Pheromone biosynthetic pathways in the moths Heliothis subflexa and Heliothis virescens

Sex pheromones of many moth species have relatively simple structures consisting of a hydrocarbon chain with a functional group and one to several double bonds. These sex pheromones are derived from fatty acids through specific biosynthetic pathways. We investigated the incorporation of deuterium-labeled tetradecanoic, hexadecanoic, and octadecanoic acid precursors into pheromone components of Heliothis subflexa and Heliothis virescens. The two species utilize (Z)11-hexadecenal as the major pheromone component, which is produced by Delta11 desaturation of hexadecanoic acid. H. subflexa also produced (Z)11-hexadecanol and (Z)-11-hexadecenyl acetate via Delta11 desaturation. In H. subflexa, octadecanoic acid was used to biosynthesize the minor pheromone components (Z)9-hexadecenal, (Z)9-hexadecenol, and (Z)9-hexadecenyl acetate. These minor components are produced by Delta11 desaturation of octadecanoic acid followed by one round of chain-shortening. In contrast, H. virescens used hexadecanoic acid as a substrate to form (Z)11-hexadecenal and (Z)11-hexadecenol and hexadecenal. H. virescens also produced (Z)9-tetradecenal by Delta11 desaturation of the hexadecanoic acid followed by one round of chain-shortening and reduction. Tetradecanoic acid was not utilized as a precursor to form Z9-14:Ald in H. virescens. This labeling pattern indicates that the Delta11 desaturase is the only active desaturase present in the pheromone gland cells of both species.     

Replication and serial passage of infectious Heliothis nucleopolyhedrosis virus in an established line of Heliothis zea cells

An established cell line derived from the ovary of adults of the cotton bollworm, Heliothis zea, supported growth of the Heliothis nucleopolyhedrosis virus (NPV). Typical NPV symptoms were obtained when infected cells were fed to neonatal bollworms; however, the cell line never produced free virions or inclusion bodies containing virions. Infectious virus was passed through the cell line 7 consecutive times, using only infected cells from the previous pass. Infectivity at the 7th serial-pass represented a dilution of >10⁻⁸ of the original inoculum.     

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.     

Genetics of sex pheromone blend differences between Heliothis virescens and Heliothis subflexa: a chromosome mapping approach

Males of the noctuid moths, Heliothis virescens and H. subflexa locate mates based on species-specific responses to female-emitted pheromones that are composed of distinct blends of volatile compounds. We conducted genetic crosses between these two species and used AFLP marker-based mapping of backcross families (H. subflexa direction) to determine which of the 30 autosomes in these moths contained quantitative trait loci (QTL) controlling the proportion of specific chemical components in the pheromone blends. Presence/absence of single H. virescens chromosomes accounted for 7-34% of the phenotypic variation among backcross females in seven pheromone components. For a set of three similar 16-carbon acetates, two H. virescens chromosomes interacted in determining their relative amounts within the pheromone gland and together accounted for 53% of the phenotypic variance. Our results are discussed relative to theories about population genetic processes and biochemical mechanisms involved in the evolution of new sexual communication systems.     

Histopathology of virus-like particles in Heliothis spp

Larvae of Heliothis spp. collected from cotton, soybean, and peanut fields in South Carolina were found to be infected with virus-like particles (VLPs). Infected larvae became pale and swollen, stopped feeding, and remained alive for 2–3 weeks. Hemolymph from these larvae was milky and contained numerous spherical bodies ranging in diameter from 2 to 10 μm. The hemolymph also contained VLPs which were oval and measured 375 × 125 nm. Infectivity tests with crude saline extracts of infected larvae demonstrated that the pathogen could be transmitted by injection but not per os. The spherical bodies contained VLPs (387 × 149 nm) surrounded by two envelopes and packed together in clusters. These VLPs were also found in fat body cells, cuticular epithelial cells, tracheal cells, and connective tissue associated with the body wall and the gut. They were not found in muscle tissue or in midgut epithelial cells. Similar VLPs have been found in Heliothis zea from Mississippi and Trichoplusia ni from California, but a positive identification of the VLPs has not been made in any of these studies. Morphologically they appear to be distinct from any other previously described insect viruses.     

Post-eclosion diuresis in adult Heliothis zea

ABSTRACT. The marked weight loss that follows adult eclosion in Heliothis zea (Boddie) (Lepidoptera: Noctuidae) was found to be the result of diuresis. This diuretic weight loss amounted to 18.2% of the emergence weight in females and 22.5% in males. Ligation between head and thorax immediately following emergence resulted in a significant reduction in this weight loss. Injection of homogenates of brain (BR) or suboesophageal ganglion (SOG) partially restored the weight loss in ligated insects, indicating potential neurohormonal regulation of this phenomenon.
Because injection of homogenates of nervous and neuroendocrine tissues other than BR and SOG did not result in increased weight loss over controls, the activity present in BR and SOG was probably a tissue-specific phenomenon. We speculate that a factor at least partially responsible for the enhanced diuretic weight loss is present in the fused BR/SOG.     

