Two sex pheromone components of the tobacco budworm moth, Heliothis virescens

Two compounds were isolated from female $\text{Heliothis}$$\text{virescens}$ (Lepidoptera: Noctuidae) extracts and identified as $\text{cis}$-9-tetradecenal and $\text{cis}$-ll-hexadecenal. Together they elicit intense male $\text{H}$. $\text{virescnes}$ response in laboratory tests and have attracted males in the field. Although $\text{cis}$-ll-hexadecenal is an $\text{H}$. $\text{zea}$ sex pheromone, no evidence was obtained for $\text{cis}$-9-tetradecenal in $\text{H}$. $\text{zea}$.     

The identification and biosynthesis of two juvenile hormones from the tobacco budworm moth (Heliothis virescens)

Brain-corpora cardiaca-corpora allata complexes from the tobacco budworm $\text{Heliothis virescens}$ produce both radiolabelled methyl (2E, 6E, 10Z)-10,11-epoxy-3, 11-dimethyl-7-ethyl-2, 6-tridecadienoate (JH I) and methyl (2E, 6E, 10Z)-10, 11-epoxy-3, 7, 11-trimethyl-2, 6-tridecadienoate (JH II) when cultured in medium containing L-[$\text{methyl}$−¹⁴C] methionine or sodium [1−¹⁴C] propionate. Degradative studies of the hormones derived from propionate show the specific incorporation of the latter into the homo-isoprenoid portions of these compounds.     

Cardiac responses to sensory stimulation in the adult tobacco budworm moth, Heliothis virescens

Electrocardiogram recordings were performed on intact Heliothis virescens moths using Ag-AgCl electrodes positioned on the body cuticular surface. Regular heart activity and its changes in response to mechanical and olfactory stimulations were analysed. Moths were also tested in their anemotactic orientation in response to olfactory stimulation. Results show that regular cardiac activity in Heliothis is cyclical and consists of the alternation of a high and a low spike-frequency period. This activity pattern is strongly influenced by sensory stimulation. Both mechanical stimulations at various intensities and olfactory stimulations with sex pheromone and 1-hexanol evoked tachycardiac effects when applied during low spike-frequency cardiac activity. In contrast, they did not affect high spike-frequency cardiac activity. It is concluded that the cardiac response in Heliothis is an effective indicator of sensory reception. It would therefore appear to represent a valid tool for testing insect reactivity. Accepted: 30 August 1997     

Metabolism of ecdysteroid by testes of the tobacco budworm,Heliothis virescens

Testes from late last stage larvae of the tobacco budworm, Heliothis virescens, were incubated with [³H]ecdysone and [³H]cholesterol. [³H]Ecdysone was converted to six other major ecdysteroids, identified by cochromatography in reverse-phase high-pressure liquid chromatography (RPHPLC); four of them were verified by normal-phase HPLC. A highly polar fraction, moderately polar ecdysteroids (20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, and 20-hydroxyecdysone) and low-polarity ecdysteroids, including 2-deoxyecdysone, were detected after incubation with [³H]ecdysone. Compounds that reacted positively to antibodies to progesterone and testosterone were detected in the low-polarity fractions. Testes were incubated in fractions corresponding to each of the major ecdysteroid peaks derived from [³H]ecdysone metabolism. Although most of the radioactive ecdysteroid fractions were further metabolized to high- and low-polarity endpoints, 88% of the [³H]20-hydroxyecdysone peak apparently remained unmetabolized. 20-Hydroxyecdysone may be the primary ecdysteroid product of testes of H. virescens. [³H]Cholesterol was not metabolized to any appreciable extent.     

Regulation of juvenile hormone esterase gene expression in the tobacco budworm (Heliothis virescens)

The tissue distribution, developmental control, and induction of juvenile hormone esterase (JHE) mRNA was examined in Heliothis virescens using an 800-base pair fragment of a JHE cDNA clone. Northern hybridization analysis of poly(A)+RNA from fat body and integument of fifth stadium larvae indicated the presence of a single JHE mRNA species having an estimated length of 3 kilobases. On Day 2 of the fifth stadium (L5D2), basal JHE mRNA levels were 3-fold higher in the integument than the fat body, which correlated with the higher specific activity of the enzyme in the integument at this time. However, JHE mRNA levels in the fat body on Day 4 of the fifth stadium were 9-fold higher than on Day 2, while mRNA levels in the integument remained the same. This endogenous increase in JHE mRNA and activity in the fat body occurred at the time of peak hemolymph JHE activity. JHE mRNA was not detected in third stadium larvae which have very low levels of JHE activity. Treatment of L5D2 larvae with the juvenile hormone mimic epofenonane resulted in a 7- and 14-fold increase in the level of JHE mRNA in the integument and fat body, respectively. The mRNA induced in both tissues was of the same estimated length as the constitutively expressed message. The data indicate that the developmental regulation and induction of JHE can occur at the level of mRNA. There is evidence that the fat body secretes more JHE than does the integument and could be the major source of hemolymph JHE.     

