Stored alcohol and fatty acid intermediates and the biosynthesis of sex pheromone aldehyde in the moth Chloridea virescens.

In most species of moths, the female produces and releases a volatile sex pheromone from a specific gland to attract a mate. Biosynthesis of the most common type of moth sex pheromone component (Type 1) involves de novo synthesis of hexadecanoate (16:Acyl), followed by modification to various fatty acyl intermediates, then reduction to a primary alcohol, which may be acetylated or oxidized to produce an acetate ester or aldehyde, respectively. Our previous work on the moth Chloridea virescens (Noctuidae) showed that females produce 90% of the major pheromone component, (Z)-11-hexadecenal (Z11-16:Ald), via a direct and rapid route of de novo biosynthesis with highly labile intermediates, and ca. 10% from an indirect route that likely mobilizes a pre-synthesized 16-carbon skeleton, possibly, (Z)-11-hexadecenoate (Z11-16:Acyl) or hexadecanoate (16:Acyl). In this paper, we use stable isotope tracer/tracee techniques to study the dynamics of the precursor alcohol (Z)-11-hexadecenol (Z11-16:OH) and stores of Z11-16:Acyl and 16:Acyl to determine their roles in biosynthesis of Z11-16:Ald. We found: (i) that intracellular Z11-16:OH is synthesized at roughly the same rate as Z11-16:Ald, indicating that translocation and oxidation of this moiety does not rate limit biosynthesis of Z11-16:Ald, (ii) intracellular Z11-16:OH consists of two pools, a highly labile one rapidly translocated out of the cell and converted to Z11-16:Ald, and a less labile one that mostly remains in gland cells, (iii) during pheromone biosynthesis, net stores of Z11-16:Acyl increase, suggesting it is not the source of Z11-16:Ald produced by the indirect route, and (iv) no evidence for the gland synthesizing stored 16:Acyl prior to (up to 2 days before eclosion), or after, synthesis of pheromone commenced, suggesting the bulk of this stored moiety is synthesized elsewhere and transported to the gland prior to gland maturation. Thus, the pheromone gland of C. virescens produces very little stored fat over its functional lifetime, being optimized to produce sex pheromone.

Some Factors Influencing Calling Behavior and Mass Emission Rate of Sex Pheromone from the Gland of the Moth Chloridea virescens.

To attract a mate, females of most moth species synthesize and emit sex pheromone from a specific gland in a behavior termed "calling". In a broad temporal sense, calling behavior and pheromone synthesis are synchronized through the overlap of their circadian rhythms. However, the limited amount of pheromone a female produces each day must be managed so that pheromone is emitted at a sufficient (to attract males) mass emission rate (MER) over the entire calling period, typically many hours. We are studying pheromone synthesis and emission in the moth Chloridea (formerly Heliothis) virescens (family Noctuidae). One way that female C. virescens manage pheromone over their calling period is by calling intermittently; the period between calling bouts allows females to replenish pheromone, and resume calling at high MERs. However, militating against replenishment is loss of pheromone through putative catabolism. In this paper, we examined three aspects pertaining to pheromone MER in C. virescens: (i) the effect of adult feeding on calling behavior, (ii) the effect of certain behavioral/physical parameters on MER, and (iii) the relative loss (putative catabolism) of pheromone in retracted (non-calling) and everted (calling) glands. We found that (i) adult feeding increases calling duration, consistent with the known concomitant increase in pheromone production, (ii) various physical factors relating to the gland, including degree of eversion (surface area), orientation to airstream, and air velocity over the gland influence MER, and (iii) putative catabolism occurs in both retracted and everted glands, but substantially less pheromone is lost in the everted gland primarily because of the high MER when the gland is first everted. Together, these data demonstrate that, over the calling period, the efficient use of pheromone for emission by female C. virescens is dependent on the interaction among synthesis, storage, catabolism, and calling behavior.

Sex pheromone biosynthesis, storage and release in a female moth: making a little go a long way.

