Construction and analysis of cDNA libraries from the antennae of male and female cotton bollworms Helicoverpa armigera (Hübner) and expression analysis of putative odorant-binding protein genes

Two high-quality cDNA libraries were constructed from female and male antennae of the cotton bollworm Helicoverpa armigera (Hübner). The titers were approximately 2.0 × 10? pfu/ml for females and 2.3 × 10? pfu/ml for males, and this complies with the test requirement. From the libraries, 1750 male ESTs and 1640 female ESTs were sequenced and further analyzed. We identified 15 olfactory genes (12 are new), and 14 of them have the characteristic six conserved cysteine residues. With the exception of OBP9, all the genes were classified as classical OBP genes. By alignment and cluster analysis, the 14 classical OBPs were divided into pheromone binding protein (PBP) genes, odorant binding protein (OBP) genes, general odorant binding protein 1 (GOBP1) genes, general odorant binding protein 2 (GOBP2) genes and antennae binding protein (ABP) genes. Among these genes, we obtained three PBP genes (PBP1-PBP3) including two new PBP genes, one new ABP gene, nine new OBP genes (OBP1-OBP9), one known GOBP1 gene and one known GOBP2 gene. Furthermore, the expression patterns of these 14 classical OBP genes were investigated in various tissues by real-time quantitative polymerase chain reaction (qPCR). The results indicated that some OBP genes are expressed differently in different sexes and tissues, but most of them are highly expressed in antennae.     

Cloning,expression and immunocytochemical localization of a general odorant-binding protein gene from Helicoverpa armigera (Hübner)

A cDNA clone coding for general odorant-binding protein2 was isolated from the antenna of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. Results of sequencing and structural analyses showed that the full-length of GOBP2Harm was 636 bp, possessing 162 amino acid residues and a signal peptide of 21 amino acids. Its predicted molecular weight and isoelectric point were 18.2 kDa and 5.21, respectively. This deduced amino acid sequence shared some common structural features with odorant-binding proteins from several moth species, including the six conserved cysteine motif, typical of insect's OBPs. Northern blot showed that GOBP2Harm is specifically expressed in the antenna of Helicoverpa armigera at similar levels in both sexes. In order to obtain sufficient GOBP2 for further determining its biochemical and physiological properties, a bacterical expression vector of GOBP2 was constructed and successfully expressed. The protein was obtained mainly as insoluble inclusion bodies, that, however, could be solubilized and refolded. The rGOBP2 was purified by affinity chromatography and gel filtration. The rGOBP2 was shown to cross-react with an anti-GOBP antiserum from Antheraea polyphemus. Finally, polyclonal antibodies against GOBP2Harm were used to mark the distribution of the protein in olfactory sensilla and were tested by immuno-electron microscopy. In the male, GOBP2Harm is mainly expressed in sensilla basiconica, while in the female, it is equally expressed in sensilla basiconica and in sensilla trichodea.     

Characterization of three pheromone-binding proteins (PBPs) of Helicoverpa armigera (Hübner) and their binding properties

Three pheromone-binding proteins of Helicoverpa armigera were cloned and expressed in Escherichia coli. In order to characterize their physiological properties, ligand-binding experiments were performed using five biologically relevant substances including sex pheromones and interspecific signals. The results showed that one of the pheromone-binding proteins, HarmPBP1, binds strongly to each of the two principal pheromone components of H. armigera, (Z)-11-tetradecenal and (Z)-9-hexadecenal, but not to the interspecific signal (Z)-9-tetracecenal. The two remaining pheromone-binding proteins, HarmPBP2 and HarmPBP3, showed only weak affinities with the ligands tested. The 3-D structure of HarmPBP1 was predicted and the docking experiments indicate that the key binding site of (Z)-9-hexadecenal to HarmPBP1 includes Thr112, Lys111, and Phe119 whereas that of (Z)-11-tetradecenal includes Ser9, Trp37, Phe36, and Phe119.     

Mechanisms of monocrotophos resistance in cotton bollworm,Helicoverpa armigera (Hübner)

Insensitive acetylcholinesterase was identified as a resistance mechanism by comparing biochemical analysis with a laboratory selected monocrotophos resistant cotton bollworm (RR: 200) and the susceptible strain. The cDNA encoding AChE was cloned by the method of RACE (rapid amplification of cDNA ends). The complete AChE gene deduced from the cDNA consisted of a putative signal peptide of 32 amino acid residues, a mature protein of 615 residues, 5' untranslated regions (UTR) of 315 bp and 3' UTR of 324 bp. The coding sequence had a high degree of homology to the AChE from other insect species reported in the GenBank. After comparing analysis of the entire AChE gene sequence from 5 resistant and 6 susceptible cotton bollworm individuals, nine mutations were identified. One of them, the Ala/Thr mutation, is likely to be responsible for the AChE insensitivity to monocrotophos.     

