Ultrastructural changes associated with pheromone production in the sex pheromone gland of Diatraea saccharalis

An ultrastructural study of the sex pheromone gland of female adult sugarcane borers suggests that pheromone is not produced during the first 2 hr after emergence but reaches a peak 48 hr after emergence. The sex pheromone gland is composed of two cell types and a number of structural changes observed to occur in the cells of the sex pheromone gland prior to pheromone production are described.     

Neural regulation of sex-pheromone glands in Lepidoptera

Substantial progress has been made toward understanding the neuroendocrine regulation of sex-pheromone glands in Lepidoptera, but several recent studies have revealed that direct contact of the pheromone gland with blood-borne factors is not necessary to induce pheromone biosynthesis and release in some species. The nervous system provides an alternate route of activation. Evidence from several species indicates that the pheromone gland is innervated and regulated by neural activity. Electrical stimulation of efferent axons arising from the terminal abdominal ganglion results in a significant increase in pheromone production, and neural stimulation furthermore evokes the rapid release of pheromone into the surrounding air. In some heliothine moths, the biogenic monoamine octopamine stimulates pheromone production, and octopamine has also been isolated from pheromone gland tissue. Moreover, the critical period for maximal octopamine action mirrors the time when peak levels of octopamine are present in the gland. These findings suggest that octopamine is involved in the regulation of pheromone biosynthesis and/or release, but its actions depend on additional factors associated with age and photoperiod. The combined evidence using anatomical, electrophysiological, and biochemical methods indicates that the pheromone gland is innervated and regulated by neurons that arise in the terminal abdominal ganglion. Indirect evidence suggests that at least some of this innervation is octopaminergic. In these respects, the pheromone gland in Lepidoptera exhibits characteristics of other neuroeffector systems in insects.     

烟夜蛾雄蛾性附腺因子对雌蛾性信 息素合成的抑制作用

烟夜蛾Helicoverpa assulta处女蛾在交配后1 h,其性信息素滴度即显著降低,72 h内未见恢复。生测结果表明,烟夜蛾性信息素合成抑制因子主要来源于雄蛾性附腺。不同日龄雄蛾性附腺提取物的抑制活性无显著差异。光暗期对其活性具显著影响,暗期中雄蛾的性附腺物质对雌蛾性信息素合成具有较强抑制作用,而光期中雄蛾的性附腺物质不具抑制活性。在暗期的不同时间处理,对处女蛾性信息素合成的抑制作用无显著差异。雄蛾性附腺提取物对雌蛾性信息素合成的抑制作用与注射剂量有明显的相关性,0.2 ME（雄蛾当量）是产生显著抑制作用的最小剂量。对交配雌蛾注射性信息素生物合成激活神经肽（PBAN）提取物后,其性信息素合成又可恢复,这说明雌蛾交配后,性信息素滴度降低的原因是由于缺少了PBAN的调控。     

Diel changes in the presence and physiological actions of octopamine in the female sex-pheromone glands of heliothine moths

Recent evidence suggests that the biogenic monoamine octopamine (OA) may be involved in the regulation of female sex-pheromone production in Lepidoptera. A radioenzymatic assay coupled with high performance liquid chromatography revealed the presence of OA in the innervated sex-pheromone gland of the corn earworm moth Helicoverpa (Heliothis) zea. Significantly more OA was found in glands just before the onset of scotophase (ca 320 fmol/gland), compared to levels at mid-photophase or just after the onset of scotophase (ca 160 fmol/gland).

Exogenous OA had several actions on pheromone production. H. zea virgin females normally do not produce pheromone during the photophase, but highly significant levels of pheromone were induced by injection of OA into intact, day-2 photophase females. Importantly, this effect was absent in older females that showed increased levels of flight and oviposition activity. A second action of OA was revealed in isolated abdomen preparations from day-2 H. virescens females. Exogenous OA stimulated highly significant increases in pheromone production if abdomens were treated at the onset of scotophase, but not if they were treated in photophase. This critical period for OA action in these reduced preparations coincided with the time when peak levels of OA were present in the pheromone gland tissue. OA is therefore sufficient to induce pheromone production, but its actions in these short-lived insects depend on factor such as age and photoperiod. Diel fluctuations in OA levels in the pheromone gland, together with the observed phermonotropic actions of this amine, support the hypothesis that OA is involved in the regulation of pheromone production in these insects.     

