein ratio and total polyphenolic content) than control trees. A similar increase in leaf nutritional value was recorded in areas of high elephant impact in the Kruger National Park, but not after simulated or natural elephant damage in Venetia, where natural elephant utilization was less intense. Time since damage (i.e. first versus second flush) had a significant influence on regrowth after pruning, as shoot and leaf length were significantly longer on trees flushing for the first time, while within-season timing of damage was important for defoliation, as late-season defoliation had a greater negative impact than mid-season defoliation. Late-season defoliation also had a negative effect on leaf carriage into the dry season, while pruning appeared to aid leaf retention. Reproductive investment was found to be unaffected by mopane caterpillar defoliation or elephant pruning, as mean pod density and pod mass on utilised trees was no different to unutilised trees. Defoliation also had no influence on a plant’s likelihood of flowering that same season, with flowering being determined more by tree height. Unlike pod production, however, mean leaf density was significantly reduced in the regrowth of defoliated trees, presumably due to the use of stored resources for reproduction prior to the onset of regrowth. Neither simulated nor natural defoliation by mopane caterpillars and pruning by elephants was found to affect the level of leaf FA in mopane trees, even though the degree of damage inflicted on trees was considerably higher than in studies on other species where increases in FA were observed. Mopane therefore appears to be extremely tolerant of herbivory in comparison to other species. A positive relationship between leaf nutritional value (higher protein and lower tannin and polyphenolic content) and FA was detected, but only when trees from all study areas (i.e. a wide range of environmental conditions) were considered simultaneously. Environmental conditions, rather than herbivory, therefore appear to have a greater stressing affect on mopane. In the absence of heavy elephant utilisation of mopane trees, tree size, rather than shoot length, leaf length, leaf FA or leaf nutritional value, was found to have the greatest influence on oviposition behaviour of mopane moths. Ovipositing moths showed a preference for the tall riverine habitat over the shorter woodland and scrub mopane. This preference for large trees was, however, not evident at the individual tree level, as even though egg mass number per tree was positively related to tree height, large trees were not utilised more than expected according to the available canopy volume in each size class (resource availability). Heavy elephant utilisation of mopane had a negative impact on the density of tall trees within an area, due to branch and stem breakage while feeding. Unsurprisingly then, mopane caterpillar egg mass abundance was also significantly reduced in these areas, even though the nutritional value of leaves was higher than in non-elephant impacted areas. Elephants therefore appear to have a negative effect on mopane caterpillar abundance, primarily due to their negative impact on the density of tall mopane trees. This megaherbivore and invertebrate do therefore interact through their shared food resource, mopane trees. / Thesis (PhD (Zoology))--University of Pretoria, 2007. / Zoology and Entomology / unrestricted

Na tentativa de reduzir a biodegradação dos compostos ativos do nim e melhorar seu efeito residual, extratos de nim foram nanoformulados com diferentes tipos de nanoparticulas e de polímeros. Estas nanoformulações foram testadas sobre Spodoptera frugiperda (J. E. Smith) e sua eficiência comparada a um produto comercial. Os polímeros poli--caprolactona (PCL), poli-- hidroxibutirato (PHB) e polimetilmetacrilato(PMMA), foram utilizados nas quantidades 0,25, 0,50 e 0,75g, e em dois tipos de nanopartículas (cápsulas e esferas). Foram utilizados Tween®80 e polivinil álcool (PVA) como agentes tensoativos. As nanoformulações foram produzidas em suspensão aquosa e em pó, sendo diluídas para aplicação. Essas nanoformulações foram aplicadas sobre dieta artificial oferecida a lagartas de segundo instar de S. frugiperda, e em plantas de milho das quais as folhas foram cortadas e fornecidas a lagartas de primeiro instar. A eficiência de diferentes nanoformulações foi testada considerando a mortalidade e o peso 10 dias após o tratamento. Quando aplicado em