Genetic basis of sex pheromone blend difference between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

The two closely related moth species, Helicoverpa armigera and H. assulta, are sympatric in China. Both species use a mixture of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) as their sex pheromones but in widely different ratios. Hybridization and backcrossing experiments between H. armigera and H. assulta were conducted and sex pheromone compositions of the parent species, their F(1) hybrids and backcrosses were compared to study the genetic basis of the production of their sex pheromone blend composition. Results show that the difference in sex pheromone blend ratios of these Helicoverpa species is mainly controlled by an autosomal locus with two alleles, with the allele from H. armigera being almost completely dominant over that derived from H. assulta.     

Structure and ultrastructure of the silk glands and spinneret of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

This study provides comprehensive documentation of silk production in the pest moth Helicoverpa armigera from gland secretion to extrusion of silk thread. The structure of the silk glands, accessory structures and extrusion apparatus are reported. The general schema of the paired silk glands follows that found for Lepidoptera. Morphology of the duct, silk press, muscle attachments and spigot are presented as a three-dimensional reconstruction and the cuticular crescent-shaped profile of the silk press is demonstrated in both open and closed forms with attendant muscle blocks, allowing advances in our knowledge of how the silk press functions to regulate the extrusion of silk. Growth of the spigot across instars is documented showing a distinctive developmental pattern for this extrusion device. Its shape and structure are related to use and load-bearing activity.     

Phenotypic variability in the Old World bollworm,Helicoverpa armigera (Hübner) populations in India

Helicoverpa armigera (Hübner) is one of the most prominent polyphagous species of the Heliothinae pest complex that inflicts severe damage to a wide range of crops in India. Knowledge regarding the population structure of the pest species, whether morphological or genetic, is considered as an essential tool in making effective management decisions. Thus here, we performed the phenotypic characterization of H. armigera populations collected from varied geographic locations across India. Studied populations differed significantly for several external morphometric traits studied at larval, pupal and adult stages. Significant differences were also observed with respect to the intensity of black pigmentation on larval body as well as adult eye and forewing colour patterns. Besides external phenotypic traits, the length of genital organs like aedeagus and valva in males, and bursa copulatrix and bursa seminalis in females also differed significantly amongst populations. The dendrogram based on selected traits showed clear cut differentiation of studied populations into two major groups, one including all the South Zone populations and the other having populations from North and Central Zones. Differences based on phenotyping in the present study indicate the possibility of the existence of different subspecies within the Indian populations of H. armigera.     

Polycalin is involved in the toxicity and resistance to Cry1Ac toxin in Helicoverpa armigera (Hübner)

Polycalin has been confirmed as a binding protein of the Cry toxins in a few Lepidoptera insects, but its function in the action mechanism of Cry1Ac and whether it is involved in resistance evolution are still unclear. In this study, Ligand blot and enzyme-linked immunosorbent assays showed that Helicoverpa armigera polycalin could specifically interact with Cry1Ac with a high affinity (K_d = 118.80 nM). Importantly, antisera blocking polycalin in H. armigera larvae decreased the toxicity of Cry1Ac by 31.84%. Furthermore, the relative gene and protein expressions were lower in Cry1Ac-resistant strain (LF60) than that in Cry1Ac-susceptible strain (LF). These findings indicated that H. armigera polycalin was a possible receptor of Cry1Ac and may be contributed to the resistance to Cry1Ac.     

Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins

Intra-specific variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) in Australia to the Cry1Ac and Cry2Ab delta-endotoxins from Bacillus thuringiensis (Berliner) (Bt) was determined to establish a baseline for monitoring changes that might occur with the use of Bt cotton. Strains of H. armigera and H. punctigera were established from populations collected primarily from commercial farms throughout the Australian cotton belts. Strains were evaluated for susceptibility using two bioassay methods (surface treatment and diet incorporation) by measuring the dose response for mortality (LC50) and growth inhibition (IC50). The variation in LC50 among H. armigera (n=17 strains) and H. punctigera (n=12 strains) in response to Cry1Ac was 4.6- and 3.2-fold, respectively. The variation in LC50 among H. armigera (n=19 strains) and H. punctigera (n=12 strains) to Cry2Ab was 6.6- and 3.5-fold, respectively. The range of Cry1Ac induced growth inhibition from the 3rd to 4th instar in H. armigera (n=15 strains) was 3.6-fold and in H. punctigera (n=13 strains) was 2.6-fold, while the range of Cry2Ab induced growth inhibition from neonate to 3rd instar in H. armigera (n=13 strains) was 4.3-fold and in H. punctigera (n=12 strains) was 6.1-fold. Variation in susceptibility was also evaluated for two age classes (neonates and 3rd instars) in laboratory strains of H. armigera and H. punctigera. Neonates of H. punctigera had the same or higher sensitivity to Bt than 3rd instars. Neonates of H. armigera were more sensitive to Cry2Ab than 3rd instars, while being less sensitive to Cry1Ac than 3rd instars. Differences in the two methods of bioassay used affected relative sensitivity of species to Bt toxins, highlighting the need to standardize bioassay protocols.     

Mechanisms of premating isolation between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

Helicoverpa armigera and Helicoverpa assulta are sympatric sibling species, and in the laboratory they can interbreed and produce viable offspring. To assess the contributions of temporal barriers and sexual barriers to premating isolation, we investigated both the temporal rhythms of calling behavior and pheromone titers of H. armigera and H. assulta females and the behavioral responses of males to conspecific and heterospecific calling females in a wind tunnel. Both H. armigera and H. assulta females called throughout the scotophase, and there was more calling during the second half of the scotophase than during the first half. Maximal pheromone titer and maximal calling activity in H. armigera synchronously occurred at the sixth hour into the scotophase, whereas, in H. assulta, the maximal pheromone titer occurred 2 h before the peak of calling. Pheromone blend ratios of the two species were opposite and, within each species, changes in the ratio within the scotophase and at different ages were relatively small. Males of both H. armigera and H. assulta responded strongly to their conspecific calling females in the wind tunnel and completed the whole courtship sequence. In contrast, they did not land and had no copulation attempts in response to heterospecific calling females. These results show that the two species do not have obvious temporal differences in calling behavior and pheromone production, and the specificity of sex pheromone blend emitted by females plays a key role in their premating isolation. In addition, we summarized the potential isolation mechanisms of H. armigera and H. assulta.     

Antifeedant activity of numb and salty taste compounds against the larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Helicoverpa armigera, an important polyphagous insect pest in agriculture, attacks more than 200 plant species of more than 30 families. Our previous study showed that the choice feeding percentages of H. armigera larvae to tobacco leaf discs treated with sweet, bitter, and hot taste substances were higher than the control leaf discs, while numb and salty substances could significantly inhibit their feeding. To quantitatively determine the synergistic effect of numb and salty substances, in this paper, the antifeeding activities of numb and salty substances and their mixtures blended in different doses or volume ratios were assayed on H. armigera larvae. The first bioassay was designed to elucidate the relative feeding preference of the larvae to the leaves from several common host species, each paired with tobacco leaf discs, and the result indicated that the most preferred host leaf by the larvae was tobacco leaf, followed by cotton and peanut leaves, suggesting that tobacco leaf was the most suitable matrix for the antifeeding bioassay, and the larval consumption of maize, pepper, or tomato leaves were significantly lower than that of tobacco leaf. The second bioassay was to test the choice feeding response of H. armigera larvae to tobacco leaf discs treated with Zanthoxylum bungeanum extracts obtained with different solvents, and the result showed that the antifeeding activity of the alcohol extracts was the strongest (93.38%), and the leaf consumption in the treatment and the control showed extremely significant difference (t = 4.23, t_0.01 = 3.25, P = 0.0022), followed by the dichloromethane extracts (47.64%), while the other three solvents (water, acetone, and n-hexane) could not extract the active antifeeding components from Z. bungeanum. The larval consumption of tobacco leaf discs treated with the alcohol extracts of Z. bungeanum and NaCl solution were significantly less than their corresponding controls. The mean larval consumption of the treated leaf discs decreased with ever-increasing dosage, and the consumption of tobacco leaf discs coated with different doses of alcohol extracts of Z. bungeanum or NaCl solution showed extremely significant difference (F_{alcohol extract of Z. bungeanum} = 3.88, F_0.01 = 3.58, P = 0.0064; F_{NaCl solution} = 54.29, F_0.01 = 3.58, P = 0.0000), with maximum antifeeding effects at a dosage of 30 μL per 1.5 cm ID leaf disc. We further tested the larval consumption of tobacco leaf discs treated with alcohol extracts of Z. bungeanum in saturated NaCl solution mixed in different volume ratios, and the result showed that the choice antifeeding percentages of the treatments with 15 μL or more Z. bungeanum alcohol extracts were higher than 90%, among which the mixture with 25:15 volume ratio of Z. bungeanum alcohol extracts and saturated NaCl solution exhibited the strongest antifeeding activity, and the mean consumed leaf area of tobacco leaf discs coated with this blend was only 0.10 mm². In the further test on feeding dose-response of the 25:15 mixture, the mean leaf consumption decreased linearly with ever-increasing dosage, with a regression equation y = ?3.9356x + 120.78(R² = 0.9998), and the 30 μL dose could completely inhibit H. armigera feeding.     