Moth pheromone research has pioneered much of our understanding of long-distance chemical communication. Two important characteristics of this communication have, however, remained largely unaddressed: the release of small quantities of pheromone by most moth species, despite potential advantages of releasing greater amounts, and the intermittency of release in some species, limiting the time of mate attraction. We addressed the proximate mechanisms underlying these characteristics by manipulating biosynthesis, storage and release of pheromone in females of the noctuid moth Chloridea virescens. We found that (i) mass release is determined by pheromone mass on the gland surface; (ii) amounts synthesized are limited by pheromone biosynthesis activating neuropeptide concentration, not precursor availability; (iii) some gland structural feature limits mass release rate; (iv) intermittent calling enables release at a mass rate greater than biosynthetic rate; and (v) at typical mass release rates, the periodicity of pheromone availability on the gland surface roughly matches the periodicity (intermittency) of calling. We conclude that mass release in C. virescens and possibly many other species is low because of constraints on biosynthesis, storage and gland structure. Further, it appears the behaviour of intermittent calling in C. virescens may have evolved as a co-adaptation with pheromone availability, allowing females to release pheromone intermittently at higher mass rates than the biosynthesis rate.

The Effect of Pheromone Synthesis and Gland Retraction on Translocation and Dynamics of Pheromone Release in the Moth Chloridea virescens.

Most species of moths use a female-produced sex pheromone to bring mates together. Typically, sex pheromone is synthesized in a specialized gland and released during the behavior of "calling", in which the ovipositor and gland are extruded, allowing pheromone to evaporate. Although there has been much study on how a gland makes specific pheromone components, we know relatively little about how it actually functions with regard to synthesis, storage and release. In this paper, we investigated three aspects of gland function in the noctuid moth Chloridea virescens (Fabricius): (i) whether translocation of pheromone from site of synthesis to release is dependent on calling or ovipositor movement, (ii) whether pheromone synthesis rate limits release and (iii) how intermittent calling (observed in this and other species) might affect the dynamics of release rate. Firstly, by manipulating the gland to simulate calling (extruded) or non-calling (retracted), we showed that pheromone translocation occurred regardless of whether the gland was retracted or extruded. Secondly, by manipulating pheromone production, we found that females that produced more pheromone had higher release rates. It was especially noticeable that females had a higher release rate at the start of calling, which dropped rapidly and leveled off over time. Together, these data suggest that intermittent calling in C. virescens (and other species) may function to allow females to replenish pheromone stores on the gland surface between calling bouts, so that brief, high release rates occur at the start of a calling bout; thus, potentially increasing a female's chances of attracting a mate.

Calling Behavior and Sex Pheromone Release and Storage in the Moth Chloridea virescens.

Female moths release sex pheromone to attract mates. In most species, sex pheromone is produced in, and released from, a specific gland. In a previous study, we used empirical data and compartmental modeling to account for the major pheromone gland processes of female Chloridea virescens: synthesis, storage, catabolism and release; we found that females released little (20-30%) of their pheromone, with most catabolized. The recent publication of a new pheromone collection method led us to reinvestigate pheromone release and catabolism in C. virescens on the basis that our original study might have underestimated release rate (thereby overestimating catabolism) due to methodology and females not calling (releasing) continuously. Further we wished to compare pheromone storage/catabolism between calling and non-calling females. First, we observed calling intermittency of females. Then, using decapitated females, we used the new collection method, along with compartmental modeling, gland sampling and stable isotope labeling, to determine differences in pheromone release, catabolism and storage between (forced) simulated calling and non-calling females. We found, (i) intact 1 d females call intermittently; (ii) pheromone is released at a higher rate than previously determined, with simulations estimating that continuously calling females release ca. 70% of their pheromone (only 30% catabolized); (iii) extension (calling)/retraction of the ovipositor is a highly effective "on/off' mechanism for release; (iv) both calling and non-calling females store most pheromone on or near the gland surface, but calling females catabolize less pheromone; (v) females are capable of producing and releasing pheromone very rapidly. Thus, not only is the moth pheromone gland efficient, in terms of the proportion of pheromone released Vs. catabolized, but it is highly effective at shutting on/off a high flux of pheromone for release.

Investigating the status of pyrethroid resistance in UK populations of the cabbage stem flea beetle (Psylliodes chrysocephala).

