The role of two plant-derived volatiles in the foraging movement of 1st instar Helicoverpa armigera (Hübner): time to stop and smell the flowers

Positive phototaxis and negative geotaxis are behaviours that 1st instar Helicoverpa armigera use to direct their foraging movement upward towards nutritious new plant growth and reproductive structures. Odours emitted by fruits or seeds can attract larvae directly via chemotaxis. In this study we clarify the effect of leaf and flower odours on foraging 1st instar H. armigera. Using a Y-tube olfactometer we tested for chemotaxis towards two plant volatiles and found larvae were not attracted. Bioassays for phototaxis towards UV, blue, green and white light showed that a green leaf volatile ((Z)-3-hexenyl acetate) and a flower volatile (phenylacetaldehyde) reduced larval phototaxis towards blue light. Feeding on a host plant reduced phototaxis towards blue and green light. We concluded that the upward movement of 1st instars on plants is largely due to phototaxis towards the blue wavelengths of skylight. Plant attributes such as volatile chemicals affect the expression of phototaxis and therefore, indirectly influence larval movement to locate food resources. Handling Editor: Anna-Karin Borg-Karlson     

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.     

Effects of larval host plants on over-wintering preparedness and survival of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Laboratory colonies of cotton bollworm larvae, Helicoverpa armigera, kept at 20 degrees C under a photoperiod of L:D=10:14 were fed on five host plants (cotton, corn, kidney bean, tobacco and tomato) and an artificial diet (control) to determine the effects of larval host quality on survival and pupal over-wintering preparedness. A separate experiment showed that diapausing pupae weighed more and contained greater nutrient stores than did non-diapausing pupae. Diapausing pupae reared on different host plants showed significant differences in terms of over-wintering reserve storage, and degree of cold-hardiness (extent of low-molecular-weight substances and SCPs), and survivorship. The more nutrients the host plant had, the more the pupae weighed and the higher the levels of total lipids and glycogen. Body water content was also significantly affected by larval food quality. The mean pupal super-cooling capacities varied significantly from -16.7 to -18.9 degrees C according to host plants the larvae feed on, and these significantly related to water content, pupal weight, lipid and glycogen content, and the levels of glycerol. Levels of trehalose, glycerol, and inositol, which were mainly low-molecular-weight substances, showed no significant differences among different host plants, except for trehalose. Pupal mortality varied from 39.7% on corn to 3.3% on the artificial diet, which was significantly related to pupal weight, total lipid content, trehalose levels, and super-cooling points. These results suggest that larval food quality can affect survival and influence the over-wintering preparedness of the cotton bollworm.     

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.     

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.     

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.     

Effects of larval host plants on over-wintering physiological dynamics and survival of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Laboratory colonies of cotton bollworm larvae, Helicoverpa armigera, were kept at 20 °C under a photoperiod of L:D = 10:14 and fed on three host plants (cotton, tobacco, kidney bean) and an artificial diet (control) to determine the dynamic effects of larval host quality on over-wintering physiology and mortality. Energy reserves (glycogen and lipid), super-cooling points (SCPs), low-molecular-weight sugars, temperature, and mortality were monitored from November 2002 to April 2003. Lipid content did not change much for each group during over-wintering, but differed according to larval host plants. Larval host plants obviously influence the amount of glycogen, as does time of year: glycogen was lowest in February and increased in early spring. During winter, the mean pupal SCPs increased the most in February, then decreased, and were also affected by larval host plant, i.e. over-wintering pupae reared on kidney bean had the highest SCPs. Levels of glycerol and inositol differed significantly among host plants and months, which peaked in February. Pupal mortality also varied according to larval host plants and time: pupae reached their highest mortality in March and showed host plant differences in January. Records show that February was the coldest month during the period we observed, which corresponded closely to changes in over-wintering characteristics.     

Development of the predator Chrysoperla carnea (Stephens) as affected by feeding on Helicoverpa armigera (Hübner) reared on transgenic Cry 2Ab tomato plants

Investigations were conducted using Helicoverpa armigera fed on transgenic tomato plants containing a synthetic Cry2ab gene aiming to evaluate its impact on the development of the predator Chrysoperla carnea. Prey consumption and development parameters of C. carnea were assessed. No obvious differences were observed in the development of C. carnea that preyed on H. armigera fed on transgenic Bt tomato plants as compared to the control. This shows obvious that transgenic Bt tomato plants can be safely used as an efficient tool for the biocontrol of H. armigera with no effect on its predator, C. carnea.     