Modeling Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) damages on cotton

We studied and modeled damage caused by Helicoverpa armigera larvae on cotton with the aim of developing a coupled crop pest model. Two damage components were studied: the voracity (quantity of fresh matter and number of organs consumed) and feeding preferences (type of organ infested). The laboratory no-choice study of voracity on excised squares and bolls revealed that an H. armigera larva consumes 2,856 mg of fresh matter throughout its larval life, with the sixth instar consuming 86% of this quantity. This consumption rate corresponded to 23.6 squares, or 7.8 bolls. We developed equations to predict the quantity of fresh matter uptake from an individual plant organ, according to the organ mass and the larval instar. The field study of feeding preference confirmed previous findings that larvae prefer squares to bolls, with this preference decreasing as the larval age increases. However, no significant relationship was noted between the age of larvae and the size of infested organs within each organ class (square or boll). We developed a logistic model to predict the probability of a larva infesting a boll rather than a square. According to this model, the relative organ availability in the field and the larval instar were found to be significant factors.     

In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae

Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5 percentage yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80 degree C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.     

Development and mechanisms of resistance to Bacillus thuringiensis endotoxin Cry1Ac in the American bollworm, Helicoverpa armigera (Hübner)

The American bollworm, H. armigera, evolved 31-fold resistance to selection pressure of B. thuringiensis endotoxin Cry1Ac within six generations. The Cry1Ac selected larvae of H. armigera showed cross-resistance to Cry1Aa and Cry1Ab both in terms of mortality and growth reduction. Studies on mechanisms of resistance to Cry1Ac showed that proteases of resistant insects degraded Cry1Ac faster than those of susceptible insects, which led to the relative unavailability of toxin of about 58 kDa for binding and perforation of midgut epithelial membrane of the target insect. Besides, resistant and susceptible populations of H. armigera differed in the binding of their receptors with Cry1Ac toxin. These results suggest the possibility of both mechanisms existing in imparting resistance. These findings mandate the necessity of B. thuringiensis resistance management for usage of B. thuringiensis either as a conventional insecticide or through transgenic crops.     

DNA synthesis in the imaginal wing discs of the American bollwormHelicoverpa armigera (Hübner)

The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instarHelicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of incubation up to 8 h and decreased later without the addition of moulting hormone. Addition of 20-hydroxyecdysone supported long term acquisition of competence for DNA synthesis in the wing discs. Both DNA synthesis and protein content were drastically reduced in plumbagin and azadirachtin-treated insects. Underin vitro conditions, plumbagin had a more pronounced inhibitory effect than azadirachtin. All the ecdysteroids tested, viz. makisterone A, 20-hydroxyecdysone and the ecdysteroidal fraction from the silver fernCheilanthes farinosa enhanced DNA synthesis     

Effect of cotton bollworm (Helicoverpa armigera Hübner) caused injury on maize grain content, especially regarding to the protein alteration

