Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts

The binding properties of Vip3A, a new family of Bacillus thuringiensis insecticidal toxins, have been examined in the major cotton pests, Heliothis virescens and Helicoverpa zea. Vip3A bound specifically to brush border membrane vesicles (BBMV) prepared from both insect larval midguts. In order to examine the cross-resistance potential of Vip3A to the commercially available Cry1Ac and Cry2Ab2 toxins, the membrane binding site relationship among these toxins was investigated. Competition binding assays demonstrated that Vip3A does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. BBMV protein blotting experiments showed that Vip3A does not bind to the known Cry1Ac receptors. These distinct binding properties and the unique protein sequence of Vip3A support its use as a novel insecticidal agent. This study indicates a very low cross-resistance potential between Vip3A and currently deployed Cry toxins and hence supports its use in an effective resistance management strategy in cotton.     

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC₅₀s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.     

The surface hydrocarbons of larval Heliothis virescens and Helicoverpa zea

Third instar larvae of Heliothis virescens and Helicoverpa zea could be distinguished based on the hydrocarbons of their surface lipids. Hydrocarbons were the major components of the surface lipids and a distinctive capillary gas chromatographic profile could be obtained from a hexane extract of the surface lipids of a single larva. Analysis of hexane extracts of the surface lipids by capillary gas chromatography-mass spectrometry (CGC-MS) showed several obvious differences between the two species: (1) in their gas chromatographic profiles; (2) in the presence of a major alkene, hentriacontene, only in H. zea; (3) in H. virescens the CGC-MS peak with a KI of approximately 2860 was 2-methyloctacosane, but in H. zea it was a mixture of 4-methyloctacosane plus 9,13- and 8,12-dimethyloctacosanes; and (4) in the methyl branch positions of dimethyl-branched alkanes with carbon backbones of C₃₁, C₃₃, C₃₅, C₄₅, C₄₇, C₄₉ and C₅₁. The methyl branch points of H. virescens dimethylalkanes were separated by seven or nine methylenes, while in H. zea the methyl branch points of the dimethylalkanes were separated by three and sometimes five methylenes.     

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.     

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five™) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth (growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five™ cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium (LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interspecific competition between two parasitoids of Helicoverpa zea: Eucelatoria bryani and E. rubentis

Multiple parasitism of Helicoverpa zea (Lepidoptera: Noctuidae) had differential effects on the gregarious endoparasitoids Eucelatoria bryani and E. rubentis (Diptera: Tachinidae). Both species were able to survive in multiparasitized hosts. However while the survival of E. bryani was not affected by the presence of E. rubentis, E. rubentis survival was reduced when competing with 24-h older E. bryani. The presence of E. rubentis did not result in a decrease in the size of E. bryani except when E. rubentis was 24-h older than E. bryani. The presence of E. bryani tended to result in smaller E. rubentis. The presence of similarly aged heterospecific competitors often resulted in prolonged development time for both species. Naïve females did not alter oviposition between unparasitized and heterospecifically parasitized fifth-instar larvae of H. zea, either by ovipositing less frequently in parasitized hosts than unparasitized ones, or by reducing clutch size in parasitized hosts. These results indicate that in H. zea, E. bryani is a superior competitor compared with E. rubentis. These factors should be considered in evaluating the potential of E. bryani and E. rubentis as biological control agents of H. zea.     

Temporal oviposition patterns of Heliothis zea and Spodoptera ornithogalli

Egg production under laboratory conditions was examined over the lifespan of Heliothis zea (Boddie) and Spodoptera ornithogalli Guenée (Lepidoptera: Noctuidae). Although H. zea oviposits singly and S. ornithogalli oviposits in masses, temporal trends were similar. Egg numbers peaked shortly after mating and then rapidly declined. Egg weights also peaked shortly after mating, but decreased gradually over time. Temporal oviposition patterns were more erratic for unmated than mated females, suggesting the importance of mating in establishing the shape of the oviposition curve.