dieta artificial foi observada redução no peso larval. Entretanto, quando aplicadas em folhas de milho, quatro nanoformulações causaram mortalidade entre 40 a 46%. Essas quatro nanoformulações foram avaliadas quanto à preferência alimentar e efeito residual. Foram conduzidos três bioensaios, nos quais as folhas de milho tratadas com as quatro nanoformulações foram oferecidas às lagartas aos 1, 3 e 7 dias após a pulverização (DAP), considerando-se mortalidade e peso das lagartas após 10 dias. Simultaneamente, foram realizados testes de preferência alimentar com dupla escolha (tratamento x controle) com discos foliares de milho aos 1, 3 e 7 DAP. A preferência alimentar foi determinada por meio de um índice de preferência, calculando-se a área foliar consumida pela lagarta após 24h. O efeito residual das nanoformulações foi menor que do produto comercial avaliado. Apenas uma nanoformulaçao e Organic Neem® apresentaram fagodeterrência a S. frugiperda, mas somente em 1 DAP. Embora as nanoformulações tenham causado mortalidade e redução de peso larval, sua eficiência foi inferior a do produto comercial. / Neem extracts were nanoformulated with several types of nanoparticles and polymers in a tentative to reduce the biodegradation of neem active compounds and improve their residual effect. These nanoformulations were tested against Spodoptera frugiperda and their efficacy compared to a commercial product. The polymers poly(-caprolactone) (PCL), poly(- hidroxibutirate) (PHB) and poly(methyl methacrylate) (PMMA) were used in the quantities 0.25, 0.50 and 0.75g and two sorts of nanoparticles (capsules and spheres). Tween®80 and poly(vinyl alcohol) (PVA) were utilized as surfactants. The nanoformulations were produced as aqueous suspension and powder and further diluted before application. These nanoformulations were sprayed on artificial diet offered to second instars of S. frugiperda, and on corn plants which had their leaves offered to first instars. The efficacy of the different formulations was tested by assessing larval mortality and weight 10 days after treatment. When applied to artificial diets, reduced larval weight was observed, but four of them caused 40 to 46% mortality when applied on corn plants. The four most effective nanoformulations were further tested to evaluate their effects on S. frugiperda larval feeding preference and their residual effect. Three bioassays were set up using corn leaves treated with these, wich were offered to larvae at days 1, 3 and 7 after spraying (DAS), having the larval mortality and weight assessed 10 days after larval exposure. Simultaneously, choice tests for larval feeding preference (treatment x control) were carried out using corn leaf discs 1, 3 and 7 DAS. Feeding preference was evaluated by using a preference index calculated after the assessment of the leaf area fed by the larva 24h after exposure. The residual effect of the neem nanoformulations was lower than the commercial product evaluated. Only one nanoformulation and Organic Neem® were feeding deterrents to S. frugiperda, but only in the first day after the treatment. Although the nanoformulations caused larval mortality and weight reduction, their efficacy was not any better than that of the commercial product tested.

As plantas desenvolveram diferentes mecanismos para reduzir o ataque de insetos, incluindo compostos protéicos de defesa, como os inibidores de proteinases (IPs). Os insetos, ao longo da evolução, desenvolveram estratégias para superar as barreiras defensivas das plantas, permitindo a sua alimentação e desenvolvimento, como a super expressão de genes de enzimas digestivas sensíveis e insensíveis aos IPs de plantas. Uma das abordagens desse trabalho foi identificar novas serinoproteinases no intestino de lagartas de Spodoptera frugiperda. Duas novas quimotripsinas e trê novas tripsinas foram identificadas e juntamente com mais 10 genes já conhecidos que codificam estas enzimas foram submetidos à análise de expressão gênica por PCR em tempo real. Entre essas duas famílias de serinoproteinases (SPs) os genes que codificam as quimotripsinas apresentam uma regulação positiva mais ampla do que aqueles que codificam as tripsinas. Estudos de modelagem molecular das quimotripsinas também foram realizados. Foram construídos modelos tridimensionais à partir de modelagem por homologia além de análises de dinâmica molecular e docagem com oito diferentes IPs do tipo Bowman- Birk. Os resultados mostram quais quimotripsinas