Stephen P. Foster

Neural inactivation of sex pheromone production in mated lightbrown apple moths, Epiphyas postvittana (Walker)

Abstract Activation of sex pheromone production in virgin female moths has been intensively studied over the last few years. In contrast, little is known about the reciprocal process, inactivation of pheromone production in mated females. In females of the lightbrown apple moth, Epiphyas postvittana, mating results in the permannent inactivation of pheromone production. Injection of mated female haemolymph or extracts of male accessory glands or whole reproductive tracts into virgins failed to induce this effect, suggesting that whatever elicits the inactivation is not transported humourally. When pairs were artificially forced apart during copulation, females without a spermatophore in their bursa copulatrix all produced levels of pheromone similar to virgins, while of the remales with a spermatophore in their bursa copulatrix, 47% produced levels similar to virgins and the remainder produced levels similar to mated females. This suggests that mechanical factors are not responsible for inducing inactivation of pheromone production. However, when the ventral nerve cord of females was transected before mating, the resultant mated females produced levels of pheromone similar to virgins, indicating that inactivation of pheromone production is induced by a neural signal, originating from the abdomen, which travels up the ventral nerve cord to a higher centre. Data are presented to support the hypothesis that the signal functions by switching off the release of pheromone biosynthesis-activating neuropeptide, or a like neuropeptide from the corpora cardiaca.

Isolation of the diterpenoids, Ent-Kauran-16α-OL and Ent-Atisan-16α-OL, from sunflowers, as oviposition stimulants for the banded sunflower moth, Cochylis hospes

Two diterpenoid alcohols, ent-kauran-16α-ol (1) and ent-atisan-16α-ol (2), were isolated from pre bloom (R3-R4 stage) sunflower heads as oviposition stimulants for the banded sunflower moth, Cochylis hospes. Fractionation of a sunflower head extract, by normal-phase flash column chromatography, resulted in an early eluting fraction exhibiting significant activity in an egg-laying bioassay. Compounds 1 and 2, along with ent-trachyloban- 19-oic acid (3) and ent-kaur-16-en-19-oic acid (4), were isolated as the major components of this fraction and identified by their NMR and mass spectra. The purified compounds were individually tested for ovipositional activity in dose-response bioassays. In these bioassays, compounds 1 and 2 gave linear dose responses, with increasing numbers of eggs laid as the dosage of either increased. Compounds 3 and 4 failed to stimulate significant egg-laying at any of the dosages tested. A factorial design bioassay, using compounds 1 and 2, showed that 1 was relatively more stimulatory than 2, and that there was no synergistic effect on oviposition when the two compounds were combined.

Sugar feeding via trehalose haemolymph concentration affects sex pheromone production in mated Heliothis virescens moths.

SUMMARY Long-distance, female-produced sex pheromones are widespread among moths. Larval feeding provides most of the nutrients for development of these insects but is not thought to influence the de novo production of the fatty-acid derived compounds used as pheromones by most species. Feeding on plant nectar (sugar) by adult moths is important for increasing female fitness and also for the pollination of many plant species. In this paper, I show that feeding on sucrose solution, as opposed to water, increases sex pheromone titre in mated, but not virgin, female Heliothis virescens. Mating caused a rapid decrease in haemolymph trehalose concentration, which was restored to near-virgin levels by sugar ingestion. When isolated mated female abdomens were cultured with different concentrations of trehalose, pheromone titre increased with increasing trehalose concentration. This effect was not observed when abdomens were cultured on saline containing the sugar rhamnose, which insects cannot metabolise to glucose. Virgins injected with the juvenile hormone (JH) analogue, methoprene, showed the same effects as mated females with respect to pheromone titre and haemolymph trehalose concentration. Thus, following mating increases in JH titre increase demand for, and lowering of, blood sugar to develop oocytes, which can be compensated for by sugar ingestion. Haemolymph trehalose concentration probably influences glycolysis in gland cells and, consequently, levels of cytosolic citrate and acetyl-CoA for pheromone biosynthesis. This increase in pheromone titre in sugar-fed, mated females may facilitate further mating and increased fecundity. Thus, exogenous sugar feeding is behaviourally and physiologically integrated with endogenous JH titre to maximise female fitness.

