Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

Genotypic and phenotypic diversity of a baculovirus population within an individual insect host

It is becoming increasingly apparent that many pathogen populations, including those of insects, show high levels of genotypic variation. Baculoviruses are known to be highly variable, with isolates collected from the same species in different geographical locations frequently showing genetic variation and differences in their biology. More recent Studies at smaller scales have also shown that virus DNA profiles from individual larvae can show polymorphisms within and between populations of the same species. Here, we investigate the genotypic and phenotypic variation of an insect baculovirus infection within a single insect host. Twenty four genotypically distinct nucleopolyhedrovirus (NPV) variants were isolated from an individual pine beauty moth, Panolis flammea, caterpillar by in vivo cloning techniques. No variant appeared to be dominant in the population. The Pafl NPV variants have been mapped using three restriction endonucleases and shown to contain three hypervariable regions containing insertions of 70-750 bp. Comparison of seven of these variants in an alternative host, Mamestra brassicae, demonstrated that the variants differed significantly in both pathogenicity and speed of kill. The generation and maintenance of pathogen heterogeneity are discussed. (c) 2005 Elsevier Inc. All rights reserved.

Optimal foraging when regulating intake of multiple nutrients

There is growing evidence that, rather than maximizing energy intake subject to constraints, many animals attempt to regulate intake of multiple nutrients independently. In the complex diets of animals such as herbivores, the consumption of nutritionally imbalanced foods is sometimes inevitable, forcing trade-offs between eating too much of nutrients present in the foods in relative excess against too little of those in deficit. Such situations are not adequately represented in existing formulations of foraging theory. Here we provide the necessary theory to fit this case, using an approach that combines state-space models of nutrition with Tilman's models of resource exploitation (Tilman 1982, Resource Competition and Community Structure, Princeton: Princeton University Press). Our approach was to construct a smooth fitness landscape over nutrient space, centred on a 'target' intake at which no fitness cost is incurred, and this leads to a natural classification of the simple possible fitness landscapes based on Taylor series approximations of landscape shape. We next examined how needs for multiple nutrients can be assessed experimentally using direct measures of animal performance as the common currency, so that the nutritional strategies of animals can be mapped on to the performance surface, including the position of regulated points of intake and points of nutrient balance when fed suboptimal foods. We surveyed published data and conducted an experiment to map out the performance landscape of a generalist leaf-feeding caterpillar, Spodoptera littoralis. (C) 2004 Tire Association for the Study of Animal Behaviour. Poblished by Elsevier Ltd. All rights reserved.

Associação de bioensaios e caracterização molecular para seleção de novos isolados de Bacillus thuringiensis efetivos contra Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Spatial analysis of the distribution of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) and losses in maize crop productivity using geostatistics

A lagarta-do-cartucho, Spodoptera frugiperda (J.E. Smith), é uma das principais pragas do milho nas Américas. O estudo de sua distribuição espacial é fundamental para a utilização de estratégias de controle, otimização de técnicas de amostragens, determinação de danos econômicos e incorporação de um programa de agricultura de precisão. em uma área cultivada com milho foram realizadas amostragens com intervalo semanal, correspondendo ao estádio vegetativo que compreende desde a germinação até o pendoamento. Foram amostradas 10 plantas ao acaso por parcela, no total de 2000 plantas em cada amostragem. A produtividade foi obtida através da colheita de todas as parcelas que eram pesadas separadamente no campo e em cada parcela foram coletadas 15 espigas aleatoriamente para estimar o comprimento e o diâmetro médio. As análises espaciais, utilizando geoestatística, mostraram que o modelo esférico apresentou o melhor ajuste às lagartas pequenas. À medida que as lagartas foram se desenvolvendo sua distribuição foi tornando aleatória, representada por um modelo ajustado por uma reta, não tendo sido detectado nenhum tipo de dependência espacial nos pontos de amostragem. A produtividade e o diâmetro e comprimento da espiga foram descritos por modelos esféricos, indicando uma variabilidade espacial nos parâmetros de produtividade na área cultivada. A geoestatística mostrou-se promissora para a aplicação de métodos precisos no controle integrado de pragas.

Não preferência para alimentação e aspectos biológicos de Spodoptera eridania em cultivares de feijão-caupi

The aim of this paper was to evaluate the no-preference for feeding and biological aspects of Spodoptera eridania fed on cowpea cultivars BR17 Gurgueia, BRS Urubuquara, BRS Nova Era, Sempre Verde, BRS Milenio and BR3 Tracuateua. In free-choice test, leaf discs were placed in Petri dishes where one third instar larvae per cultivar was released, whereas in no-choice test one leaf disc was placed per Petri dish where one caterpillar per cultivar was released, evaluating their attractiveness after 1, 3, 5, 10, 15, 30, 60, 120, 360 and 720 minutes, as well as the leaf area consumed. Randomized blocks and complete randomized blocks design were used for free-choice and no-choice tests, respectively, with six treatments and 10 replications. The evaluation of the biological parameters of S. eridania was carried out in Petri dishes where recently hatched caterpillars were transferred in the proportion of one per dish, and the leafs of the cultivars were offered to them during the whole larval period, and we evaluated: periods and viabilities of larvae and pupae, overall viability, weight of larvae and pupae, sex ratio, longevity and overall cycle. Complete randomized design was used with six treatments and 30 replications. In no- preference for feeding free-choice test the cultivars Sempre Verde and BR17 Gurgueia were the most and the least consumed, respectively. Regarding the effects of cowpea cultivars on larval viability we can infer that BRS Urubuquara and Sempre Verde show antibiosis-type resistance to S. eridania.

