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.     

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.     

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.     

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.     

Gossypol toxicity and detoxification in Helicoverpa armigera and Heliothis virescens

Gossypol is a polyphenolic secondary metabolite produced by cotton plants, which is toxic to many organisms. Gossypol's aldehyde groups are especially reactive, forming Schiff bases with amino acids of proteins and cross-linking them, inhibiting enzyme activities and contributing to toxicity. Very little is known about gossypol's mode of action and its detoxification in cotton-feeding insects that can tolerate certain concentrations of this compound. Here, we tested the toxicity of gossypol and a gossypol derivative lacking free aldehyde groups (SB-gossypol) toward Helicoverpa armigera and Heliothis virescens, two important pests on cotton plants. Larval feeding studies with these two species on artificial diet supplemented with gossypol or SB-gossypol revealed no detectable toxicity of gossypol, when the aldehyde groups were absent. A cytochrome P450 enzyme, CYP6AE14, is upregulated in H. armigera feeding on gossypol, and has been claimed to directly detoxify gossypol. However, using in vitro assays with heterologously expressed CYP6AE14, no metabolites of gossypol were detected, and further studies suggest that gossypol is not a direct substrate of CYP6AE14. Furthermore, larvae feeding on many other plant toxins also upregulate CYP6AE14. Our data demonstrate that the aldehyde groups are critical for the toxicity of gossypol when ingested by H. armigera and H. virescens larvae, and suggest that CYP6AE14 is not directly involved in gossypol metabolism, but may play a role in the general stress response of H. armigera larvae toward plant toxins.     

Allatotropin-like peptide in Heliothis virescens: tissue localization and quantification

The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence and distribution in H. virescens. An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against Manse-AT was used to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used for whole-mount immunocytochemistry to localize Manse-AT-immunoreactivity in the central nervous system. Several groups of Manse-AT-immunoreactive cells were discovered in the brain, subesophageal ganglion, and thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in various locations in the central nervous system suggests that these peptides may have other as yet unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by changes in the amounts of AT acting on the CA.     

Vitellogenin and egg production in the moth,Heliothis virescens

The characteristics of vitellogenin (Vg) and the relationship between Vg production and egg production in the tobacco budworm, Heliothis virescens, were studied. The relationship between Vg production and juvenile hormone (JH) and the impact of mating on Vg and egg production were also investigated. Vg appears in the hemolymph of H. virescens about 6 h after moth eclosion. Vg may be separated into two apoproteins (ApoVg-I and ApoVg-II) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights were calculated to be 156,065 ± 800 for ApoVg-I and 39,887 ± 323 for ApoVg-II. SDS-PAGE analysis revealed that the female hemolymph Vg polypeptides appear to be identical to those from eggs but are absent in male hemolymph. Vg concentration was significantly higher in mated females than in virgin females of the same age at 48 h after emergence. Rates of egg production increased as Vg production increased; rates of egg production in mated females were significantly higher than those of virgin females at 48, 72, 96, and 120 h postemergence. Vg production is dependent on JH, because hemolymph from decapitated females lacked Vg while that of decapitated females treated with synthetic JH had Vg at levels comparable to similarly aged, normal H. virescens females. Hemolymph JH titers in mated females were significantly higher compared with those in virgin females at all sampling periods. The high JH level in mated females may explain the high Vg and egg production in mated H. virescens. Arch. Insect Biochem. Physiol. 34:287–300, 1997. © 1997 Wiley-Liss, Inc.     

Microplitis croceipes teratocytes cause developmental arrest of Heliothis virescens larvae

Microplitis croceipes teratocytes placed into nonparasitized Heliothis virescens larvae survived in the absence of a parasitoid larva and caused developmental changes in the host. Expressions of these changes included delayed larval mortality, incomplete larval-pupal ecdysis, or delayed pupation. Two day old 4th stadium H. virescens larvae were more sensitive to injected teratocytes than were 5th stadium larvae. Three day old teratocytes were more effective than were 6 day old teratocytes. The degree of response was related to the number of injected teratocytes. For example, 750 three day old teratocytes (the approximate number from a single parasitoid egg) caused delayed larval mortality in 96% of the treated larvae whereas 175 three day old teratocytes caused delayed larval mortality in only 33% of the treated larvae. Even a dose of 80 teratocytes resulted in 15% incomplete larval-pupal ecdysis compared to 0% for controls. Treatment with hemocyte-and teratocyte-free hemolymph from parasitized larvae, hemocytes from nonparasitized H. virescens, unfertilized M. croceipes eggs, Cotesia congregata teratocytes, or Micrococcus lysodeikticus cells all had very little effect either on larval growth or development time.     

