Hormonal regulation of sex pheromone biosynthesis in the turnip moth,Agrotis segetum

Pheromone production in the female turnip moth, Agrotis segetum, is under the control of a brain factor. This factor was demonstrated to be a proteinaceous substance termed pheromone biosynthesis activating neuropeptide-like substance (PBAN-like substance). The sex pheromone of Swedish A. segetum includes (Z)-5-decenyl acetate, (Z)-7-dodecenyl acetate, and (Z)-9-tetradecenyl acetate as major components. Decapitation of a female decreased pheromone production significantly. Pheromone production was restored by injection of homogenates of either male or female brain-suboesophageal ganglion or the corpora cardiaca alone. Pheromonotropic activity was also found in homogenates of the female thoracic ganglion and abdominal ganglion that were obtained during scotophase. Injection of female brain and thoracic ganglion homogenates made from insects during the scotophase induced two and four times as much Z7-12:OAc, respectively, as injection with similar homogenates from photophase. As little as one-eighth female equivalent (FE) brain homogenate was sufficient to increase the amount of Z7-12:OAc. The effect of brain homogenate on pheromone titer reached its maximum after 30 min. The activity of the PBAN-like substance present in female brain extracts was not correlated to the age of the donor. Injection of hemolymph collected during either photophase or scotophase into decapitated females did not increase the pheromone titer. The target site of the PBAN-like substance was not the pheromone gland, and the ventral nerve cord was not involved in the transportation of the PBAN-like substance, which implies a mode of action different from what has been reported in other moths. Brain homogenates obtained during photophase from females of African A. segetum, Spodoptera littoralis, or Ostrinia nubilalis as well as synthetic Bombyx-PBAN also induced pheromone production in decapitated Swedish female A. segetum. © 1995 Wiley-Liss, Inc.     

Electrophysiological and behavioural evidence for a fourth sex pheromone component in the turnip moth, Agrotis segetum

Abstract. In addition to the pheromone components (Z)-5-decenyl, (Z)-7-dodecenyl and (Z)-9-tetradecenyl acetate (Z5-10:OAc, Z7-12:OAc and Z9-14:OAc), it has previously been shown that the sex pheromone gland of the turnip moth, Agrotis segetum (Lepidoptera: Noctuidae Schiff) contains 10:OAc, 12:OAc, Z5-12:OAc, Z9-12:OAc, 11–12:OAc, Z5-14:OAc, Z7-14:OAc and Z11-16:OAc. To find out whether any of these additional compounds is involved in the sex pheromone communication in A. segetum, a comprehensive electro-physiological and behavioural investigation was conducted. Single-sensillum recordings on male antennae revealed three subtypes of sensilla among the previously so-called Z5-10:OAc sensilla. One subtype was identified having one receptor neurone (A) that responded to Z5-10:OAc with a large spike amplitude and another neurone (B) that responded to (Z)-5-decenol (Z5-10:OH) with a small spike amplitude. In another subtype the B neurone responded to Z5-12:OAc and sometimes also to 27-12:OAc and 10:OAc, in addition to responding to Z5-10:OH. In a third subtype the A neurone responded to all acetates identified from the female pheromone gland, whereas the small spike amplitude neurone was tuned to Z5-10:OH. A flight tunnel assay showed that blends composed of nine, eight or seven compounds were equivalent to the previously identified three-component pheromone blend in eliciting male behavioural responses. In field trapping tests, blends of eleven, nine or seven compounds did, however, catch significantly more moths than the three-component blend. Further assays showed that only 25- 12:OAc could significantly increase the catch numbers when added to the three-component blend, and thus qualified as a fourth pheromone component in A. segerum. The behavioural significance of additional female-produced acetates — for which males possess antennal receptors — is suggested, but may be impossible to confirm because of ‘diminishing returns’ when trying to refine a multicomponent pheromone further.     

Dienic analogs of (Z)-5-decenyl acetate, a pheromone component of the turnip moth, Agrotis segetum: Synthesis, conformational analysis and structure-activity relationships

Structure-activity relationships for (Z, E)-dienic analogs of (Z)-5-decenyl acetate, a pheromone component of the turnip moth, Agrotis segetum, have been studied by electrophysiological single-cell recordings and molecular mechanics calculations. The biological activities of the dienic analogs are highly sensitive to the position of the additional (E) double bond. The experimental observations are well rationalized by the use of a receptor-interaction model in which the biological activity is determined by conformational energies required to mimic spatial relationships in the natural pheromone component. A biologically active conformation for this compound is suggested.     

