RESUMO: Para avaliar o efeito tóxico de formulações comerciais de óleo de neem, Azadirachta indica A. Juss, pré-pupas (PP), pupas jovens (PJ) e pupas velhas (PV) da Diatraea saccharalis F. (Lepidoptera: Crambidae) foram pulverizadas com o extrato diluído em água destilada, em concentrações de 0,0, 0,3, 0,5, 1,0 e 2,0%. O neem provocou diferentes efeitos sobre a mortalidade de pupas, dependendo da concentração. As pupas que não conseguiram emergir em adultos apresentaram anormalidades múltiplas. Quanto às pupas que emergiram em adultos (grupos PJ e PV), foi calculada a sua longevidade, que não diferiu da do grupo controle. As anormalidades encontradas em adultos estão relacionadas com a mortalidade em todos os tratamentos com exceção da concentração de 1,0%. A fecundidade foi avaliada de acordo com a oviposição de adultos fêmeas do grupo PJ, que não apresentaram anormalidades; dentro dos ovos depositados foi avaliado o número de ovos não desenvolvidos. Os resultados demonstraram redução no número de ovos depositados e aumento na porcentagem de ovos não desenvolvidos. Esses resultados mostraram que o óleo de neem tem elevado potencial para o controle do estágio mais resistente da broca da cana-de-açúcar, além de reduzir o aparecimento das fases subsequentes. Portanto, formulações comerciais de óleo de neem apresentam um efeito tóxico em pupas e adultos de D. Saccharalis.

ABSTRACT: To evaluate the toxic effect of commercial formulations of neem oil, Azadirachta indica A. Juss, pre-pupae (PP), young pupae (YP) and old pupae (OP) of Diatraea saccharalis F. (Lepidoptera: Crambidae) were sprayed with the diluted extract in distilled water at concentrations of 0.0, 0.3, 0.5, 1.0 and 2.0%. The neem extract caused concentration-dependent effects on mortality of pupae, and the pupae that failed to emerge in adults had multiple abnormalities. The longevity of pupae that emerged in adults (YP and OP group) did not differ from the control group. The abnormalities found in adults were related to mortality in all treatments, except at the concentration of 1.0%. Fertility was assessed according to the oviposition of adult females from the YP group that showed no abnormalities, through the evaluation of the number of deposited eggs and the rate of undeveloped eggs. The results showed a reduction in the number of eggs laid and an increase in the percentage of undeveloped eggs. These results show that neem oil has a high potential to control the toughest stage of the sugarcane borer and also reduces the further development. Therefore, commercial formulations of neem oil have a toxic effect on pupae and adults of D. saccharalis.

Diatraea saccharalis (Fabricius) has cryptonephridial type Malphigian tubules (MT). This type of MT is characterized by the penetration of the distal part of the MT into the external walls of the rectum, which is usually lined with a perinephral membrane. The MT is divided into three differentiated regions: proximal, middle and distal. In this study, our objective was to compare the nuclear activities of each one of the three regions of the D. saccharalis MT by using a nuclear basophilic technique and critical electrolyte concentration with a toluidine blue stain at pH 4.0. This method allows differentiation of DNA/protein complexes in in situ and in vitro chromatin. MT chromatin structure in D. saccharalis is variable. Fifth instars have a more decondensed chromatin than fourth instars. The distal tubule region was the most decondensed region of the MT. Our data show an elevated genetic activity of the MT in the pre-metamorphosis period. The distal region of the MT has the highest observed activity, which may be associated with the re-absorption of useful components and the excretion of waste materials.

Diatraea saccharalis, the main pest of sugarcane, has been controlled by Cotesia flavipes. Very little is known about the effect of parasitism on the host organs, including the midgut. The Lepidoptera midgut epithelium is composed of columnar, goblet, regenerative, and endocrine cells. Spherites have been described in columnar and regenerative cells of several Lepidoptera species, and presented a lot of functional meaning. We identified spherites in the midgut epithelial cells of non-parasitized D. saccharalis larvae analyzed the effect of parasitism on spherite morphology and distribution along the length of the midgut. Midgut fragments of both non-parasitized and parasitized larvae were processed for transmission electron microscopy. All the midgut epithelial cells showed spherites, but they were not preferentially located in a particular part of the cells. Parasitized larvae had more spherites, mainly in the columnar cells, than non-parasitized larvae. This observation was associated with an ionic imbalance within the insect host. Spherites were more abundant in the anterior midgut region than in other regions, which suggests that this region is involved in ion transport by intracellular and/or paracellular route. The morphological variability of spherites in the cells of parasitized larvae was related to the developmental stages of these structures.

The migration rate of esterases and their substrate specificity for 4-methylumbelliferyl esters (acetate, propionate, and butyrate) and alpha- and beta-naphthyl esters were analyzed in Diatraea saccharalis by starch gel electrophoresis. Substrate preference of esterases was observed with Est-2 and Est-8 isozymes showing substrate specificity for 4-methylumbelliferyl esters and Est-4 isozyme showing specificity for 4-methylumbelliferyl butyrate and alpha-naphthyl butyrate. Allele variation was detected at the Est-3 locus. Two alleles, Est-3F and Est-3S, were identified in pupae with fluorogenic and ester-naphthyl substrates. Chi-square analysis showed no differences between the observed genotypic frequencies and those expected on the basis of Hardy-Weinberg frequencies for the Est-3 locus (chi² = 2.4; p < 0.01). The negative value for the Wright's fixation index (F = -0.2096) calculated for the D. saccharalis population maintained under laboratory conditions indicates an excess of heterozygotes, however, the observed Hardy-Weinberg equilibrium indicates that in the laboratory the population of D. saccharalis behaved as if the moth were randomly mating in nature. The high level of heterozygosity at the Est-3 locus indicates also that this esterase may be a good genetic marker for studies of natural D. saccharalis populations.