The plant sesquiterpene germacrene D specifically activates a major type of antennal receptor neuron of the tobacco budworm moth Heliothis virescens

Plants release hundreds of volatiles that are important in interactions with insects or other organisms. However, knowledge is scarce as to which of the compounds are detected by the organism's olfactory receptor neurons. In the present study, single receptor neurons on the antennae of the tobacco budworm moth, Heliothis virescens, were screened for their sensitivities to naturally produced plant volatiles by the use of gas chromatography linked to electrophysiological recordings from single cells (GC-SCR). Plant volatiles, collected by aeration of host and non-host plants, were tested on each receptor neuron via parallel GC-columns. Thus, simultaneous recordings of the gas chromatogram and the neuron responses to each component were obtained. One type of receptor neuron, appearing in 80% of all experiments, responded with high sensitivity and selectivity to one particular component, present in host as well as non-host mixtures. The component, identified as a sesquiterpene hydrocarbon by linked gas chromatography-mass spectrometry, was isolated from a sesquiterpene fraction of cubebe oil and identified by NMR as germacrene D. The purified compound was then re-tested via gas chromatography on the same receptor neuron type, verifying the identification. A weaker response to another sesquiterpene hydrocarbon was also recorded.     

Modes of action of fraxinellone against the tobacco budworm, Heliothis virescens

Fraxinellone significantly reduced the relative growth rate, food consumption rate as well as the efficiency of conversion of ingested food into biomass of Heliothis virescens when incorporated into artificial diets at concentrations of 4.31 × 10⁻⁵ mol/L and above. After being fed with fraxinellone-treated diets for 24 h, the larval midguts of H. virescens possess significantly lower activities of α-amylase and non-specific proteases and higher activities of cytochrome P450s. In vitro , the compound did not inhibit the activities of α-amylase and non-specific proteases extracted from the larval midguts. Clear evidence of post-ingestive toxicity of fraxinellone to midgut cells was observed under an electron microscope. The modes of action of the compound against insects were discussed.     

Movement of proteins across the digestive system of the tobacco budworm, Heliothis virescens

Bovine serum albumin (BSA) and anti‐BSA polyclonal antibody were used as model polypeptides to examine the movement of foreign proteins across the insect digestive system and their accumulation in hemolymph of fourth stadium tobacco budworms, Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Hydrateable meal pads were developed in these studies as a method for easily introducing compounds into the insect digestive system. When insects were allowed to feed continuously on hydrated meal pads containing 0.8 mg of anti‐BSA per gram diet, the level of antibody found in hemolymph was 2.4 ± 0.1 and 3.4 ± 0.1 µg ml^?1 (average  1 SEM) after 8 and 16 h, respectively, as determined by enzyme‐linked immunosorbant assay (ELISA). Continuous feeding on hydrated meal pads containing the same concentration of BSA produced hemolymph concentrations of 1.5 ± 0.1 and 1.6 ± 0.1 µg ml^?1 hemolymph at 8 and 16 h, respectively. Western blot analyses demonstrated that BSA and anti‐BSA both retained their primary and multimeric structure and that anti‐BSA maintained its antigenic activity in the meal pads and after movement from meal pads into the hemolymph. When 1 µg of anti‐BSA or BSA was injected into the hemocoel of fourth instars, the concentrations decreased with time and 120 min after injection were 20% and 0.6% of the original concentration, respectively. When added at the same concentration to plasma in vitro, the decrease was 81.5% and 57.5%, respectively, at 2 h. The accumulation of native anti‐BSA and BSA protein in insect hemolymph is the result of their rate of movement across the gut and their rate of turnover in hemolymph. Movement of anti‐BSA and BSA across the digestive system was also noted in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), Acheta domesticus (L.) (Orthoptera: Gryllidae), and Gromphadorhina portentosa (Schaum) (Blattaria: Blattellidae). Anti‐BSA and BSA were not detected in the hemolymph of Manduca sexta (L.) (Lepidoptera: Sphingidae) after feeding.     