Diferentes diâmetros de gotas e equipamentos para aplicação de inseticida no controle de Pseudoplusia includens

It was aimed to evaluate the spectrum and uniformity of droplets in function of sprayers, spraying volumes and rates of chemical insecticide on the mortality of Pseudoplusia includens in laboratory. The work was carried out at UNESP, Jaboticabal, SP, Brazil. The following treatments were applied over the caterpillars: two equipments (atomizer and hydraulic nozzle); two spray volumes (17 and 50 L ha(-1) for the atomizer and 50 and 100 L ha(-1) for the hydraulic nozzle); and two dosages of the insecticide endosulfan (0.5 and 1.0 L pc ha(-1)), in fully randomized plots, in a factorial scheme 2x2x2 and 1 untreated check. The caterpillars mortality was evaluated until the 6(th) day after the application of treatments. The spectrum of the droplets was evaluated in a particle size analyzer in real time determining the diameter and spectrum of droplets sprayed through the laser beam of the analyser. It was verified that the spray volume can be reduced by 17 L ha(-1) without losses on the P. includens control. The doses of 0.5 L pc ha(-1) (recommended for Anticarsia gemmatalis) did not satisfactorily control the caterpillar soybean looper. The atomizer produces droplets of higher uniformity (SPAN: 0.52) and lower percentage of droplets susceptible to drift (3.3%) compared to hydraulic nozzle (SPAN: 1.34 e % droplets <= 100 mu m: 15.2).

Efeito da aplicação de extratos aquosos em couve na alimentação de largatas de Ascia monuste orseis

Os efeitos de extratos aquosos de amêndoas de Azadirachta indica e frutos de Sapindus saponaria, aplicados em discos de folhas de couve (Brassica oleracea var. acephala) nas concentrações de 0,0117% e 1,0342% (p/v), respectivamente, foram estudados sobre a alimentação das lagartas de Ascia monuste orseis. Avaliou-se a atratividade e o consumo de área foliar de lagartas de primeiro e terceiro ínstar durante 24 horas, em condições de laboratório (T = 25 ± 2ºC, UR = 60 ± 10% e fotofase = 12 horas). Nos testes com e sem chance de escolha, para lagartas de primeiro ínstar e teste sem chance de escolha para lagartas de terceiro ínstar, não houve diferença quanto à atratividade das lagartas. No teste com chance de escolha para lagartas de terceiro ínstar, houve menor atratividade das lagartas pelos discos de folhas tratadas com S. saponaria, diferindo da testemunha. No decorrer de 24 horas de avaliações, pode-se observar 58,3% de lagartas atraídas na testemunha, não diferindo de A. indica e diferindo de S. saponaria, com 39,3% e 2,4% das lagartas atraídas, respectivamente. Quanto ao consumo de área foliar, o extrato de S. saponaria diminuiu o consumo de lagartas, em todos os testes realizados. Quando não tinham opção de escolha para se alimentar de folhas sem os extratos, as lagartas consumiram as folhas tratadas nas concentrações testadas, porém em menor quantidade. Os extratos testados neste experimento demonstram ter efeitos sobre a alimentação das lagartas de A. monuste orseis, possivelmente com propriedades deterrentes e/ou supressoras de alimentação.

Não-preferência para oviposição e alimentação de Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) por cultivares de amendoim.

This study aimed to select peanut cultivars resistant to Spodoptera frugiperda, selecting non-oviposition and non-feeding preference in choice and no-choice tests. Peanut cultivars with upright growth habit (IAC 5, IAC 8112, IAC 22 and IAC Tatu ST) and runner growth habit (IAC 503, IAC 505, IAC 147, IAC 125, IAC Caiapo and IAC Runner 886) were evaluated. Tests of non-oviposition preference were conducted with newly emerged adults. Adults were fed a 10% honey solution, remaining in cages for 4 days until the start of the evaluations. The evaluations consisted of quantifying the number of eggs and egg masses, followed by calculating the number of eggs per egg mass. To perform non-feeding preference tests, leaf discs from different peanut cultivars were placed in disposable Petri dishes followed by releasing of a third-instar caterpillar for each cultivar. The attractiveness at 1, 5; 10, 15, 30, 60, 120, 360, 720 and 1,440 minutes was evaluated by counting the caterpillars that were feeding on cultivars. The leaf area consumed was evaluated at the end of the experiment for each cultivar. In the tests of non-oviposition and non-feeding preference, with choice or no-choice, none of the cultivars of the upright growth and runner growth habit influenced the oviposition or food of S. frugiperda. Thus, the peanut cultivars showed no resistance of the non-feeding and non-oviposition types.