Sex pheromone titre in the glands of Spodoptera litura females: circadian rhythm and the effects of age and mating

The present study investigates the effects of age and mating status on the circadian variations of gland sex pheromone titre in female Spodoptera litura Fabricius. Similar to other nocturnal moths, S. litura females exhibit circadian variations of gland sex pheromone contents, with higher levels during scotophase and lower levels during photophase. The sex pheromone titre in the glands peaks during the first scotophase after eclosion and sharply declines afterwards. Higher pheromone contents during scotophase may facilitate female reproductive activities, and the negative relationship between pheromone titre and female calling is likely the result of pheromone release during female calling. Interestingly, the present study demonstrates that mated S. litura females have significantly higher sex pheromone titre in their pheromone glands (PGs) than virgin females. This finding contrasts with all previous studies of other insect species, in which mating generally reduces the sex pheromone titre in female PGs. In S. litura, mating and male accessory gland fluids can suppress female calling behaviours and re‐matings. These results suggest that the suppression of female calling behaviours by mating and male accessory gland fluids may significantly reduce the release of sex pheromones and thus result in higher sex pheromone titre in the PGs of mated females.     

白蚁信息素研究进展

白蚁是最古老的社会性昆虫, 其社会性的维持需要信息素的相互作用。本文回顾了近年来国内外白蚁信息素研究的最新进展, 内容涉及白蚁踪迹信息素、 性信息素、 告警信息素和促食信息素的功能、 化学成分及产生信息素的外分泌腺。白蚁分泌信息素的腺体主要有背板腺、 腹板腺、 后腹板腺、 额腺和唾腺。绝大多数白蚁信息素是挥发性物质。白蚁在化学通讯上存在节俭策略, 即同一种化合物由不同的白蚁种类的不同外分泌腺分泌, 可具有不同的功能。总结了各类信息素在白蚁物种间、 同一物种的品级间和性别间的异同和作用方式, 强调了白蚁信息素的反应阈值、 最佳浓度、 有效期和物种特异性对其功能的影响。目前对白蚁信息素的研究尚处于起步阶段, 其研究成果对等翅目系统发育研究和白蚁防治具有重要的意义。文章最后展望了白蚁信息素在白蚁防治上的应用前景。     

二个不同发育阶段茶银尺蠖雌蛾性腺超微结构的比较研究

本文利用扫描电镜和透射电镜分别对不同发育阶段的茶银尺蠖Scopula subpunctaria Herrich-Schaeffer雌蛾性信息素腺体进行了观察和研究,对探索信息素的合成途径提供科学依据。结果表明,雌蛾性信息素腺体位于第8、9/10腹节的节间膜上,由其表皮下方的单层上皮细胞组成,并几乎覆盖整个节间膜形成一个近乎完整的环状。成熟雌蛾(3日龄)性腺的超微结构照片显示性腺细胞具有发达的微绒毛、质膜内褶、大量的脂滴、细胞间的运输孔道以及细胞桥粒等结构组织。而在未成熟雌蛾(羽化5h内)性腺细胞内,这些结构均明显缺失或发育不完整。     

Development of functionally competent cabbage looper moth sex pheromone glands

Unlike some moths, pheromone production in Trichoplusia ni is not regulated by a pheromone activating neuropeptide. Rather, competency to produce pheromone apparently is linked with changes in the ecdysteroid titer that occur late in metamorphosis. In contrast to adult pheromone glands, glands from pharate adults 2 days before eclosion were non-competent, and (1) had undetectable levels of the pheromone, (Z)-7-dodecenyl acetate, and pheromone-specific intermediates, (2) showed little or no conversion of radiolabeled substrate to product in enzyme assays of fatty acid synthetase, Δ11 desaturase, and acetyltransferase, and (3) failed to incorporate radiolabeled acetate into pheromone in gland culture. Glands 1 day before adult eclosion exhibited low titers of pheromone and the intermediate, (Z)-11-hexadecenoate, and showed low levels of radiolabeled acetate incorporation into pheromone in gland culture. By the time of adult eclosion, the gland was fully competent. Precocious development of pheromone gland competency was induced by removing the head and thorax from pupae 2 days before adult eclosion. This effect appears to result from the reduction of ecdysteroid, since it was blocked by the administration of 20-hydroxyecdysone. This ability to manipulate the development of the pheromone gland was restricted to a critical period, since removal of head and thorax from younger pupae did not induce pheromone gland competency, and administration of 20-hydroxyecdysone to older pupae did not block its onset. In addition to differences in competency, early pharate and adult glands exhibited dissimilarities with respect to (1) the types of proteins synthesized in gland culture, and (2) the types of proteins translated from mRNA in vitro.     