Habituation of sexual response in male Heliothis moths

It has been generally hypothesized that habituation mediates the effects of pheromone‐based disruption strategies used in the management of moth pests. The current study demonstrates that pheromone‐mediated sexual response in the tobacco budworm moth, Heliothis virescens (F.) (Lepidoptera: Noctuidae), can in fact be modulated by conditions consistent with the production of habituation. An ethogram was used to measure response strength in a wind tunnel experiment where male moths were allowed to respond freely to one of two different blends of female pheromone in 16 trials over 4 days. Post‐test measures were collected to investigate stimulus specificity and long‐term effects. In conditions appropriate to the formation of habituation, habituation will develop and disrupt male sexual response to female sex pheromone. Males repeatedly exposed to plumes of synthetic pheromone blends display a habituated response lasting up to 96 h. Habituation rate and spontaneous recovery of response strength are greater with less intense stimuli. Additionally, males habituated to one blend express no habituation of sexual response when exposed to a different blend. This indicates a high degree of stimulus specificity, which could facilitate outbreeding, and that moths attend to the configuration of the pheromone blend, not simply to its elements.     

Pharmacological analysis of ovarial patency in Heliothis virescens

The insect oocyte sequesters nutritive proteins during patency, which is facilitated as a result of intercellular spaces occurring between follicular epithelial cells under the influence of juvenile hormone (JH). Patency was analyzed in the moth, Heliothis virescens, using a pharmacological approach, in which we used different JH homologues and chemicals that specifically target elements of two second-messenger pathways in vertebrates, the cAMP-dependent and inositol triphosphate/diacylglycerol signaling pathways. JH I and JH III evoked dose-dependent patency in H. virescens oocyte follicles, which was suppressed by the Na/K-ATPase inhibitor, ouabain. Patency was observed in follicular epithelial cells treated with either protein kinase C activator, PDBu, or protein kinase A activator, 8-Br-cAMP, by itself. The protein kinase C inhibitor, H-7, preferentially suppressed patency evoked by JH III, whereas the protein kinase A inhibitor, H89, preferentially suppressed that evoked by JH I. Additionally, patency was triggered by the adenylate cyclase activator, NKH 477, or peptide Gs-protein activator, cholera toxin, alone. Patency evoked by JH I was suppressed by the adenylate cyclase inhibitor, SQ 22,536, and GPAnt-2, a peptide antagonistic to Gs proteins that stimulates adenylate cyclase. Neither of these latter inhibitors, however, affected JH III-evoked patency. These results suggest that, in the process of patency in H. virescens ovarial follicles, JH I predominantly signals via the cAMP-dependent second messenger system, whereas JH III acts via the inositol triphosphate/diacylglycerol signaling pathway. Moreover, stimulation of patency by cholera toxin alone and inhibition of JH I-evoked patency by GPAnt-2, strongly suggest that JH I acts on the follicular epithelial cells via activation of G-protein, and-possibly-via G(s)-protein coupled receptor.     

Establishment of early instar Heliothis zea on soybeans

Early instar Heliothis zea (Boddie) (Lepidoptera: Noctuidae) established on all stages of soybean (Glycine max [L.] Merrill) tested when larvae were confined to plants, although establishment decreased after podset stage and as seeds matured. In tests where larvae were allowed to disperse freely on plants, establishment also decreased as maturity increased, but was much lower in each stage than when confined to plants. Neonate larvae settled more quickly on terminals and expanding trifoliates than on mature trifoliates based on initiation of feeding and movement away from initial sites of placement. The ability of neonate larvae placed a short distance from soybean plants to reach a host was affected by high soil and ambient temperature. The results of these experiments suggest that larval host establishment and spin-down behavior play a major role in the late instar population distribution among soybean fields and that maturity of the host strongly influences that behavior.