Reidentification of pheromone composition of Sparganothis sulfureana (Clemens) and evidence of geographic variation in male responses from two US states

GC-EAD analyses of pheromone gland extracts of calling female Sparganothis sulfureana revealed at least 6 compounds that consistently elicited antennal responses from male antennae. In addition to the major pheromone compound, (E)-11-tetradecenyl acetate (E11–14:OAc), which was previously reported, the other compounds were found to be (E)-9-dodecenyl acetate (E9–12:OAc), (Z)-9-dodecenyl acetate (Z9–12:OAc), (Z)-9-tetradecenyl acetate (Z9–14:OAc), (Z)-11-tetradecenyl acetate (Z11–14:OAc), and (E)-11-tetradecenol (E11–14:OH). Tetradecyl acetate, hexadecyl acetate and hexadecenyl acetates were also present in the extracts, but elicited no EAG response from male antennae. Wind tunnel tests demonstrated that males from New Jersey responded equally well to a blend containing five pheromone components in relative to the pheromone glands of calling females. Different male-response profiles from field-trapping tests conducted in the states of Wisconsin and New Jersey were observed, respectively. Significantly higher numbers of male S. sulfureana were caught in New Jersey in traps baited with the binary blend of E11–14:OAc (30 μg) with 1% of Z11–14:OAc, but males from Wisconsin responded equally well to traps containing blends of E11–14:OAc with 0–10% of Z11–14:OAc. The addition of more than 10% of Z11–14:OAc to the primary pheromone compound reduced male captures significantly in both states. Male catches were doubled by adding E9–12:OAc and E11–14:OH to the most attractive binary blend in both states. The trapping test with caged live virgin female moths showed that males in Wisconsin preferred females from the local population than those from New Jersey. The differences in male responses observed may indicate the existence of pheromone polymorphism in this species.     

Relationship between sex pheromone elicited behaviour and response of single olfactory receptor neurones in a wind tunnel

Responses from pheromone‐specific receptor neurones in male Agrotis segetum (Denis & Schiffermüller) (Lepidoptera: Noctuidae) were recorded in a laboratory wind tunnel. Stimuli were: (1) rubber septum dispensers loaded with single components or a four‐component pheromone blend, (2) excised glands from female A. segetum, (3) constrained A. segetum females with extruded glands. Dose–response curves for three neurone‐types with different specificity were established. The neurones were specifically tuned to respond to either one of the two pheromone components (Z)‐5‐decenyl acetate and (Z)‐7‐dodecenyl acetate, or to the behavioural antagonist (Z)‐5‐decenol. In parallel, a behavioural dose–response curve with males flying upwind to a four‐component pheromone blend was established. There was a clear correlation between behavioural arrestment of upwind flight and maximum spiking activity in Z5–10:OAc‐specific neurones. The pheromone release rates of individual females and synthetic dispensers were compared. A load of 50–200 ng of Z5–10:OAc on a rubber septum elicited approximately the same neural response as one female gland.     

Identification and behavioral evaluation of sex pheromone components of the Chinese pine caterpillar moth, Dendrolimus tabulaeformis

Background

The Chinese pine caterpillar moth, Dendrolimus tabulaeformis Tsai and Liu (Lepidoptera: Lasiocampidae) is the most important defoliator of coniferous trees in northern China. Outbreaks occur over enormous areas and often lead to the death of forests during 2–3 successive years of defoliation. The sex pheromone of D. tabulaeformis was investigated to define its chemistry and behavioral activity.

Methodology/Principal Findings

Sex pheromone was collected from calling female D. tabulaeformis by headspace solid phase microextraction (SPME) and by solvent extraction of pheromone glands. Extracts were analyzed by coupled gas chromatography/mass spectrometry (GC-MS) and coupled GC-electroantennographic detection (GC-EAD), using antennae from male moths. Five components from the extracts elicited antennal responses. These compounds were identified by a combination of retention indices, electron impact mass spectral matches, and derivatization as (Z)-5-dodecenyl acetate (Z5-12:OAc), (Z)-5-dodecenyl alcohol (Z5-12:OH), (5Z,7E)-5,7-dodecadien-1-yl acetate (Z5,E7-12:OAc), (5Z,7E)-5,7-dodecadien-1-yl propionate (Z5,E7-12:OPr), and (5Z,7E)-5,7-dodecadien-1-ol (Z5,E7-12:OH). Behavioral assays showed that male D. tabulaeformis strongly discriminated against incomplete and aberrant blend ratios. The correct ratio of Z5,E7-12:OAc, Z5,E7-12:OH, and Z5,E7-12:OPr was essential for optimal upwind flight and source contact. The two monoenes, Z5-12:OAc and Z5-12:OH, alone or binary mixtures, had no effect on behavioral responses when added to the optimal three-component blend.