Structural organization of the sex pheromone gland in Helicoverpa zea in relation to pheromone production and release

Morphological location of the sex pheromone producing area in the ovipositor of the female corn earworm Helicoverpa zea, was correlated with gas chromatographic analysis of the extracted pheromone. Histological studies showed that the pheromone gland occupied an almost complete ring of specialized columnar cells between the 8th and 9th abdominal segments. Ultrastructure of the pheromone gland cells revealed distinct features such as microvilli, pockets of granular material, intercellular canals with abundant desmosomes. Apparent changes in some of these features are associated with phases of pheromone production and non-production. Examination of the tissue with low temperature scanning electron microscopy showed the presence of excreted droplets at the tips of cuticular hairs in the glandular area during the period of pheromone production.     

Stimulation of sex pheromone production by PBAN-like substance in the pine caterpillar moth, Dendrolimus punctatus (Lepidoptera: Lasiocampidae)

Sex pheromone production in the female pine caterpillar moth, Dendrolimus punctatus is controlled by a PBAN-like substance located in the head of female moth. Pheromone titer was significantly decreased by decapitation of female moth, and restored by injection of either Hez-PBAN or head extract prepared from male or female moth. Stimulation of pheromone production by head extract followed a dose-dependent pattern from 0.5 to at least 4 head equivalent. A gland in vitro assay was used to study the relationship between gland incubation time and pheromone production as well as calcium involvement in the stimulation of pheromone production by head extract. Maximum pheromone production was occurred at 60 min after pheromone gland was incubated with two equivalents of head extracts. In vitro experiments showed that the presence of calcium in the incubation medium was necessary for stimulation of pheromone production. The calcium ionophore, A 23187, alone stimulated pheromone production. The pheromone components (Z,E)-5,7-dodecadienol and its acetate and propionate were produced in these experiments but in addition to the aldehyde, (Z,E)-5,7-dodecadienal was also found. This indicates that females are capable of producing four oxygenated functional groups. The PBAN-like substance control of the pheromone biosynthetic pathway was investigated by monitoring the incorporation of the labeled precursor into both pheromone and pheromone intermediates.     

Synthetic rates of key stored fatty acids in the biosynthesis of sex pheromone in the moth Heliothis virescens

Using a tracer–tracee approach, we fed 1-d-old virgin Heliothis virescens U-¹³C-glucose and analyzed the key labeled fatty acids, (Z)-11-hexadecenoate, hexadecanoate and octadecanoate, known to be intermediates in pheromone biosynthesis, by mass isotopomer distribution analysis. This method allowed determination of enrichment, and fractional (FSR) and absolute (ASR) synthetic rates. As expected, FSRs and ASRs for all three moieties were greater in the scotophase than photophase. However, in whole gland extracts, FSRs and ASRs of (Z)-11-hexadecenoate and hexadecanoate were much lower than those of the major pheromone component, (Z)-11-hexadecenal, determined previously. Since pheromone is made via these acids, we postulated that pheromone was produced directly and very rapidly via a small pool of acyl CoA thioesters of these acids and that the pool of acids we analyzed in our whole gland extract was largely a ‘dead end’ pool of excess acids (i.e., not converted directly to pheromone) stored in glycerolipids. We tested this by fractionating the whole glandular extract and analyzing the glycerolipid fraction. FSRs and ASRs for the two acids in the glycerolipid fraction were similar to those for the whole gland extract, confirming our postulate. Thus, most acetate produced in the pheromone gland is converted rapidly and directly to pheromone, while excess fatty acids are stored in glycerolipids and remain relatively inaccessible for pheromone production, at least over the two periods studied. Precursor enrichment of octadecanoate was substantially lower than that determined for the two 16-carbon acids and pheromone component. This suggests that hexadecanoate is the principal product of the multi-enzyme complex fatty acid synthase in the gland, and that octadecanoate is formed by subsequent chain elongation of hexadecanoate.     