The cotton bollworm (Helicoverpa armigera Hübner), which migrated in the Carpathian-basin from Mediterraneum in the last decades, is becoming an increasingly serious problem for maize producers in Hungary. In several regions the damage it causes has reached the threshold of economic loss, especially in the case of the sweet maize cultivation. The aim of the research was to determine the changing of ears weights and in-kernel accumulation and alteration in grain as a function of cotton bollworm mastication.Our investigation confirmed that there is an in-kernel and protein pattern change of maize grain by cotton bollworm. Our results proved the significant damaging of each part of ears by cotton bollworm masticating (the average weight loss of ears: 13.99%; the average weight loss of grains: 14.03%; the average weight loss of cobs: 13.74%), with the exception of the increasing of the grain-cob ratio. Our examinations did not prove the water loss - that is the "forced maturing" - caused by the damage. Decreasing of raw fat (control: 2.8%; part-damaged: 2.6%; damaged: 2.4%) and starch content (control: 53.1%; part-damaged: 46.6%; damaged: 44.7%) were registered as a function of injury. In contrast, the raw protein content was increased (control: 4.7%; part-damaged: 5.3%; damaged: 7.4%) by maize ear masticating. The most conspicuous effect on protein composition changing was proved by comparison of damaged grain samples by SDS PAGE. Increased amounts of 114, 50, 46 and 35 kDa molecular mass proteins were detected which explained the more than 50% elevation of raw protein content. The statistical analysis of molecular weights proved the protein realignment as a function of the pest injuries, too.     

Changes in haemolymph constituents of American bollworm, Helicoverpa armigera (Hübner), infected with nucleopolyhedrovirus

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucleopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-à-vis immunity in insect is discussed.     

Effect of volatiles emitted from wilted Populus nigra L. leaves on the mating and oviposition of Helicoverpa armigera (Hübner)

Wilted black poplar, Populus nigra ‘Italica’ L., leaves are very attractive to a vast number of noctuid moth species. This provides an opportunity for the development of effective trapping methods for the integrated management of pest species, such as Helicoverpa armigera, a major global and economically important insect pest.In the present study, we investigated the (1) nocturnal attraction patterns of H. armigera males and females to wilted P. nigra leaves; (2) effects of P. nigra volatiles on the mate-searching behavior of males through laboratory serial-chamber bioassays and field trapping; and (3) effects of P. nigra volatiles on the ovipositional choice and reproductive performance of females. Females and males, when tested alone, could be attracted by wilted P. nigra leaves, and the time periods of the first two attraction peaks were largely overlapped between sexes. Streams consisting of wilted P. nigra leaves and virgin females were not more attractive than virgin females alone, regardless of the stream sequence in a serial chamber. However, a stream of virgin females passed through wilted P. nigra leaves was more attractive than wilted P. nigra leaves alone. The addition of P. nigra extracts and its major aromatic components to the sex lure of H. armigera did not attract more moths than the sex lure alone. The volatiles from wilted P. nigra leaves were significantly more attractive to ovipositing females than those from cotton, tomato, and corn leaves, but equally attractive to tobacco leaves. Females exposed to volatiles from different leaves (P. nigra, cotton, and tobacco) showed similar fecundities. In summary, the attraction of moths to wilted P. nigra leaves may be attributable to multiple mechanisms, including the adsorption of sex pheromones, ovipostional attraction, and possible feeding attraction.     

棉铃虫[Helicoverpa armigera(Hübner)]蛾复眼视网膜电位研究

运用视网膜电位（Ｅｌｅｃｔｒｏｒｅｔｉｎｏｇｒａｍ , ＥＲＧ） 技术研究了棉铃虫［ Ｈｅｌｉｃｏｖｅｒｐａ ａｒｍｉｇｅｒａ（Ｈüｂｎｅｒ）］ 成虫在暗适应过程中对单色光和复合光（ 即白光） 刺激的光感受性变化, 并初步分析了ＥＲＧ 的波形成分组成及影响因素。结果显示：（１） 依ＥＲＧ 振幅大小（ 峰－ 峰值） ,棉铃虫蛾的光谱敏感曲线在３４０ － ６０５ｎｍ 波谱内有三个大小不等的峰,其中第一峰位于绿－ 黄光区的５６２ｎｍ ,第二峰位于蓝光区的４８３ｎｍ ,第三个峰在近紫外区的４００ｎｍ ,显示其复眼至少具有三种感受器类型。（２） 一定光强度范围内, 一定日龄的棉铃虫蛾的ＥＲＧ 值随单色光和白光刺激光强度的增强而增大, 至一定光强度时增加变缓,呈近似Ｓ 型曲线式样。表明该蛾复眼具有较强的光强度自调节和适应机制。（３） 棉铃虫蛾复眼的ＥＲＧ 波形成分由４ 个部分组成：开光反应、正相电位、持续负电位和闭光反应,波长、光强度及光刺激时程对其各成分有不同影响。