Feeding and hemolymph trehalose concentration influence sex pheromone production in virgin Heliothis virescens moths

Previously, we demonstrated that sex pheromone production in mated female Heliothis virescens moths is dependent upon hemolymph trehalose concentration (HTC), which is influenced by activities such as the feeding of adults on sucrose. In this paper we demonstrate, for the first time, that this effect also occurs in starved (i.e., sugar-stressed) virgin females. Females allowed to feed on sugar for 6 days, following eclosion, had significantly greater titers than females that had fed only on water (i.e., were starved). No differences in pheromone titer were observed between sugar- and water-fed females at shorter (1 or 3 days) periods following eclosion. The relatively short-term effects of HTC on sex pheromone titer of virgins, were demonstrated by feeding experiments, in which starved (for 4 days) virgins fed on 10% sucrose solution had significantly greater HTC and pheromone titers than ones fed only on water; an increase in HTC was apparent within an hour, while the increase in pheromone titer was apparent within 2.5h, of sugar feeding. Starvation also showed similar effects on titers of pheromone gland fatty acids (pheromone intermediates) and HTC. Over 6 days of starvation, fatty acid titers and HTC declined gradually. After feeding on sucrose, titers of hexadecanoic, (Z)-9-hexadecanoic, (Z)-11-hexadecanoic and (Z)-9-octadecanoic, acids, as well as HTC, increased significantly 24h later, but titers of octadecanoic and (Z,Z)-9,12-octadecanoic (linoleic) acids did not. Lepidoptera cannot biosynthesize polyunsaturated acids, but the lack of change in octadecanoic acid titer suggests this acid may not participate in pheromone biosynthesis. In addition to these short-term changes in pheromone and fatty acid production, mediated by HTC, a longer-term effect of age, regardless of HTC, on pheromone titer was observed. Overall, these results are consistent with hemolymph trehalose and glandular fatty acids acting as twin metabolite reservoirs for pheromone biosynthesis. Hemolymph trehalose, able to be refilled through feeding on exogenous sugars, has a one-way flow of metabolites for synthesis of glandular free fatty acids (FFAs) and pheromone, while glandular glycerolipids provide a reversible reservoir for metabolites, accepting surplus FFAs when glandular concentrations are high, and providing FFAs for pheromone biosynthesis when concentrations are low.

Isolation of three diterpenoid acids from sunflowers, as oviposition stimulants for the banded sunflower moth, Cochylis hospes.

The banded sunflower moth (BSFM), Cochylis hospes Walshingham (Lepidoptera: Cochylidae) is a specialist insect, the larvae of which feed on sunflowers, Helianthus spp., and a few other species of Compositae. It is one of the most important pests of sunflower in the USA. Previous work on H. annuus, the cultivated sunflower, revealed two diterpenoids that function as oviposition stimulants for female BSFM, and that other, more polar compounds also stimulated oviposition. Using a bioassay-guided approach, we isolated three additional diterpenoids, grandifloric acid (1), 15β-hydroxy-ent-trachyloban-19-oic acid (2), and 17-hydroxy-16α-ent-kauran-19-oic acid (3), from polar fractions of pre-bloom sunflower head extracts. In laboratory bioassays, purified natural samples of each of these compounds stimulated oviposition by female BSFM. Structure–activity relationships of the five diterpenoids known to stimulate oviposition by female BSFM are discussed.

Change in reductase activity is responsible for senescent decline in sex pheromone titre in the lightbrown apple moth, Epiphyas postvittana (Walker).

Sex pheromone titre in the tortricid moth Epiphyas postvittana follows a pattern commonly observed in other species of moths: an increase to a peak some time after eclosion (2-3days), and then a slow decline as the female ages. Previous work has shown that this decline is not regulated by the pheromone biosynthesis activating neuropeptide PBAN. Using in vivo and in vitro enzyme assays, and fatty acid methyl ester (FAME) analyses of pheromone precursors in the gland, we have investigated this senescent decline in pheromone titre. The enzyme assays have shown that in older females the fatty acid reductase and fatty acid synthesis enzyme systems decrease in activity (relative to younger females), whereas other enzyme systems involved in pheromone biosynthesis, including limited beta-oxidation (2-carbon chain-shortening), (E)-11-desaturation, and acetylation (by an acetyl transferase) remain unchanged in their activity. Of the two enzymatic processes involved, the more important one contributing to the decline appears to be the fatty acid reductase. This is consistent with FAME analyses of pheromone glands in old and young females, which show little difference in levels of saturated FAME, but a significant increase in the level of the putative precursor, (E)-11-tetradecenoate, of the sex pheromone component (E)-11-tetradecenyl acetate. Thus, this decline in fatty acid reductase activity results in a buildup of the precursor as the female ages. The near ubiquity of fatty acid reductases in moth sex pheromone systems suggests that this may be a common mechanism for the senescent decline of sex pheromone titre in moths.

Permeability Barriers to Embryo Cryopreservation of Pectinophora gossypiella (Lepidoptera: Gelechiidae)

ABSTRACT The aim of this study was to develop a method to cryopreserve the embryos of the pink bollworm moth, Pectinophora gossypiella (Saunders). Previously developed dipteran cryopreservation protocols were not directly adaptable to use with the embryos of this lepidopteran species. Physiochemical and electron microscope observations revealed substantial differences in the structure of the chorion, wax layer, and vitelline membrane complex when comparing the cryopreservable embryonic stages of P. gossypiella and dipteran embryos. Thus, the initial steps dealing with dechorionation and permeabilization were ineffective and had to be altered. Exposure to the sodium hypochlorite-based chorion removal step decreased P. gossypiella embryo viability to a very low level. Survival increased and permeability was evident when an alkane wash was used as the first step in the procedure. After the alkane treatment with a surfactant yielded the maximum exchange of cryoprotectant with water as evidenced by a significant lowering of the supercooling point of the cryoprotectant-loaded embryos. The remainder of the cryopreservation and storage recovery protocol for P. gossypiella was similar to those developed for dipteran embryos. Survival of recovered, hatched embryos to adulthood was ≈7%.