The cabbage stem flea beetle, Psylliodes chrysocephala L. is a major pest of winter oilseed rape in several European countries. Traditionally, neonicotinoid and pyrethroid insecticides have been widely used for control of P. chrysocephala, but in recent years, following the withdrawal of neonicotinoid insecticide seed treatments, control failures have occurred due to an over reliance on pyrethroids. In line with previous surveys, UK populations of P. chrysocephala were found to exhibit high levels of resistance to the pyrethroid lambda-cyhalothrin. This resistance was suppressed by pre-treatment with the cytochrome P450 inhibitor PBO under laboratory conditions, suggesting that the resistance has a strong metabolic component. The L1014F (kdr) mutation in the voltage-gated sodium channel, which confers relatively low levels (10-20 fold) of resistance to pyrethroids, was also found to be widespread across the UK regions sampled, whereas the L925I (s-kdr) mutation was much less common. The current survey also suggests that higher levels of pyrethroid resistance have spread to the North and West of England, and that resistance levels continue to remain high in the South East.

Production and Distribution of Aldehyde and Alcohol Sex Pheromone Components in the Pheromone Gland of Females of the Moth Chloridea virescens.

Aldehydes are components of many moth sex pheromones, and are thought to be produced from analogous alcohols by oxidase(s) in the cell membrane or the gland cuticle. This implies that the two types of components are produced and/or stored in different parts of the gland: alcohols in cells and aldehydes in cuticle. Few studies have investigated the distribution of components in moth pheromone glands. Using rinse/extract sampling, stable isotope tracer/tracee methods, and decapitation/ pheromone biosynthesis activating neuropeptide stimulation, we studied production and distribution of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-hexadecenol (Z11-16:OH) in the gland of Chloridea virescens (formerly Heliothis virescens). The rinse, which likely sampled the surface and outer cuticle, contained large amounts of aldehyde and small amounts of alcohol. By contrast, the residual extract, which likely sampled cells and less solvent-accessible (inner) cuticle, had large amounts of alcohol and small amounts of aldehyde. When a tracer (U-13C-glucose) was fed to females, the aldehyde had higher isotopic enrichment than the alcohol in the rinse, but not in the residual extract, showing that in the rinse pool, Z11-16:Ald was, on average, synthesized before Z11-16:OH. This is consistent with greater aldehyde than alcohol flux through the cuticle. While our results are consistent with cell/cuticle synthesis sites for alcohol/aldehyde components, we cannot rule out both being synthesized in gland cells. We propose two alternative conceptual models for how site of production, cuticular transport and catabolism/metabolism might explain the relative masses of Z11-16:Ald and Z11-16:OH translocated to the pheromone gland surface in female C. virescens.

Differential Pheromone Sampling of the Gland of Female Heliothis Virescens Moths Reveals Glandular Differences in Composition and Quantity.

By differentially sampling the pheromone gland of females of the moth Heliothis virescens, we explored differences in pheromone on the surface, or outer distal layer(s) of the gland, and that located more proximally. For this, we used two sampling approaches, (i) a solid phase microextraction fiber rub followed by solvent extraction of residual pheromone (SPME rub/extract), and (ii) rapid solvent rinsing followed by solvent extraction of residual pheromone (rinse/extract). The SPME rub showed differences in component ratio between the dorsal and ventral gland surfaces. The rinse sampled a greater amount of pheromone than the SPME rub, sampling the whole gland surface as well as likely deeper into the gland. Compared to the other samplings, pheromone in the rinse was depleted in the minor component; consequently, the corresponding residual extract was highly enriched in the minor component. Further rinses of the gland yielded only small amounts of pheromone, with a similar component ratio as the first rinse, suggesting that the residual pheromone was less accessible and required extraction in solvent to be liberated. Sampling over the photoperiod showed that the more volatile minor component was depleted (relative to the major component) on the surface/outer cuticle over the period when females called. Together, these data suggest that the pheromone is stored, at least in part, on and in the gland cuticle and that distinct pools may be transported to different topographic regions. Females fed with a stable isotope tracer, incorporated label into pheromone in the gland very rapidly, with the labeled pheromone appearing on the gland surface ca. 1 min later.

The Dynamics of Pheromone Gland Synthesis and Release: a Paradigm Shift for Understanding Sex Pheromone Quantity in Female Moths.

Moths are exemplars of chemical communication, especially with regard to specificity and the minute amounts they use. Yet, little is known about how females manage synthesis and storage of pheromone to maintain release rates attractive to conspecific males and why such small amounts are used. We developed, for the first time, a quantitative model, based on an extensive empirical data set, describing the dynamical relationship among synthesis, storage (titer) and release of pheromone over time in a moth (Heliothis virescens). The model is compartmental, with one major state variable (titer), one time-varying (synthesis), and two constant (catabolism and release) rates. The model was a good fit, suggesting it accounted for the major processes. Overall, we found the relatively small amounts of pheromone stored and released were largely a function of high catabolism rather than a low rate of synthesis. A paradigm shift may be necessary to understand the low amounts released by female moths, away from the small quantities synthesized to the (relatively) large amounts catabolized. Future research on pheromone quantity should focus on structural and physicochemical processes that limit storage and release rate quantities. To our knowledge, this is the first time that pheromone gland function has been modeled for any animal.