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Résumé La production d'ufs d'Heliothis zea (Boddie) et de Spodoptera ornithogalli Guenée (Lepidoptera: Noctuidae) durant toute la vie adulte a été examinée au laboratoire. Bien que H. zea dépose ses ufs isolément et que S. ornithogalli les dépose en groupes, les courbes temporelles d'oviposition chez des individus fécondés sont similaires. Le nomber d'ufs culmine peu après l'accouplement, puis décline rapidement. Le poids des ufs culmine aussi peu après l'accouplement, puis décroît graduellement. Des femelles fécondées produisent environ deux fois autant d'ufs que les femelles non fécondées, quoique la longévité ne diffère pas significativement entre les deux groupes. Des femelles non fécondées montrent des distributions temporelles plus irrégulières que des femelles fécondées, en ce qui concerne le nombre des ufs et leur poids. Ces irrégularités ont été attribuées à la tendance des femelles non fécondées à retenir leur ufs, ce qui suggère que l'accouplement exerce une influence sur la forme de la courbe temporelle d'oviposition. Des femelles d'H. zea contenant un, deux ou trois spermatophores n'ont pas produit des nombres d'ufs significativement différents.

     

Receptor-mediated endocytosis of storage proteins by the fat body of Helicoverpa zea

The selective uptake of storage proteins by the fat body of the corn earworm Helicoverpa zea is mediated by a membrane-bound receptor protein. In this study, the major storage proteins of this insect species, arylphorin and very high density lipoprotein, were directly labeled with colloidal gold-particles of different size. After the fat body had been incubated with the labeled storage proteins, the distribution of these proteins was examined by electron microscopy. Both storage proteins were found at the extracellular side of coated pits and within coated vesicles. Moreover, fusion products of several coated vesicles such as endosomes and multivesicular bodies contained both proteins in their lumen. Ultimately, the proteins accumulated in electron-dense storage granules. Equal numbers of either storage protein were present in each organelle, supporting the notion that a single receptor mediates the uptake of both proteins. In contrast, only small numbers of gold-labeled immunoglobulin G molecules were found in the organelles, indicating that the protein uptake is specific for storage proteins. The results show that storage protein uptake in this lepidoteran species occurs in a process of receptor-mediated endocytosis that is similar to the well-established uptake of specific proteins into mammalian tissues.     

Damage to lima beans by Helicoverpa zea larvae treated as different instars with a nucleopolyhedrovirus

Larvae of the corn earworm,Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), were caged assecond, third, or fourth instars on lima bean,Phaseolus lunatus L., plants in the fieldwith or without treatment of the plants withlethal concentrations of the H. zeanucleopolyhedrovirus. The virus treatmentprevented over 90% of damage to pods and beansif larvae were second or third instars whenplaced in the cages. Damage to pods and beanswas reduced by 73 and 86%, respectively, ifthe larvae were fourth instars when placed onthe plants. When insects survived to the endof the test (which they did only on controlplants), numbers of pods or beans damaged byeach surviving insect was not affected by thestadium in which the larva was when it wasplaced on the plant. This result indicates thatmost damage was done by fourth or laterinstars. If treatments are to effectivelyprevent damage to lima bean, they should thusbe applied before the insects reach the fourthinstar.     

The distribution of pheromone-biosynthesis-activating neuropeptide (PBAN) immunoreactivity in the central nervous system of the corn earworm moth,Helicoverpa zea

Summary Production of sex pheromone in several species of moths has been shown to be under the control of a neuropeptide termed pheromone-biosynthesis-activating neuropeptide (PBAN). We have produced an antiserum to PBAN from Helicoverpa zea (Lepidoptera: Noctuidae) and used it to investigate the distribution of immunoreactive peptide in the brain-suboesophageal ganglion complex and its associated neurohemal structures, and the segmental ganglia of the ventral nerve cord. Immunocytochemical methods reveal three clusters of cells along the ventral midline in the suboesophageal ganglion (SOG), one cluster each in the presumptive mandibular (4 cells), maxillary (12–14 cells), and labial neuromeres (4 cells). The proximal neurites of these cells are similar in their dorsal and lateral patterns of projection, indicating a serial homology among the three clusters. Members of the mandibular and maxillary clusters have axons projecting into the maxillary nerve, while two additional pairs of axons from the maxillary cluster project into the ventral nerve cord. Members of the labial cluster project to the retrocerebral complex (corpora cardiaca and cephalic aorta) via the nervus corpus cardiaci III (NCC III). The axons projecting into the ventral nerve cord appear to arborize principally in the dorsolateral region of each segmental ganglion; the terminal abdominal ganglion is distinct in containing an additional ventromedial arborization in the posterior third of the ganglion. Quantification of the extractable immunoreactive peptide in the retrocerebral complex by ELISA indicates that PBAN is gradually depleted during the scotophase, then restored to maximal levels in the photophase. Taken together, our findings provide anatomical evidence for both neurohormonal release of PBAN as well as axonal transport via the ventral nerve cord to release sites within the segmental ganglia.Abbreviations A aorta - Br-SOG brain-suboesophageal ganglion complex - CC corpus cardiacum - PBS phosphate-buffered saline - PLI PBAN-like immunoreactivity - TAG terminal abdominal ganglion - VNC ventral nerve cord     