The Use of Mass Isotopomer Distribution Analysis to Quantify Synthetic Rates of Sex Pheromone in the Moth Heliothis virescens

Although there has been much investigation of the steps involved in sex pheromone biosynthesis in moths, little is known about the kinetics of biosynthesis in vivo, primarily because there are few techniques suitable for studying the small amounts of pheromone produced without perturbing a female moth’s normal physiology. In this paper, female Heliothis virescens moths fed on U-13C-glucose were subjected to mass isotopomer distribution analysis, enabling calculation of fractional (FSR) and absolute (ASR) synthetic rates of the main pheromone component, (Z)-11-hexadecenal, at two different photoperiodic times: during the scotophase (when adults are sexually active) and during the photophase (when adults do not engage in mating behavior). FSRs differed substantially at the two times with, as expected, the greater rate occurring during the scotophase. After determining Z11-16:Ald pool sizes, ASR through the scotophase was calculated to be roughly 20 times greater than ASR in the photophase. These differences are consistent with the release/non-release of the pheromone biosynthesis-activating neuropeptide. This approach should facilitate determination of more quantitative measures of semiochemical production in moths and other sugar-feeding insects that synthesize semiochemicals from glycolytic metabolites.

Sex pheromones in mate assessment: analysis of nutrient cost of sex pheromone production by females of the moth Heliothis virescens

ABSTRACT It has been postulated that sex pheromones, in addition to their role in mate recognition and/or finding, may also serve a role in assessment of mate quality. For this, a sex pheromone must give honest information about a signalers quality, with honesty ensured by a direct metabolic or indirect fitness cost to the signaler. Using a stable isotope tracer–tracee method, we characterized the nutrient pools that fuel sex pheromone production in females of the moth Heliothis virescens, as well as the relative importance of larval- and adult-acquired nutrients to this process. Females used three pools for de novo biosynthesis of sex pheromone, hemolymph trehalose, glycogen (via trehalose) and fat, and produced ca. 25% of pheromone directly from stored (previously synthesized) precursor fatty acids. Pheromone was produced roughly equally from carbohydrate and fat. Adult feeding was very important for pheromone biosynthesis, with a maximum of 65% of de novo biosynthesized pheromone produced from a single adult feed (carbohydrate). Although these nutrient pools are shared with other reproductive physiologies, notably oocyte production, it is unlikely that pheromone production imposes a significant metabolic cost on females, because (i) the amount of nutrients used for pheromone production is negligible compared with that available, (ii) the hemolymph trehalose pool is readily replaceable throughout the adult life, and (iii) in mated females, carbohydrate shortages result in reduced allocation to pheromone. Summary: Pheromone biosynthesis uses insignificant quantities of the nutrients available and therefore imposes negligible metabolic cost to the moth Heliothis virescens.

Increased allocation of adult-acquired carbohydrate to egg production results in its decreased allocation to sex pheromone production in mated females of the moth Heliothis virescens.

Females of most species of moths produce a volatile sex pheromone that attracts conspecific males over distance. In females of the polyandrous moth Heliothis virescens, feeding on carbohydrate (e.g. nectar) supplies precursor, via hemolymph trehalose, for both sex pheromone and egg production. With limited carbohydrate acquisition these two reproductive physiologies might compete for hemolymph trehalose, resulting in an allocation deficit to either sex pheromone or egg production. Using virgin and mated females, which have low and high egg maturation rates, respectively, we fed females a limited diet of 13C-labeled glucose daily and, using mass isotopomer distribution analysis, determined allocations of adult-acquired carbohydrate (AAC) to newly synthesized pheromone and ovarian and egg fats, our proxies for allocation to egg production. With increased number of feeds, AAC enrichment of hemolymph trehalose increased, as expected. This led to mated females increasing their proportional allocation of AAC to ovarian and egg fats, but decreasing their proportional allocation of AAC to pheromone production. By contrast, virgins increased their proportional allocation of AAC to pheromone production with increased feeds, consistent with increasing AAC enrichment of hemolymph trehalose. These results show that with limited AAC intake, enhanced egg maturation in mated females results in reduced AAC allocation to pheromone production; this does not occur in virgins because of their lower egg maturation rate. This physiological competition for AAC corresponded with decreased pheromone production in mated moths to levels unlikely to attract mates. Therefore, the availability and/or allocation of AAC may be a proximate mechanism underlying the incidence of polyandry in this and other species of moths.