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

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 signaler's 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.

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 (13)C-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.

Amplification of a cytochrome P450 gene is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.

The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs), have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A) suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s) may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold) of a single P450 gene (CYP6CY3); and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2-16-fold), and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.

Permeability barriers to embryo cryopreservation of Pectinophora gossypiella (Lepidoptera: Gelechiidae).

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 approximately 7%.

Survey of resistance to four insecticides and their associated mechanisms in different genotypes of the green peach aphid (Hemiptera: Aphididae) from Chile.

The green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) is amajor pest of agriculture worldwide that has proved to be particularly adept at evolving insecticide resistance. Several mechanisms that confer resistance to many insecticide types have been described in M. persicae. We measured the resistance status of nine multilocus genotypes (MLGs) of this aphid species collected in Chile. MLGs were identified using microsatellite markers, and these MLG clonal populations were measured for the presence of modified acetylcholinesterase (MACE), kdr and super kdr mutations, and enhanced carboxyl esterase activity. Toxicological bioassays were used to estimate aphid LC50 when treated with metamidophos (organophosphate), pirimicarb (dimethyl carbamate), cyfluthrin (pyrethroid), and imidacloprid (neonicotinoid). Two MLGs presented >20-fold resistance to pirimicarb, which was associated with the MACE mutation in the heterozygous condition. The kdr mutation was found in only four MLGs in the heterozygous condition and they showed resistance ratios (RR) to cyfluthrin of less than sevenfold. The super kdr mutation was not detected. Enhanced carboxyl esterase activity was predominantly found in the susceptible (S) to first level of resistance (R1) with RR to metamidophos less than eight-fold. Finally, RR to imidacloprid was also less than eight-fold in all MLGs tested. A few MLGs with resistance to pirimicarb were found, while susceptibility to cyfluthrin, metamidophos and imidacloprid was still predominant. A significant positive correlation between imidacloprid tolerance with pirimicarb resistance was detected, as well as between imidacloprid and metamidophos tolerance. With the increase in the use of neonicotinoid insecticides, better rotation of insecticides with different modes of action will be necessary to prevent further development of M. persicae insecticide resistance in Chile.

Reinvestigation of sex pheromone biosynthesis in the moth Trichoplusiani reveals novel quantitative control mechanisms.

Many species of moths have a common control mechanism for synthesizing sex pheromone: the circadian release of pheromone biosynthesis-activation neuropeptide (PBAN) that switches pheromone synthesis on/off during the day. One apparent exception to this is the noctuid moth Trichoplusia ni (Hübner), in which pheromone synthesis appears continuous through the photoperiod, with circadian release of PBAN controlling emission rate of pheromone during calling. Sex pheromone biosynthesis was reinvestigated in T. ni using stable isotope tracer-tracee and gland sampling techniques to ascertain how pheromone quantities in gland cells and on the gland cuticular surface varied and were controlled. It was found that (i) carbohydrate from adult female feeding is used to synthesize sex pheromone, (ii) most of the stored acetate ester pheromone component(s) is contained in gland cells, (iii) a large pool of pheromone is synthesized and stored through the photoperiod with a slow turnover rate, (iv) although pheromone is synthesized throughout the photoperiod, its rate can vary, influenced by release of PBAN and possibly by an unidentified head factor, with both affecting carbohydrate uptake into the acetyl CoA pheromone precursor pool, and (v) as suggested previously, PBAN also influences translocation of pheromone out of the cell to the cuticular surface, possibly by causing breakdown of intracellular lipid droplets storing pheromone molecules. This work suggests that the quantitative synthesis and emission of pheromone in T. ni, and possibly other moths, is regulated by multiple complementary biochemical mechanisms.

Mutation of a nicotinic acetylcholine receptor ß subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.