apresentam as maiores afinidades aos inibidores testados de maneira geral e individual, inferidos à partir da estimativa de energia livre do sistema. Também foi encontrada uma serina extra próxima ao sítio catalítico de três quimotrispsinas modeladas que pode interferir na afinidade dessas enzimas já que este aminoácido apresenta perda de área acessível ao solvente quando complexada ao IP de soja testado. Os resultados de expressão gênica e grau de sensibilidade foram comparados e não se observou qualquer relação entre esses parâmentros. Isso sugere que as lagartas da espécie S. frugiperda combinam diferentes estratégias adaptativas como o aumento de expressão de todas as suas quimotripsinas independentemente do grau de sensibilidade das enzimas. / Plants have developed different mechanisms to reduce insect attack, including defence proteins such as proteinase inhibitors (PIs). In turn, insects have evolved strategies to overcome these plant defence mechanisms, such as the hyperexpression of PI-sensitive and insensitive digestive enzymes, allowing the insect to thrive. One of the aims of this work was to identify new serine proteinases (SPs) in the gut of the fall armyworm larvae, Spodoptera frugiperda. Two new chymotrypsins and three new trypsins were identified, and together with 10 previously identified genes, the genes that encode these enzymes were subjected to real-time PCR and gene expression analysis. Between these two families of serine-proteinases the genes that encode chymotrypsins show a greater positive regulation then those encoding the trypsins. Molecular modelling studies of the chymotrypsins were carried out, and 3D models were generated using homology modelling, which were then further refined by dynamic molecular and docking analyses with 8 different Bowman-Birk type PIs. The results demonstrate which chymotrypsins possess the highest affinities to the tested inhibitors in a general and individual manner, inferred from the estimated free energies. A serine residue in very close proximity to the catalytic site was present in three of chymotrypsins investigated, which may be affecting the enzymes affinity since the residue has a reduced accessible area to the solvent when complexed to the soya PI tested. The genetic expression patterns and the degree of PI-sensitivity were also compared and no relation between the parameters was found. This suggests that the larvae of the species S. frugiperda combine different adaptive strategies like the increase in expression of its entire chymotrypsin arsenal regardless of the degree of PI-sensitivity of the enzymes.

O primeiro passo para entender as funções da microbiota do trato digestivo na biologia dos insetos consiste na identificação dos membros destas comunidades. Neste trabalho, as bactérias presentes no mesêntero de lagartas da broca da cana-de-açúcar, Diatraea saccharalis e da lagarta-do-cartucho-do-milho, Spodoptera frugiperda, provenientes de diferentes localidades, espécies ou variedades de plantas hospedeiras, foram identificadas por isolamentos em meio de cultura TSA (Triptona de Soja Agar) e por técnicas moleculares que independem do cultivo dos microrganismos. As lagartas de D. saccharalis foram coletadas em nove variedades de cana-deaçúcar nos municípios paulistas de Bocaina, Tanabi e Luís Antônio. S. frugiperda foram coletadas diretamente no campo em milho em Piracicaba-SP e populações de campo e de laboratório foram também alimentadas com dieta artificial, milho, algodão, sorgo e arroz. Foram utilizadas 90 lagartas de D. saccharalis e 40 de S. frugiperda sendo a microbiota de cada lagarta avaliada individualmente. As bactérias isoladas foram testadas quanto à capacidade de degradação de celulose, hemicelulose, lignina e bagaço de cana. A caracterização molecular foi realizada por DGGE (Gel de eletroforese em gradiente desnaturante) usando-se a região 16S do RNA ribossômico amplificado de DNA metagenômico extraído do mesêntero de 57 lagartas de D. saccharalis e 26 de S. frugiperda. Posteriormente, 6 destas amostras de DNA metagenômico de cada uma das espécies de insetos foram clonadas e as 12 bibliotecas de clones foram sequenciadas. A comunidade de bactérias cultiváveis das duas espécies de insetos é composta por representantes dos filos Firmicutes, Proteobacteria e Actinobacteria. A afiliação filogenética das seqüências das bibliotecas de clones de rRNA 16S revelou a ocorrência, além destes filos, de Bacteroidetes em D. saccharalis e