Regulation of pheromone biosynthesis in the "Z strain" of the European corn borer, Ostrinia nubilalis

The regulation of pheromone biosynthesis by the neuropeptide PBAN in the Z strain of the European corn borer, Ostrinia nubilalis, was investigated using labeled intermediates. Injection of radiolabeled acetate showed PBAN did not influence the de novo synthesis of saturated fatty acids in the gland. When deuterium-labeled myristic acid was topically applied to the gland, females injected with PBAN produced more labeled pheromone than did control females, indicating that PBAN controls one of the later steps of pheromone biosynthesis. Although more myristic acid was Delta11-desaturated in the gland in the presence of PBAN, this was counterbalanced by less Delta11-desaturation of palmitic acid, indicating that desaturase activity did not change overall. This change in flux of myristic acid through to pheromone was shown to be caused by increased reduction of fatty acid pheromone precursors occurring in the presence of PBAN.     

棉铃虫和烟青虫性信息素腺体脂肪醇和乙酸酯转化的比较研究

棉铃虫Helicoverpa armigera和烟青虫H. assulta属于可同域发生的近缘种昆虫,通过产生比例相反的两种性信息素化合物——顺9-十六碳烯醛和顺11-十六碳烯醛维持种间生殖隔离。本研究应用外源不饱和脂肪醇及乙酸酯在棉铃虫和烟青虫性信息素腺体进行在体转化,利用气相色谱法分析转化产物,从酶学角度探讨了上述两近缘种昆虫性信息素腺体组分差异的形成原因。实验结果表明,两种昆虫信息素腺体表皮伯醇氧化酶对外源顺9-十六碳烯醇、顺11-十六碳烯醇和反10-十六碳烯醇无催化专一性,说明末端氧化过程对于醛类性信息素组分特定比例的形成不起作用。棉铃虫性信息素腺体组织具有较高的乙酸酯酶活性,可水解外源乙酸酯,但烟青虫性信息素腺体乙酸酯酶活性很低。这些发现对于进一步了解两种昆虫的生殖隔离机制有重要参考价值。     

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.     

Evidence for two-step regulation of pheromone biosynthesis by the pheromone biosynthesis-activating neuropeptide in the moth Heliothis virescens

The control of pheromone biosynthesis by the neuropeptide PBAN was investigated in the moth Heliothis virescens. When decapitated females were injected with [2-(14)C] acetate, females co-injected with PBAN produced significantly greater quantities of radiolabeled fatty acids in their pheromone gland than females co-injected with saline. This indicates that PBAN controls an enzyme involved in the synthesis of fatty acids, probably acetyl CoA carboxylase. Decapitated females injected with PBAN showed a rapid increase in native pheromone, and a slower increase in the pheromone precursor, (Z)-11-hexadecenoate. Total native palmitate and stearate (both pheromone intermediates) showed a significant decrease after PBAN injection, before their titers were later restored to initial levels. In contrast, the acyl-CoA thioesters of these two saturated fatty acids increased during the period when their total titers decreased. When a mixture of labeled palmitic and heptadecanoic (an acid that cannot be converted to pheromone) acids was applied to the gland, PBAN-injected females produced greater quantities of labeled pheromone and precursor than did saline-injected ones. The two acids showed similar time-course patterns, with no difference in total titers of each of the respective acids between saline- and PBAN-injected females. When labeled heptadecanoic acid was applied to the gland alone, there was no difference in titers of either total heptadecanoate or of heptadecanoyl-CoA between PBAN- and saline-injected females, suggesting that PBAN does not directly control the storage or liberation of fatty acids in the gland, at least for this fatty acid. Overall, these data indicate that PBAN also controls a later step involved in pheromone biosynthesis, perhaps the reduction of acyl-CoA moieties. The control by PBAN of two enzymes, near the beginning and end of the pheromone biosynthetic process, would seem to allow for more efficient utilization of fatty acids and pheromone than control of only one enzyme.     