Electrophysiological responses of female Helicoverpa armigera (Hübner) (Lepidoptera; Noctuidae) to synthetic host odours

Studies were conducted to investigate the electroantennographic (EAG) responses of adult female Helicoverpa armigera to a range of known and putative kairomone components. The studies show that at a given dose the EAG responses elicited by a series of straight-chain aliphatic primary alcohols were not dependent on volatility since butan-1-ol and pentan-1-ol elicited EAG responses that were significantly smaller than those elicited by hexan-1-ol. The amplitudes of responses to hexan-1-ol were found to be dose dependent with a dose of 10(-1) μmol at source in a non-volatile solvent eliciting the largest response. Similarly, changes in functionality in a range of C(6) straight-chain aliphatic compounds significantly changed the amplitude of response elicited, with aldehydes eliciting smaller responses than the related primary alcohols and saturated compounds eliciting higher responses than related unsaturated compounds. Of the range of nine host plant-produced terpenoids tested, ocimene and beta-phellandrene elicited the highest responses and of the six aromatic compounds tested phenylacetaldehyde and benzaldehyde elicited the largest responses, at the doses tested. The significance of these findings for analysis of floral odours by gas chromatography linked to electroantennography as a means of identifying kairomone components attractive to H. armigera are discussed.     

Karyomorphological effects of two new oil formulations on Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

The cotton bollworm Helicoverpa armigera is a serious pest of many economically important crops. Since this pest has become resistant to the conventional synthetic insecticides, newer compounds and formulations are being developed against this insect pest. Many natural compounds isolated from the plants were tested against this pest. Among them Hyptis suaveolens and Melochia corchorifolia showed insecticidal properties against H. arnigera. Based on bioefficacy studies, caryophyllene and β-sitosterol were isolated from H. suaveolens and M. corchorifolia respectively. The isolated natural compounds were further developed as formulations in various combinations with neem (Azadirachta indica) and karanj (Pongamia pinnata) oils. The present study indicated that the formulations influenced the karyomorphology of H. armigera.     

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.     

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.     

Molecular cloning and bacterial expression of pheromone binding protein in the antennae of Helicoverpa armigera (Hübner)

A cDNA clone coding for pheromone binding protein was isolated from the antennae of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. The full-length of H. armigera pheromone binding protein (HarmPBP) was 952 bp, possessing 162 amino acid residues including a signal peptide of 20 amino acids. Its predicted molecular weight and isoelectric point were 18.26 kDa and 5.23, 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, a typical characteristic of insect's odorant-binding proteins. Northern blot showed that HarmPBP is specifically expressed in the antennae of Helicoverpa armigera and more abundantly expressed in male than female. During the antennal development, HarmPBP is first expressed about 4 days prior to adult eclosion and rises to a plateau 2 days prior to adult eclosion. In order to obtain sufficient PBP for further determining its biochemical and physiological properties, a bacterical expression vector of PBP was constructed and successfully expressed in Escherichia coli. The recombinant PBP was shown to cross-react with an anti-PBP antiserum from Antheraea polyphemus. Polyclonal antibodies against HarmPBP were used to mark the distribution of the protein in olfactory sensilla. Very strong labeling was observed in the sensillum lymph of the hair lumen and of the sensillum-lymph cavity. In the male, HarmPBP is expressed in sensilla trichodea and not in sensilla basiconica, while in the female, it is expressed both in sensilla basiconica and sensilla trichodea.     

Oxidases responsible for resistance to pyrethroids sensitize Helicoverpa armigera (Hübner) to triazophos in West Africa

Helicoverpa armigera (Hübner) is the major insect pest of cotton in Africa, Turkey, Asia, India, Indonesia and Australia. Populations recently developed resistance to pyrethroids in West Africa via the overproduction of cytochrome P450 (oxidases) increasing pyrethroid metabolism. One way to overcome pyrethroid resistance is to use compounds that show negative cross-resistance to pyrethroids. Triazophos is one of these compounds: it is slightly more toxic against pyrethroid resistant larvae of H. armigera than against susceptible ones. Overproduced oxidases transform the non active triazophos into its active form, triazophos-oxon, since this form was significantly more often found in larvae from pyrethroid resistant strain (23%) than in susceptible strain (15%). This suggests that oxidases, which provide resistance by degradation of pyrethroids in the resistant individuals, also activate triazophos in its toxic oxon form resulting in a negative cross-resistance.     

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.     

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.     

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.     

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.