Conclusions/Significance

The fact that deviations from the optimal ratio of 100∶100∶4.5 of Z5,E7-12:OAc, Z5,EZ7-12:OH, and Z5,E7-12:OPr resulted in marked decreases in male responses suggests that biosynthesis of the pheromone components is precisely controlled. The optimal blend of the sex pheromone components of D. tabulaeformis worked out in this study should find immediate use in monitoring this pest in Chinese forests.     

Functional characterization of sex pheromone receptors in Spodoptera frugiperda,S. exigua,and S. litura moths

Moths possess an extremely sensitive and diverse sex pheromone processing system, in which pheromone receptors (PRs) are essential to ensure communication between mating partners. Functional properties of some PRs are conserved among species, which is important for reproduction. However, functional differentiation has occurred in some homologous PR genes, which may drive species divergence. Here, using genome analysis, 17 PR genes were identified from Spodoptera frugiperda, S. exigua, and S. litura, which belong to 6 homologous groups (odorant receptor [OR]6, 11, 13, 16, 56, and 62); of which 6 PR genes (OR6, OR11, OR13, OR16, OR56, and OR62) were identified in S. frugiperda and S. exigua, and 5 PR genes were identified in S. litura, excluding OR62. Using heterologous expression in Xenopus oocytes, we characterized the functions of PR orthologs including OR6, OR56, and OR62, which have not been clarified in previous studies. OR6 orthologs were specifically tuned to (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), and OR62 orthologs were robustly tuned to Z7-12:OAc in S. frugiperda and S. exigua. The optimal ligand for OR56 was Z7-12:OAc in S. frugiperda, but responses were minimal in S. exigua and S. litura. In addition, SfruOR6 was male antennae-specific, whereas SfruOR56 and SfruOR62 were male antennae-biased. Our study further clarified the functional properties of PRs in 3 Spodoptera moth species, providing a comprehensive understanding of the mechanisms of intraspecific communication and interspecific isolation in Spodoptera.     

Simultaneous mating disruption of two Grapholita species in apple orchards

Two Grapholita species (G. molesta and G. dimorpha) cause serious losses in apple production by means of direct fruit damage and destruction. These two congeners occurred at similar seasons in the same orchards. Furthermore, the two species occurred in similar population sizes in apple orchards, though G. molesta occurred much more in total occurrence density than did G. dimorpha in a pear orchard. The two species share two major sex pheromone components, (Z)-8-dodecenyl acetate (Z8-12:Ac) and (E)-8-dodecenyl acetate (E8-12:Ac), but differ with regard to the relative mixture ratio necessary to create conditions of maximal attraction. To develop a simultaneous mating disruption (SMD), a mass ratio of 95:5 of Z8-12:Ac and E8-12:Ac was selected due to its high cross-calling activity and subsequent high mating disruption against both species. When the mixture was applied to orchards, it resulted in significant mating disruption against both species. However, mating disruption efficacy was slightly higher in G. dimorpha than in G. molesta. Application dose and dispenser type affected efficacy duration of SMD. In addition, the high density occurrence of G. molesta significantly reduced the SMD efficacy. Using a wax-type of dispenser, an application of SMD in 100?g per hectare dispersed in early spring recorded >90% mating disruption efficacy through the entire growing season against both Grapholita species.     

New Synthesis of the Both Enantiomers of (Z)-5-(1-Decenyl)-oxacyclopentan-2-one,the Pheromone of the Japanese Beetle

The both enantiomers of (Z)-5-(1-decenyl)oxacyclopentan-2-one were synthesized starting from resolved acetylenic intermediates. The (R)-form of this lactone is the pheromone of Popillia japonica. The optical purities of the final products were about 90%.     

Development of Sex Pheromone Trap for Monitoring Matsumuraeses falcana (Walshingham) (Lepidoptera: Tortricidae)

This study was carried out to find out an effective formulation for monitoring the soybean podborer, Matsumuraeses falcana (Walshingham), using its sex pheromone in Korea. A 1 : 1 : 1 mixture of (E)-8-dodecenyl acetate (E 8–12: Ac), (E,E)-8,10-dodecadienyl acetate (E 8 E 10–12: Ac) and (E,Z)-7,9-dodecadienyl acetate (E 7 Z 9–12: Ac) was an effective composition in monitoring M. falcana. A binary mixture of E 8–12: Ac and E 8 E 10–12: Ac was essential to attract M. falcana male moths. E 7 Z 9–12: Ac showed a synergistic effect on the attractiveness of 1 : 1 mixture of E 8–12: Ac and E 8 E 10–12: Ac. These chemicals alone showed little or no attractiveness to M. falcana. The sticky-typed wing trap baited with sex pheromone captured significantly more males than the water-pan trap. Trap catches within a soybean field were much more than those in perimeter trap. The trap-baited with sex pheromone gave a distinct fluctuation pattern of male catches throughout the flight seasons. Therefore, it can be expected that pheromone-baited trap provides a simple tool for monitoring the soybean podborer, M. falcana in Korea.     