Bacteroidetes e Acidobacteria em S. frugiperda. Somente bactérias isoladas de D. saccharalis apresentaram resposta positiva nos testes de degradação in vitro para celulose, hemicelulose, lignina e bagaço de cana. A capacidade destes microrganismos de degradar celulose foi maior do que hemicelulose e poucos produziram enzimas para degradar lignina. Somente duas bactérias apresentaram resposta positiva aos testes de degradação para todas estas fontes de carbono. As seqüências de rRNA 16S destas se agrupam com representantes de Phyllobacterium trifolii e Bacillus subtilis. O maior número de sequências de bactérias cultiváveis pertence aos gêneros Bacillus (26,4 e 32.4% em D. saccharalis e S. frugiperda, respectivamente), Enterococcus (12,1 e 13%) e Microbacterium (18 e 12%). As análises das seqüências obtidas do DNA metagenômico revelaram que Klebsiella esteve presente em todas as amostras analisadas de D. saccharalis. As comunidades de bactérias em S. frugiperda apresentaram maior variação em função do alimento. Entretanto, Ralstonia e Hydrogenophilus estiveram presentes na maioria das lagartas. As variações das estruturas das comunidades bacterianas de S. frugiperda e de D. saccharalis estão diretamente relacionadas com a origem das populações e o tipo de alimentação dos insetos. A presença constante de algumas bactérias na maioria dos insetos indica que estas possam ser indispensáveis, exercendo alguma função vital para o inseto ou que apresentam alguma relação vantajosa para o hospedeiro. / The identification of members of the microbe communities is the first step to understand their roles on insect biology. We have characterized the midgut bacterial community of fifth ínstar caterpillars of sugarcane borer, Diatraea saccharalis, and of fall armyworm, Spodoptera frugiperda, through isolation on tryptic soy agar medium and through culture-independent molecular techniques. D. saccharalis caterpillars were collected on nine sugarcane varieties in Bocaina, Tanabi and Luís Antônio cities in São Paulo State. S. frugiperda were collected directly from corn fields in Piracicaba-SP and field and laboratory populations were reared on artificial diet, corn, cotton, sorghum and rice. Ninety D. saccharalis caterpillars and 40 S. frugiperda caterpillars were used. The microbiota community of each caterpillar was evaluated individually. The ability of bacterial isolates to degrade cellulose, hemicellulose, lignin and sugarcane bagasse was assessed. The molecular analyses were based on Denaturing Gradient Gel Electrophoresis (DGGE) of 16S ribosomal RNA sequences amplified from metagenomic DNA extracted from 57 individual midgut of D. saccharalis and 26 from S. frugiperda. Latter, 6 of these metagenomic DNA of each insect species were cloned and the 12 clone libraries were sequenced. The cultivable bacterial community of these two insect species is composed by representatives of the phyla Firmicutes, Proteobacteria and Actinobacteria. The phylotypes from the clone libraries of 16S rRNA were comprised by Bacteroidetes in D. saccharalis and Bacteroidetes and Acidobacteria in S. frugiperda, besides the three phyla found by culturing. Only bacteria isolated from D. saccharalis had ability to degrade cellulose, hemicelluloses, lignin and sugarcane bagasse in the in vitro tests. The ability to degrade cellulose was more common than the ability to degrade hemicelullose and just a few isolates had the ability to degrade lignin. The only two bacteria presenting a positive response to degradation of all carbon sources were clustered with Phyllobacterium trifolii and Bacillus subtilis. The greatest number of cultivable bacteria sequences belongs to genera Bacillus (26.4 and 32.4%, in D. saccharalis and S. frugiperda, respectively), Enterococcus (12.1 and 13%) and Microbacterium (18 and 12%). Sequence analysis from metagenomic DNA revealed the genera Klebsiella in all samples from D. saccharalis. Bacterial community varied according to S. frugiperda host plant; however, both Ralstonia and Hydrogenophilus genera were present in the digestive tracts of insects from most host plants. Community structure variation of S. frugiperda and D. saccharalis is related directly to insect population origin and type of insect food source. The constant presence of some bacteria in the majority of insects indicates that those microorganisms might either be indispensable, playing a vital role to the insect, or have some symbiotic or commensal relation with the insect.

