Molecular characterization of brinjal shoot and fruit borer, Leucinodes orbonalis (Guenée) (Lepidoptera: Crambidae) based on mitochondrial marker cytochrome oxidase I and their phylogenetic relationship.

Shoot and fruit borer, Leucinodes orbonalis is an important insect pest infesting brinjal or eggplant in India. Molecular characterization of nine different populations belonging to various brinjal growing regions was done using Cytochorome C Oxidase I (COI) gene. Nucleotide analysis of genetic diversity and phylogenetic analysis of the COI indicate that the L. orbonalis from different geographical regions are homogenous. The results showed less nucleotide diversity (π = 0.007895) and overall mean distance (0.008±0.003). Topologies of neighbour-joining (NJ) trees indicate all the populations belong to single major clade. Therefore, it is inferred that there was no significant molecular diversity within L. orbonalis of different geographical locations of India with respect to COI.

Purification of elastase-like chymotrypsin from cardamom shoot and Capsule borer [corrected]

An elastase-like chymotrypsin was purified by aprotinin-agarose affinity chromatography from the midgut extract of cardamom shoot and capsule borer, Conogethes punctiferalis. The purified enzyme had a Vmax of 687.6 +/- 22.1 nmole pNA released/min/mg protein, Km of 0.168 +/- 0.012 mM with SAAPLpNA as substrate and gave a single band on SDS-PAGE with a molecular mass of 72.1 kDa. Casein zymogram revealed one clear zone of proteolytic activity, which corresponded to the band obtained with SDS-PAGE indicating that this could be a single-polypeptide enzyme.

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 4°C and the supernatants were used in enzymatic assays at 30°C, 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 50°C, a temperature that reduces enzyme stability to 80 and 60 percent 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.

Effect of Baculovirus spodoptera isolates in Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) larvae and their characterization by RAPD

The total of 22 Baculovirus isolates surveyed in different corn producing regions in Brazil were used against fall armyworm, Spodoptera frugiperda (J.E. Smith). The viruses were purified and their suspensions were used to feed fall armyworm larvae from 4th and 5th instar. The mortality rate was checked daily and the infected larvae were frost after death, what generally occurred between the 5th and 7th day after virus ingestion. The 22 Baculovirus isolates were used in six concentrations (from 10³ to 10(8) polyhedra/ml) and one check treatment with water. Mortality rate, larval period, pupal period, pupa weight and lethal concentration (LC50) were determined for all isolates. Significant differences were found among all isolates and different concentrations, also interaction between isolate x virus concentration for all characteristics evaluated, except for pupal period. Amplification patterns of 54 RAPD markers, being 41 polymorphic among the isolates, were used to evaluate the genetic distance and its correlation with the fall armyworm larvae mortality rate. The genetic diversity calculated by the Jaccard's coefficient using the molecular data allowed a division of the isolates into two groups, with a high level of confidence. These groups did not present any association with the mortality rate caused by the isolates or with their geographical distribution. However, a RAPD fragment OPW04.2280 was highly associated with the larvae mortality rate and with LC50, explaining 23 and 65 percent of the phenotypic variation for these traits among the isolates, respectively.

Foram utilizados 22 isolados de vírus amostrados em diferentes regiões produtoras de milho do Brasil. Os vírus foram purificados e suas suspensões fornecidas a lagartas sadias do 3° e 4° ínstar de Spodoptera frugiperda (J.E. Smith). A mortalidade foi avaliada diariamente, e as lagartas infectadas foram congeladas logo após sua morte, o que em geral ocorreu do 5° ao 7° dia após ingestão do vírus. Os isolados foram usados em seis concentrações (10³ a 10(8) poliedros/ml) e uma testemunha (água). Os percentuais de mortalidade, duração do período larval e período pupal, peso de pupa e a concentração letal (CL50) foram determinados para todos os isolados. Foram observadas diferenças significativas entre todos os isolados e concentrações testadas para todos os parâmetros avaliados, e também foi constatada a presença da interação isolado x concentração, exceto para período pupal. Os padrões de amplificação de 54 marcadores RAPD, sendo 41 polimórficos, foram utilizados para avaliar a distância genética e a sua correlação com os índices de mortalidade das lagartas. A divergência genética calculada pelo coeficiente Jaccard utilizando os dados moleculares permitiu dividir os isolados em dois grupos, com um elevada confiabilidade. O agrupamento não apresentou associação com a taxa de mortalidade causada pelos isolados ou com sua distribuição geográfica. No entanto, um fragmento de RAPD OPW04.2280 apresentou-se altamente associado com a mortalidade das lagartas e com a CL50, explicando 23 por cento e 65 por cento da variação fenotípica para essas características entre os isolados virais, respectivamente.