Técnica de medição de área foliarconsumida por insetos através de digitalizador de imagens

Este trabalho teve por objetivo verificar a viabilidade do uso de digitalizador de imagens manual, acoplado a um microcomputador, para a avaliação do consumo de folhas de soja, por lagartas de 5o instar de Anticarsia gemmatalis Hübner (Lep.: Noctuidae), em comparação com o método de pesagem e do planímetro, baseando-se na eficiência dos métodos e no tempo gasto para a avaliação. Os testes foram realizados utilizando-se folhas de soja `IAC 8' e lagartas criadas em dieta artificial. Foram realizados 2 tipos de teste: 1o) oferecimento de folíolos inteiros de soja às lagartas e, 2o) oferecimento de disco de folhas de área conhecida. No 1o teste comparou-se o método de pesagem com o digitalizador de imagens (scanner); no 2o experimento foram comparados o método do planímetro com o digitalizador de imagens que emprega o programa PCXAREA. Os resultados obtidos demonstraram que não existem diferenças nas medições de folíolos e discos de soja consumidos por A. gemmatalis quando comparados os métodos tradicionais (planímetro e pesagem) e o de digitalização de imagens. A medição com o digitalizador reduziu o tempo de avaliação em 88,5% e 87%, em relação ao planímetro e método de pesagem, respectivamente, sendo plenamente viável a sua utilização.

Palmistichus elaeisis (Hymenoptera: Eulophidae) parasitizing pupae of Citioica anthonilis (Lepidoptera: Saturniidae) collected on Piptadenia gonoacantha (Fabaceae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Velocidade do fluxo de ar em barra de pulverização no controle químico de Anticarsia gemmatalis, Hübner e percevejos na cultura da soja

A lagarta Anticarsia gemmatalis (Hübner) e os percevejos fitófagos são pragas importantes na cultura da soja no Brasil. Este trabalho objetivou avaliar o controle desses insetos após o tratamento com inseticidas, sob diferentes velocidades do fluxo de ar junto à barra de pulverização nessa cultura. O experimento foi desenvolvido em Botucatu, na cultura da soja [Glycine max (L.) Merrill], var. Conquista (safra 2007/08), no delineamento experimental de blocos ao acaso (quatro velocidades do fluxo de ar: 0, 9, 11 e 29 km h-1), mais testemunha, totalizando cinco tratamentos e quatro repetições. No estádio de desenvolvimento vegetativo (V10) realizou uma aplicação do inseticida deltametrina na dosagem de 6,5 g do i.a. ha-1 para o controle de lagartas e no estádio de desenvolvimento reprodutivo (R6) aplicou o inseticida tiametoxam associado com lambda-cialotrina na dosagem de 25,38 + 19,08 g do i.a. ha-1 para o controle de percevejos. A aplicação foi feita com um pulverizador Advanced Vortex 2000, com pontas de pulverização jato cônico JA2, conferindo um volume de calda de 200 L ha-1. As avaliações antes e após a aplicação foram realizadas pelo método de batidas no pano. Avaliaram-se os danos causados por percevejos, considerando-se a porcentagem de danos às sementes, ao poder germinativo e à produtividade. No geral, o número médio de lagartas e percevejos foram significativamente menores nas parcelas tratadas em relação ao obtido na testemunha. Não houve diferença de produtividade entre os tratamentos. A porcentagem de emergência e sementes picadas por percevejos foram significativamente menores nos tratamentos que receberam o controle em comparação à testemunha.

Avaliação preliminar da viabilidade de produção in vitro de um isolado brasileiro de baculovírus Spodoptera frugiperda MNPV

In vivo production of viral biopesticides is the major source of viral insecticides currently in the marketplace. However, this system presents limitations during production scale-up. For the Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV), the insect used for replication has cannibalistic characteristics, thus production is even more difficult. Insect cells are commonly used for in vitro baculovirus production. Most of these cell lines are derived from Lepidoptera species. The Sf21 cell line is derived from Spodoptera frugiperda caterpillar ovarian tissue, and its clonal isolate Sf9 has been used for biopesticide production due to its ease of growth in suspension cultures. In this work, the in vitro production capabilities of a Brazilian SfMNPV isolate obtained from cornfields was evaluated. Comparison of polyhedra production was carried out using both Sf21 and Sf9 cells, based on volumetric and specific yields. Both cell lines were cultivated in Hyclone medium supplemented with different fetal bovine serum concentrations (2,5 and 5%). The best results were obtained using Sf9 cells supplemented with 5% serum. These results were further confirmed quantitively through kinetic parameter estimation for both cells lines and different serum concentrations. After seven successive passages, this system still presented high specific polyhedra production

Brachymeria pandora (Crawford) (Hymenoptera: Chalcididae) as a New Parasitoid of Thyrinteina leucocerae (Rindge) (Lepidoptera: Geometridae) in Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)