Os seres vivos estão constantemente influenciando e sendo influenciados pelo meio ambiente. Esta interação pode afetar diretamente o comportamento e a sobrevivência destes seres vivos, incluindo os insetos. O objetivo deste trabalho foi descrever os efeitos de fatores bióticos e abióticos sobre o comportamento e sobrevivência de Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) e de seus parasitoides de ovos Telenomus remus Nixon 1937 (Hymenoptera: Scelionidae), Trichogramma atopovirilia Oatman & Platner 1983 e T. pretiosum Riley 1879 (Hym.: Trichogrammatidae). Os resultados mostraram que adultos de S. frugiperda realizam suas atividades reprodutivas em períodos bem definidos de sua vida, preferencialmente desde o início até meados da escotofase. Fêmeas de T. remus realizaram parasitismo de ovos de S. frugiperda tanto durante o dia quanto durante a noite. T. atopovirilia foi, dentre as três espécies de parasitoides estudadas, a que apresentou a mais ampla faixa térmica para desenvolvimento ótimo, podendo apresentar elevado potencial de controle de S. frugiperda em regiões quentes como frias. A sobrevivência e os comportamentos de forrageamento de T. remus, T. atopovirilia e T. pretiosum foram afetados pelas competições intra e/ou interespecíficas, sendo os efeitos destas competições peculiares para cada uma das espécies. A competição intra-específica interna afetou a sobrevivência de T. atopovirilia, mas não a de T. pretiosum nem a de T. remus. As competições interespecíficas internas afetaram a sobrevivência destas três espécies, sendo a ordem de parasitismo e o intervalo entre as oviposições determinantes para o resultado da competição. Observou-se emergência das duas espécies de Trichogramma a partir de um único ovo multiparasitado. A espécie T. atopovirilia, dentre as três espécies estudadas, foi a que apresentou maior probabilidade de vencer na competição interna interespecífica. Estes resultados sugerem que pistas químicas (ex.: feromônios) liberadas por adultos de S. frugiperda podem ser utilizadas como cairomônios por fêmeas de T. remus durante o período noturno, viabilizando o fenômeno de forésia, e que a temperatura e competição afetam a sobrevivência e/ou o comportamento de T. remus, T. atopovirilia e T. pretiosum, devendo ser consideradas na escolha da espécie ou combinação de espécies a ser liberada em campo visando ao controle populacional de S. frugiperda. / Living beings often influence and are influenced by the environment. Such interactions can directly affect the behavior and survivorship of insects. The aim of this study was to describe the effects of biotic and abiotic factors on the behavior and survivorship of Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) and its egg parasitoids Telenomus remus Nixon 1937 (Hymenoptera: Scelionidae), Trichogramma atopovirilia Oatman & Platner 1983, and T. pretiosum Riley 1879 (Hym.: Trichogrammatidae). The results showed that adults of S. frugiperda perform reproductive activities during well-defined periods of their life, preferably from the beginning until the middle of scotophase. Females of T. remus parasitized S. frugiperda eggs in both photophase and scotophase. T. atopovirilia was the species with the widest temperature range for optimal development, and therefore with a high potential for controlling S. frugiperda populations in different temperature conditions. Survivorship and foraging behaviors of T. remus, T. atopovirilia, and T. pretiosum were affected by intra- and/or interspecific competition, with particular effects on each parasitoid species. Intrinsic intraspecific competition affected T. atopovirilia survivorship, but did not affect T. pretiosum or T. remus. Intrinsic interspecific competition affected survivorship of all three parasitoid species. The parasitism sequence and oviposition interval were critical for the result of this competition. Emergence of two Trichogramma species from a single multiparasitized host egg was observed. Of the three parasitoid species, T. atopovirilia showed the highest likelihood of winning in intrinsic interspecific competition. These results suggest that chemical cues (e.g., pheromones) released by adults of S. frugiperda can be used as kairomones by females of T. remus during the scotophase, enabling the phenomenon of phoresy. Moreover, temperature and competition affect the survivorship and/or behavior of T. remus, T. atopovirilia and T. pretiosum and should be considered when selecting the species or combination of species for field release to control S. frugiperda populations.