Effects of azadirachtin on the nutrition and development of the tobacco budworm,Heliothis virescens (Fabr.) (Noctuidae)

The plant chemical azadirachtin was administered, either in artificial diet or by oral injection, to fifth instar larvae of the tobacco budworm, Heliothis virescens (Fabr.). At a dietary concentration of 0.03125 ppm, azadirachtin significantly reduced the amount of diet consumed and the weight gained by the larvae. Higher dietary concentrations (0.25 and 0.5 ppm) were necessary to reduce the efficiency of larval conversion of digested and ingested food, respectively. However, the approximate digestibility increased at the dietary concentration of 0.25 ppm.Orally injected azadirachtin (0.25 and 0.5 μg) delayed moulting to the pupal stage, produced defective pupae or adults, and inhibited development to the adult stage. Higher doses (5.0 and 10.0 μg) reduced the pre-pupal weight loss normally associated with pupation, and completely inhibited pupation. At the critical dose of 1.0 μg (the minimal dose that disrupted development to the pupal stage), azadirachtin had less of an effect on older than on younger larvae. Larvae injected on the first day of the fifth instar failed to pupate, whereas approx 40% of those injected on subsequent days pupated.The results suggest that azadirachtin affects H. virescens in a manner similar to other tested species of insects. The significance of these results, especially regarding hormonal events in the insects, is discussed.     

The relationship of hemolymph osmotic pressure to spermatogenesis in the tobacco budworm,Heliothis virescens

The hemolymph osmotic pressure of male Heliothis virescens last instar larvae and pupae can be correlated with the state of spermatogenesis: intermediate (approx. 325 mOsm/kg) osmotic pressures are found in pre-meiotic animals, low (approx. 300 mOsm/kg) osmotic pressures characterize meiosis and elongation, and high (approx. 370 mOsm/kg) osmotic pressures, characterize the tests of diapausing pupae, where mature sperm have disappeared and only pre-meiotic sperm are found. In vitro studies show that, as the osmotic pressure of the medium is increased, spermatogenesis is inhibited and the survival of pre-meiotic cysts is enhanced. It is proposed that the osmotic pressure of the hemolymph plays a role in spermatogenesis and in the preservation of immature cysts during diapause.     

Characterization of esterases associated with profenofos resistance in the tobacco budworm, Heliothis virescens (F.)

The utility of microplate and electrophoretic assays for detection of biochemical and physiological mechanisms underlying resistance to profenofos in the tobacco budworm, Heliothis virescens (F.), was assessed. Esterase (EST) activities were significantly higher in profenofos-resistant than -susceptible larvae, and activities were highly correlated (r(2) = 0.87) with resistance to profenofos. Both qualitative and quantitative variation was observed in electrophoretic gels stained with alpha- and beta-naphthyl acetates. Staining of ESTs was more intense with resistant larvae than those from a susceptible strain. In addition, a band (designated A') was expressed in larvae from profenofos-resistant strains, but not in larvae from an insecticide-susceptible strain. The frequency of expression of A' increased following selection with profenofos and was detected in 100% of the individuals from a profenofos-selected strain. The appearance of this band coincided with the decreased expression of a second band (designated A). A similar pattern (overexpression of A' and underexpression of A) also was observed in larvae from field-collected strains. Finally, reduction in the activity or the sensitivity of acetylcholinesterase to inhibition by chlorpyrifos oxon was observed in laboratory-selected and field-collected larvae that expressed resistance to profenofos. These results suggest that microplate and electrophoretic assays can be utilized as complementary tools for detecting and monitoring profenofos resistance in H. virescens.     

Saxitoxin binding to neural membranes from pyrethroid susceptible and resistant tobacco budworm moths Heliothis virescens

1. [³H]Saxitoxin, a sodium channel probe, was found to bind reversibly with high affinity to a single class of noninteracting sites in membrane preparations from whole head homogenates of tobacco budworm, Heliothis virescens (F.), moths from a pyrethroid susceptible strain and from a pyrethroid resistant strain with nerve insensitivity and metabolic resistance mechanisms.2. No significant interstrain differences were detected in saturation (K_D, B_max) and kinetic (k₊₁, k₋₁) experiments.3. Also, five pyrethroids, two formamidines, and DDT had no significant effect on [³H]saxitoxin binding when tested at concentrations of 1 × 10⁻⁴ and 1 × 10⁻⁸ M.4. It appeared that a decreased density in sodium channels in resistant strain moths as compared with that in susceptible strain moths was not a factor in pyrethroid-induced resistance in this particular strain of tobacco budworm.     

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.     

Ecdysteroid production by testes of the tobacco budworm,Heliothis virescens,from last larval instar to adult

Testes of Heliothis virescens were examined in vitro for the ability to produce ecdysteroids. Production was detected in testes removed from larvae at mid and late periods in the last instar, in testes from pupae after the third day of purpal development, and in testes taken from young adult males. The testes in the isolated abdomens do not fuse during later stages of spermatogenesis as they do in intact insects. Spermatocysts in testes of larval abdomens isolated from the upper body early in the last instar developed to mature sperm despite low haemolyph and testis ecdysteroid titres. Gonadectomized animals developed and moulted normally, indicating that ecdysteroids from the testes are not essential to general development.     