Hormonal control of egg maturation in the corn earworm, Heliothis zea

At eclosion, the ovaries of female Corn earworm Heliothis zea do not contain mature eggs. Virgin-unfed females produced approximately 400 mature eggs in 8 days; mating or feeding doubled this number, and mating plus feeding more than tripled it. Females allatectomized or decapitated at day O matured few eggs. Egg production was restored by implantation of active corpora allata (CA) or by treatment with the juvenile hormone (JH) analogue methoprene at day 0. 20-Hydroxyecdysone, on the other hand, had no effect. Females in which the CA had been denervated or in which the median neurosecretory cells of the brain had been ablated at day O produced fewer eggs than sham-operated animals. These results indicate that egg maturation is controlled by JH and that continuous input from the brain is required for sustained CA activity for maintaining a high rates of egg maturation.The rate of JH biosynthesis by CA in vitro was determined with a radiochemical assay. The major hormones produced were JH-II and JH-III with small quantities of JH-I. The rates of JH synthesis were similar in all experimental groups which may indicate that the in vitro rate of JH synthesis does not reflect the actual state of CA activity in the female.     

Model for genetic control of Heliothis virescens

Summary A model was developed which corrects and extends an earlier one proposed for the control of the tobacco budworm, Heliothis virescens (F.), through hybrid male sterility. Population suppression is effected through the release into natural populations of the backcross progeny of a hybrid between H. virescens and a related species. Thereafter, the system perpetuates itself in nature through continual backcrossing of the fertile backcross females to native H. virescens males. When the proportion of backcross hybrid females in the total population is large enough to draw off the insemination potential of the native males, the native females fail to replace themselves. The present model demonstrated that the ratio of released backcross hybrids to natural H. virescens remains constant in a closed population. Furthermore it was shown that the release ratio necessary to achieve extinction of a closed population is related to the number of females that a male can inseminate and to the population growth rate. Release ratios required to slow natural population growth and to lessen the impact damage of releases on crop plants were also examined. Effects of selection against the backcross females on the predictions of the model were explored.     

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.     

Antennal lobe projection destinations of Helicoverpa zea male olfactory receptor neurons responsive to heliothine sex pheromone components

We used single sensillum recordings to define male Helicoverpa zea olfactory receptor neuron physiology followed by cobalt staining to trace the axons to destination glomeruli of the antennal lobe. Receptor neurons in type A sensilla that respond to the major pheromone component, (Z)-11-hexadecenal, projected axons to the cumulus of the macroglomerular complex (MGC). In approximately 40% of these sensilla a second receptor neuron was stained that projected consistently to a specific glomerulus residing in a previously unrecognized glomerular complex with six other glomeruli stationed immediately posterior to the MGC. Cobalt staining corroborated by calcium imaging showed that receptor neurons in type C sensilla sensitive to (Z)-9-hexadecenal projected to the dorsomedial posterior glomerulus of the MGC, whereas the co-compartmentalized antagonist-sensitive neurons projected to the dorsomedial anterior glomerulus. We also discovered that the olfactory receptor neurons in type B sensilla exhibit the same axonal projections as those in type C sensilla. Thus, it seems that type B sensilla are anatomically type C with regard to the projection destinations of the two receptor neurons, but physiologically one of the receptor neurons is now unresponsive to everything except (Z)-9-tetradecenal, and the other responds to none of the pheromone-related odorants tested.     

The effect of age of parents on the progeny of diapaused and non-diapaused Heliothis zea

The effect of parental age at oviposition on the offspring of diapaused and non-diapaused females of Heliothis zea (Boddie) (Lepidoptera: Noctuidae) was examined under laboratory conditions. Diapause was induced in the laboratory by reducing day lengths and temperatures. The number of eggs per female, duration of the egg stage, percent hatch, duration of the larval stage, percent pupation and four-day pupal weights of progeny from diapaused and non-diapaused adults were determined. Significantly reduced egg production and increased duration of egg and larval stages occurred in offspring of diapaused females, but no effect on hatching and pupation percentages and pupal weights were observed. The possible effects of these differences on reproductive potential and length of generation are discussed.