Testosterone-dependent primer pheromone production in the sebaceous gland of male goat

To test the hypothesis that the primer pheromone responsible for inducing the "male effect" is produced in the sebaceous gland androgen dependently, we examined the correlation between morphological changes of sebaceous glands and the pheromone activity in skin samples taken from castrated goats that had been treated with testosterone. Five castrated goats were implanted s.c. with testosterone capsules to maintain physiological levels of plasma testosterone for four weeks. Skin samples were obtained from the head region on Day 0 (the day of testosterone implant), Day 7, Day 14, Day 28 (the day of testosterone removal), Day 36, Day 42, and Day 56. Matched blood samples were also collected for measurement of testosterone concentration. The pheromone activity of the ether-extracts of the upper dermal layer containing sebaceous glands was assessed by its stimulatory effect on the hypothalamic GnRH pulse generator, which was monitored for changes of specific multiple unit activity (MUA) in ovariectomized estradiol-primed goats as described previously. The sebaceous gland enlarged during the testosterone treatment but reduced in size after testosterone removal. The pheromone activity first appeared in 2 out of 5 goats on Day 7 and in all the 5 goats by Day 28. Fourteen days after testosterone removal (Day 42), the pheromone activity was no longer detectable in any of the 5 goats. In short, the sebaceous gland size and the pheromone activity shifted almost in parallel. The present results provide strong support for the view that the primer pheromone is produced testosterone dependently in the sebaceous gland of the male goat.     

Age-dependent changes in structure and function of the male labial gland in Bombus terrestris

The cephalic region of the labial gland in the buff-tailed bumblebee, Bombus terrestris, consists of numerous acini (formed by associated secretory cells and a central lumen) and connecting ducts. Age-dependent changes in secretion production (both qualitative and quantitative) are associated with changes in the amount of rough endoplasmic reticulum (RER), Golgi apparatus, and smooth endoplasmic reticulum (SER). The main secretory organelle is RER in the youngest individuals (pharate, and less-than-a-day old males), Golgi apparatus in 1-day-old males, and SER in males older than 2 days. Secretory cell death starts at 5 days of age, with maximal longevity to 10 days. Pheromone production starts immediately after eclosion, with pheromone quantities increasing until day 7. 2,3-dihydrofarnesol, the main component of the male-marking pheromone, appears in 1-day-old male glands, and reaches a maximum at 7 days of age, when its presence in the gland starts to decrease gradually. Older glands contain compounds not present in young ones. Variation in pheromone quantity and composition are reflected sensitively in the response of the queen antennae. Though queen antennae responded to gland extracts of all ages examined, maximum sensitivity was observed in response to extracts of glands 2-10 days old, while extracts of older glands gradually lose their effectiveness. Both major and minor components of the labial gland secretion extract elicited queen antennal responses suggesting that the pheromone is a multicomponent blend. Age-dependent changes in pheromone production, accumulation and tuning of pheromone activity are all synchronized approximately with male flight from the hive.     

The effects of topical application of various fatty acids on pheromone and glandular lipid biosynthesis in the moth Heliothis virescens

Binary mixtures of deuterium-labeled palmitic acid and an excess of different fatty acids were applied to the sex pheromone gland of female Heliothis virescens and the effects on the terminal steps of pheromone biosynthesis, including incorporation of fatty acids into the glandular lipids, observed. Relative to labeled palmitic acid applied alone, application of all the binary mixtures resulted in decreased levels of the labeled pheromone component, (Z)-11-hexadecenyl acetate (Z11-16:OAc), but there was generally no decrease in the amounts of labeled pheromone precursor, (Z)-11-hexadecenoate, nor labeled palmitate in the glandular lipids. These data suggest that the excess of fatty acid in the gland inhibits Delta11-desaturation. However, in the case of excess myristoleic acid, the amount of labeled (Z)-11-hexadecenoate increased significantly, suggesting that this acid inhibited fatty acid reduction. Dose-response tests with certain of the fatty acids were consistent with the above interpretations and further indicated that the gland had a high capacity for rapidly activating and incorporating excess fatty acids into the glandular lipids. Finally, application of the various fatty acids resulted in increased levels of these acids in the gland and, in the cases of myristoleic, palmitoleic and myristic acids, it also resulted in increased levels of the corresponding aldehydes, which had previously been detected in the gland of female H. virescens. This suggests that the fatty acid reductase in H. virescens is not highly specific for the major component, and that the final ratio of pheromone components is determined in part by the availability of their corresponding fatty acids in the gland.