BACKGROUND: Myzus persicae is a globally important aphid pest with a history of developing resistance to insecticides. Unusually, neonicotinoids have remained highly effective as control agents despite nearly two decades of steadily increasing use. In this study, a clone of M. persicae collected from southern France was found, for the first time, to exhibit sufficiently strong resistance to result in loss of the field effectiveness of neonicotinoids. RESULTS: Bioassays, metabolism and gene expression studies implied the presence of two resistance mechanisms in the resistant clone, one based on enhanced detoxification by cytochrome P450 monooxygenases, and another unaffected by a synergist that inhibits detoxifying enzymes. Binding of radiolabeled imidacloprid (a neonicotinoid) to whole body membrane preparations showed that the high affinity [3H]-imidacloprid binding site present in susceptible M. persicae is lost in the resistant clone and the remaining lower affinity site is altered compared to susceptible clones. This confers a significant overall reduction in binding affinity to the neonicotinoid target: the nicotinic acetylcholine receptor (nAChR). Comparison of the nucleotide sequence of six nAChR subunit (Mpα1-5 and Mpß1) genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR ß1 subunit of the resistant clone, causing an arginine to threonine substitution (R81T). CONCLUSION: Previous studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. The discovery of the mutation at this position and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides and also provides further validation of exisiting models of neonicotinoid binding and selectivity for insect nAChRs.

Signal honesty through differential quantity in the female-produced sex pheromone of the moth Heliothis virescens.

Over the last 50 years, female-produced sex pheromones of moths have been subjected to intensive study. Most work has focused on their role in mate recognition, and little on any role they may have in mate assessment. This is largely because it has been assumed that female, rather than male, moths are "choosy", and invest larger amounts of carbon in eggs than in pheromone. Recently, we found that pheromone production in the moth Heliothis virescens depended on hemolymph trehalose concentration, and that sugar-stressed females produced less pheromone than unstressed ones. In this paper, we demonstrate, for the first time in moths, that a female-produced pheromone signal can allow H. virescens males to assess sugar resources (quality) of a female. This signal honesty is based on quantitative, rather than qualitative (component ratio), differences in pheromone, produced and released by sugar-stressed and unstressed females. Increasing marginal cost of pheromone production, as sugar resources are depleted, may ensure signal honesty.

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-(13)C-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.

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), 15beta-hydroxy-ent-trachyloban-19-oic acid (2), and 17-hydroxy-16alpha-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.

Identification of sex pheromone components of the hessian fly, Mayetiola destructor.

Coupled gas chromatographic (GC)-electroantennographic detection (EAD) analyses of ovipositor extract of calling Hessian fly, Mayetiola destructor, females revealed that seven compounds elicited responses from male antennae. Four of the compounds-(2S)-tridec-2-yl acetate, (2S,10Z)-10-tridecen-2-yl acetate, (2S,10E)-10-tridecen-2-yl acetate, and (2S,10E)-10-tridecen-2-ol-were identified previously in female extracts. Two new EAD-active compounds, (2S,8Z,10E)-8,10-tridecadien-2-yl acetate and (2S,8E,10E)-8,10-tridecadien-2-yl acetate, were identified by GC-mass spectroscopy (MS) and the use of synthetic reference samples. In a Y-tube bioassay, a five-component blend (1 ng (2S)-tridec-2-yl acetate, 10 ng (2S,10E)-10-tridecen-2-yl acetate, 1 ng (2S,10E)-10-tridecen-2-ol, 1 ng (2S,8Z,10E)-8,10-tridecadien-2-yl acetate, and 1 ng (2S,8E,10E)-8,10-tridecadien-2-yl acetate) was as attractive to male Hessian flies as a similar amount of female extract (with respect to the main compound, (2S,10E)-10-tridecen-2-yl acetate). The five-component blend was more attractive to male flies than a three-component blend lacking the two dienes. Furthermore, the five-component blend was more attractive than a blend with the same compounds but that contained one tenth the concentration of (2S,8E,10E)-8,10-tridecadien-2-yl acetate (more accurately mimicking the ratios found in female extract). This suggests that the ratios emitted by females might deviate from those in gland extracts. In a field-trapping experiment, the five-component blend applied to polyethylene cap dispensers in a 100:10 microg ratio between the main component and each of the other blend components attracted a significant number of male Hessian flies. Also, a small-plot field test demonstrated the attractiveness of the five-component blend to male Hessian flies and suggests that this pheromone blend may be useful for monitoring and predicting Hessian fly outbreaks in agricultural systems.