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Résumé Des expériences ont été réalisées dans deux types de conditions (avec ou sans possibilité de dispersion) pour déterminer si le taux d'installation des chenilles de premier stade de H. zea Bod. dépendait d'une mortalité liée au stade développement de Glycine max L. L'installation était considérée comme réussie quand les chenilles avaient atteint le second stade. Bien qu'il fût possible de distinguer les variétés de soja tant par le taux d'installation des chenilles que par les dégâts provoqués, les plantes les plus jeunes étaient plus colonisées par des chenilles que les témoins plus âgés. Toutes les variétés de soja à n'importe quel stade étaient acceptées lorsque la dispersion des chenilles était impossible. Avec possibilité de dispersion, les chenilles ont aussi accepté tous les stades phénologiques, mais en moins grands nombres que lorsque la dispersion était impossible. Le comportement de dispersion était beaucoup plus fréquent sur les plantes à port indéterminé que sur celles à port déterminé. Les possibilités de réinstallation après dispersion des chenilles de premier stade ont été limitées.

     

Physiology of interspecific chemical communication in Heliothis moths

Abstract Electroantennograms were recorded from the antennae of adult male and female corn earworms, Heliothis zea (Boddie). A total of seventeen female moth sex pheromone components from several species were tested. Of these, two components elicited significantly greater responses than the other fifteen. These were (Z)-11-hexadecenal, a conspecific component, and (Z)-9-tetradecenal, a component found in the pheromone blend of a sympatric species H.virescens (F.) that inhibits attraction of H.zea males. The results from dose-response and selective adaptation studies indicate that there are separate populations of receptors for these two chemical signals on the antenna of male H.zea. The more sensitive population is selective for (Z)-11-hexadecenal, while the less sensitive one responds to (Z)-9-tetradecenal. These findings provide a physiological basis by which H.zea males can distinguish the interspecific repellent from the conspecific pheromone blend. It is likely that this discrimination contributes to reproductive isolation between these two species.     

Fat body stimulation of ecdysone synthesis in Heliothis zea

Prothoracic glands of Heliothis zea pupae require both a humoral factor and prothoracicotropic hormone (PTTH) to synthesize ecdysone. The humoral factor is absent when pupae are maintained at diapause-sustaining temperatures. Thus, pupae remain in diapause despite the release of PTTH at or before larval-pupal ecdysis.Tissue implantation experiments revealed that a diapause-terminating factor is present in the fat body of non-diapausing pupae. Other tissue implantation experiments showed that, when diapausing pupae were transferred from 19 to 27°C, diapause-terminating activity appeared first in the fat body and then the fat body into the haemolymph. HPLC separation of the haemolymph and fat body fractions followed by bioassay demonstrated that fractions containing diapause-terminating activity eluted from both tissues within 28–30 min. These results suggest that the factors found in the fat body and haemolymph may be the same compound.Evidence from ecdysone radioimmunoassay experiments ruled out the possibility that the diapauseterminating activity was due to either free or conjugated ecdysteroids. Corresponding in vitro experiments in which the prothoracic glands were cultured with brain extracts versus fat body and haemolymph fractions also indicated that the haemolymph/fat body factor was not PTTH.     

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.     

Carbohydrate digestion in the adult moth Heliothis zea

Extracts from the ventriculus and the salivary glands of the adult corn earworm, Heliothis zea, were tested for carbohydrase activity. The hydrolysis that occurred among the 12 carbohydrate substrates tested and the evidence from thin-layer chromatography indicated only one carbohydrase, a β-fructosidase, from the salivary glands and two, a β-fructosidase fnd an α-glucosidase, from the ventriculus. Optimum pH was 6·5 for the β-fructosidase from both the ventriculus and the salivary glands and 5·5 for the α-glucosidase. The K_m for the β-fructosidase from the salivary glands was 112 mM.     

Protocol for assessing soybean antixenosis to Heliothis virescens

Larvae of Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) often infest soybean crops, Glycine max (L.) (Fabaceae), causing significant yield losses in important soybean-producing regions. The use of soybean varieties resistant to lepidopteran larvae is a major approach in soybean integrated pest management. However, standardization and optimization of bioassays that are used to screen genotypes for insect resistance are essential for high-throughput phenotyping. Methodologies for screening were assessed to determine the most effective method for discriminating levels of antixenosis to H. virescens in soybean plants. Feeding and oviposition preference assays were performed to determine optimal densities of larvae and adults, and optimal plant structures and growth stages for conducting assays. In addition, trichome densities, and fiber and lignin contents were quantified in plant structures of soybean cultivars differing in resistance. Resistance levels of cultivars were best differentiated using nine neonate larvae and two 6-day-old larvae, and by using young leaves of plants at the vegetative stage. This was likely due to the more pronounced differences in lignin and fiber contents in young leaves of vegetative-stage plants. Density of adult pairs, plant structure, and growth stage did not affect ability to distinguish differences in oviposition preference by H. virescens. Higher numbers of eggs were found on the leaves, which were the plant structures that exhibited the lowest trichome densities. The protocol developed in this work will benefit future evaluations of soybean genotypes for antixenosis against H. virescens.     

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.