Diapause and development in the tobacco budworm,Heliothis virescens: A comparison of haemolymph ecdysteroid titres

The signal to induce diapause in H. virescens comes early in development (prior to the third instar in most insects), but the signal to break diapause can come shortly after entrance into diapause at pupation. Haemolymph ecdysteroid titres in both diapause-bound and non-diapause-bound Heliothis virescens larvae were similar in the first two thirds of the last-larval instar, when similar changes in morphology and behaviour occurred. However, the number of stepwise increases in titre and the timing of the steps was different in the two groups of larvae. Haemolymph ecdysteroid titres in the last third of the instar were approx, five times higher in non-diapause than in diapause-bound larvae. In diapausing pupae, haemolymph ecdysteroid titres dropped to levels found in larvae which had completed two thirds of the last instar. When diapausing pupae were warmed to break diapause, haemolymph ecdysteroid titres rose again. However, 2 of the 4 high ecdysteroid levels detected in pupae developing after diapause break were considerably lower than those detected for non-diapause pupae.     

Sperm storage by females of the polyandrous noctuid moth Heliothis virescens

Female tobacco budworm moths, Heliothis virescens, generally mate with more than one male, receiving from each mate both fertilizing sperm (eupyrene) and nonfertilizing anucleate sperm (apyrene), which is thought to play a role in sperm competition. One male typically gains sperm precedence, but it is not consistently the last or the first male to mate. I investigated the mechanism of this variable pattern of paternity by examining the patterns of storage of both types of sperm in the female's spermatheca as a function of multiple mating and male phenotype. The number of stored apyrene sperm varied with mating history, being greatest in twice-mated females and least in females mated to one nonvirgin male. In contrast, only one ejaculate's worth of eupyrene sperm was stored regardless of female mating history (once or twice mated). Thus, while they store two complements of apyrene sperm, twice-mated females apparently store only one ejaculate's worth of eupyrene sperm. This biased pattern of sperm storage may contribute to the variable pattern of paternity observed in this species. Eupyrene sperm storage also correlated positively with female size, male age and spermatophore size. Finally, a new sperm storage site was identified and described. It is a bulged region in the seminal duct. Copyright 2000 The Association for the Study of Animal Behaviour.     

Sodium channels in central neurons of the tobacco budworm,Heliothis virescens: basic properties and modification by scorpion toxins

Voltage-activated sodium channels in central neurons of larval and adult Heliothis virescens were characterized using whole-cell patch clamp techniques. Macroscopic currents showing rapid activation and inactivation kinetics were uniformly sensitive to tetrodotoxin (IC(50)=1.9nM). Currents began to activate at voltage steps to -45mV and reached half maximal at -30mV. Fast inactivation was evident at voltages as negative as -75mV and reached half maximal at -50mV. Full recovery from inactivation occurred within 1 to 2ms. Currents in larval neurons exhibited similar properties to those of adult neurons, except for the rate of fast inactivation (t(1)), which was significantly slower in larval neurons. The biophysical properties of sodium channels remained unchanged for up to 3days in culture. Two insecticidal neurotoxins, LqhalphaIT and AaIT, produced distinctly different modifications of H. virescens sodium channels. LqhalphaIT slowed channel inactivation, while AaIT specifically shifted voltage-dependent activation to more negative potentials. Overall, the results indicate that sodium channels in H. virescens neurons exhibit biophysical characteristics similar to those of vertebrates, yet possess pharmacological uniqueness with respect to scorpion toxin modification.     

The expression in tobacco plants of Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF) alters growth and development of the tobacco budworm,Heliothis virescens

The production and characterisation of transgenic tobacco plantsexpressing a precursor of a regulatory peptide from Aedesaegypti (Trypsin Modulating and Oostatic Factor, Aea-TMOF) whichinterferes with the development of tobacco budworm larvae is described. Tobaccoplants were transformed with a synthetic gene containing 6 TMOF units spaced bydibasic residues, Arg-Arg, as potential post-translational cleavage sites.Peptide extracts from transgenic plants had TMOF activity and inhibitedin vitro the biosynthesis of serine proteases. Thisactivity was consistently present in T1 plants and absent in control plants.Tobacco budworm larvae, fed with transgenic leaves showed a reduced growth ratecompared to those fed with control plants. The low rather than acute toxicityofthis low impact gene is discussed in the context of alternative integrated pestmanagement strategies.