Regulation of Heliothis virescens prothoracic glands by Cardiochiles nigriceps polydnavirus

Heliothis virescens (F.) Larvae parasitized by the endophagous braconid Cardiochiles nigriceps Viereck fail to attain the pupal stage. This developmental alteration is caused by both an inactivation of prothoracic glands of last-instar larvae and an altered ecdysone metabolism. Decrease in ecdysteroidogenesis in vitro was already evident in glands explanted from larvae that have attained the early cell formation stage (day 4 of fifth instar), 6 h after parasitoid oviposition. Ecdysteroidogenesis nearly ceased by 24 h after parasitoid oviposition. The degree of this biosynthetic depression increased as the time between parasitization and gland dissection increased. A time-course study allowed us to determine if both the degree of phosphorylation of regulatory target proteins, the rate of general protein synthesis and ecdysteroidogenesis decreased in concert over time. The results provide further evidence in support of the hypothesis that these cellular activities in prothoracic gland cells are functionally correlated in steroidogenic responses. Treatment with calyx fluid and venom of C. nigriceps duplicates the parasitism-induced inactivation of host prothoracic glands. A 6-h conditioning in vitro of pupally committed host prothoracic glands with these parasitoid female reproductive secretions resulted in a significant depression of their ecdysteroid production. However, glands lost their sensitivity to calyx fluid and venom treatment when explanted from hosts that had already attained the cell formation stage. This was further supported by the fact that nearly all the host larvae parasitized on day 4 of fifth instar (cell formation stage) pupated, while parasitization on day 3 resulted in only 11% pupation. The coupled trioxsalen/UV irradiation treatment of C. nigriceps calyx fluid and venom eliminated their negative effect on biosynthetic activity in vitro by host prothoracic glands. This result indirectly demonstrates that C. nigriceps polydnavirus is the major regulating factor involved in the host prothoracic gland inactivation. Arch. Insect Biochem. Physiol. 38:1–10, 1998. © 1998 Wiley-Liss, Inc.     

Phenoloxidase activity in the haemolymph of parasitized and unparasitized Heliothis virescens

Hymenopterous parasitoids of Heliothis virescens are able to evade the immune mechanisms of their host, especially the ability of the host to encapsulate foreign objects within a haemocyte capsule. This encapsulation is often accompanied by melanization. Our present findings show that hosts parasitized by Microplitis croceipes, Cardiochiles nigriceps and Campoletis sonorensis show changes both in the amount of melanin formed when their haemolymph is exposed to air and in the total protein as determined by the Folin phenol reagent. Furthermore, these changes are promoted by each of the three parasitoids in a different way. However, none of these parasitoids was able to alter the levels of host phenoloxidase as measured by the ability of the haemolymph to convert 4-methylcatechol to its quinone. The hymenopteran egg must therefore escape encapsulation in Heliothis in a way other than through inhibition of phenoloxidase, since the melanization reactions in the host's haemolymph are apparently unimpaired, particularly during the egg stage, by either the egg or venom of any one of the three parasitoids.     

Iron Levels Change in Larval Heliothis virescens Tissues Following Baculovirus Infection

Inductively coupled plasma mass spectrometry and (59)Fe radiotracers were used to investigate changes in levels of Fe in the tissues of 4th instar Heliothis virescens larvae following infection with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) or with Autographa californica multiple nucleopolyhedrovirus. Baculovirus infection led to significant changes in hemolymph Fe levels late in infection. (24)Na radiotracer ingested by 4th instar larvae was rapidly cleared to nearly undetectable levels 6 h post-ingestion. In contrast, (59)Fe radiotracer fed to 4th instar larvae declined within the first few hours of ingestion and then remained constant at approximately 60% of the initial tracer activity. While Fe radiotracer levels among larval tissues changed, whole insect tracer levels did not decline from 6 to 60 h post-ingestion. Tissues from HzSNPV larvae had higher radiotracer levels in the hemolymph and midgut 36 and 60 h post-infection. The protein-bound/free ratio of (59)Fe was significantly higher in baculovirus infected hemolymph than in uninfected hemolymph at 60 h post-infection, indicating that Fe released from damaged cells is protein-bound. In both studies, hemolymph Fe levels were higher in HzSNPV infected larvae. This first study of tissue Fe levels during viral infection of an insect clearly demonstrates that Fe homeostasis is substantially disrupted.     

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.