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Résumé Nous avons examiné au laboratoire l'influence de l'age des parents au moment de la ponte sur la descendance des femelles d'Heliothis zea Boddie diapausantes ou non. La réduction de la longueur du jour et de la température avait induit la diapause au laboratoire. L'étude a porté sur le nombre d'oeufs par femelle, la durée de l'incubation des oeufs, le taux d'éclosion, la durée du développement larvaire, le taux de nymphose et le poids des chrysalides de 4 jours chez les descendants d'adultes diapausantes ou non. Chez les descendants des femelles diapausantes, la production d'oeufs est significativement réduite; de même, l'incubation des oeufs et le développement larvaire sont significativement prolongés; mais on n'a observé aucun effet tant sur les taux d'éclosion et de nymphose que sur le poids des chrysalides. La discussion porte sur les influences envisageables de des différences dans le potentiel reproducteur et la durée des générations.

     

Toxicity and anticholinesterase activity of the fungal metabolites territrems to the corn earworm, Helicoverpa zea

The toxicity and anticholinesterase activity of tremorgenic fungal metabolites, territrems, to the corn earworm, Helioverpa zea (Boddie) (Lepidoptera, Noctuidae) were examined. In oral toxicity studies, territrem A significantly inhibited growth by 40% at 25 ppm and by 89% at 250 ppm. Territrem B and an epoxy-derivative significantly inhibited growth by ca. 45% at 250 ppm. Piperonyl butoxide administered orally synergized the toxicity of the territrems tested. In topical toxicity studies, the epoxy-derivative caused 26% mortality and 25% growth retardation at 10 mg/gm insects. Territrem A and B were not significantly lethal, but did reduce growth by 15–20% at 10 mg/gm insect. Paraoxon tested in the same way caused 100% mortality at 25 ppm orally and 10 mg/gm topically. However, all territrems tested in vitro as inhibitors of H. zea head acetylcholinesterase were at least comparable to or were more active than paraoxon. Topically administered epoxy-territrem B also inhibited H. zea head acetylcholinesterase. The potential for development of new insecticides from a territrem lead structure is discussed.     

Immunolocalization of a candidate pheromone receptor in the antenna of the male moth, Heliothis virescens

Pheromone recognition in insects is thought to involve distinct receptor proteins in the dendritic membrane of antennal sensory neurons. We have generated antibodies directed against a peptide derived from the sequence of the candidate pheromone receptor HR13 from Heliothis virescens. The antibodies specifically labelled the cell bodies of a distinct neuron population housed in male-specific pheromone-sensitive sensilla. Combining antibody staining with in situ hybridization the reactive cells were found to express the HR13 gene. In addition, dendrites projecting into sensilla hairs as well as the axonal processes of immunoreactive cells were labelled. Labelling of axons has allowed visualization of their fasciculation within antennal segments and permits tracking of axons as they merge into the antennal nerve. The HR13 protein was first detected 1 day before eclosion. Thus, the distribution of HR13 protein in the antennal neurons of the male moth strongly suggests a role of the HR13 receptor in recognition of pheromones.     

An analytical model for genetic control of Heliothis virescens incorporating the effects of sterile males

Summary A recent computer simulation model by Levins and Parker (1983) indicated that mass releases of male-sterile Heliothis hybrid moths could cause genetic suppression of the tobacco budworm, Heliothis virescens, without the risk of significant crop damage. We present an analytical model to explore the behavior of the Levins-Parker model. Our model shows that the length of time between matings for females when they mate with wild type fertile males to that when they mate with hybrid sterile males is extremely important to the efficacy of a suppression program. Release ratios needed to suppress a natural pest population were examined across a range of biological parameters.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

Antagonistic Effect of (Z)-11-Hexadecen-1-ol on the Pheromone-Mediated Flight of Helicoverpa zea (Boddie) (Lepidoptera:Noctuidae)

Flight-tunnel experiments were conducted using Helicoverpa zea males to determine whether or not (Z)-11-hexadecen-1-ol (Z11-16:OH), a compound emitted by another heliothine moth species, Heliothis subflexa, is a behavioral antagonist when admixed with the two-component pheromone blend of H. zea. Males were less likely to fly upwind all the way to the source when 0.3% Z11-16:OH was present in the blend. Even 0.1% Z11-16:OH caused differences in the flight behavior of H. zea males; they steered more off the windline than males responding to the pheromone blend alone, resulting in more oblique track angles. Thus Z11-16:OH appears to act antagonistically, along with another compound, (Z)-11-hexadecen-1-ol acetate (Z11-16:Ac), when it is added to the H. zea pheromone blend.