Structure and function of antennal lobe neurons in the male turnip moth,Agrotis segetum (Lepidoptera: Noctuidae)

Interneurons with dendritic branches in the antennal lobe of the male turnip moth, Agrotis segetum (Schiff., Lepidoptera: Noctuidae), were investigated with intracellular recording and staining methods. Seventeen projection neurons that transmit information from the antennal lobe to higher centers in the brain displayed dendritic arbors in the male specific macroglomerular complex (MGC) and responded to chemical components of the female sex pheromone used in species-specific sexual communication. Most of the projection neurons responded to several of the pheromone components tested, and a precise correlation between the location of the dendritic arborization and the physiological response could not be demonstrated. One MGC-projection neuron fit the definition of blend specialist. It did not respond to the individual components of the behaviorally active pheromone blend, but showed a strong response to the components when combined in the species-specific blend. Some of the projection neurons also showed clear responses to phenylacetaldehyde, a flower-produced compound and/or to (E)-2-hexenal, a common green-leaf volatile. In eight neurons, the axonal projection could be followed to the calyces of the mushroom body, and subsequently to the inferior lateral protocerebrum.Four local interneurons were characterized both morphologically and physiologically. Each neuron arborized extensively throughout the antennal lobe, and each responded to one or several of the pheromone compounds, and/or to one or both of the plant-produced compounds. One of the local interneurons responded exclusively to the pheromone blend, but not to the individual components.Abbreviations AL antennal lobe - AN antennal nerve - CB cell body - E2H (E)-2-hexenal - IACT inner antennocerebral tract - ILPR inferior lateral protocerebrum - LH lateral horn of the protocerebrum - LN local interneuron - MB mushroom body - MGC macroglomerular complex - OACT outer antennocerebral tract - PAA phenylacetaldehyde - PN projection interneuron - RN receptor neuron - Z5-10:OAc (Z)-5-decenyl acetate - Z5-10:OH (Z)-5-decenol - Z5-12:OAc (Z)-5-dodecenyl acetate - Z7-12:OAc (Z)-7-dodecenyl acetate - Z9-14:OAc (Z)-9-tetradecenyl acetate     

Sex attractant for Diloba caeruleocephala (L.), (Lep., Dilobidae) - (Z)-8-tridecenyl acetate

For seven homologous compounds, the highest electro-antennography responses from male Diloba caeruleocephala (L.) were elicited by ( Z )-5-decenyl acetate ( Z )-7-dodecenyl acetate and ( Z )-8-tridecenyl acetate. The ( Z )-8-tridecenyl acetate caused significant attraction of males of this species in the field, in both Bulgaria and Hungary. ( Z )-10-pentadecenyl acetate, when added as a 10% admixture to the latter, acted as an attractant inhibitor. In Bulgaria, the flight period of this species, as established by means of pheromone traps, commenced at the beginning of September and lasted until the middle of October.     

Temporal pattern of sex pheromone release by female Trichoplusia ni

The release rate of the pheromone component Z-7-dodecenyl acetate was determined for individual female Trichoplusia ni by using small glass tubes filled with Porapak Q to extract pheromone from air in the immediate vicinity of the everted gland. The change in release rate as a function of time was determined by taking sequential 5 min. samples from individual 4-day-old females through each pheromone release period over an entire night. The release rate was found to decline exponentially from a mean of 22 ng per min. initially to 12 ng per min. at the end of 20 min., the average length of a pheromone release period. Pheromone collections were also made from females of different ages, using a single tube of Porapak per female to collect pheromone for an entire night. The nightly mean release rate increased significantly with age, although the time spent releasing pheromone per night decreased significantly with age (to 6 days old).     

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.     

Sex pheromone components of female Cornutiplusia circumflexa

Capillary gas chromatography indicated the presence of (Z)-7-dodecenyl alcohol and (Z)-7-dodecenyl acetate in the abdominal tip extracts of female Cornutiplusia circumflexa (L.) (Lepidoptera: Noctuidae: Plusiinae) moths. Gas chromatography combined with mass spectrometry and dimethyl disulfide derivatization confirmed the structure of these two principal pheromone components. In a flight-tunnel bioassay, a 5:1 blend of alcohol and acetate elicited the complete courtship sequence by males. The response was comparable to that evoked by a live virgin female, but males spent significantly longer time at calling females than at the synthetic source. The 5:1 blend was also attractive to male C. circumflexa in the field, as indicated by catches in traps baited with this mixture. (Z)-7-dodecenyl alcohol alone was slightly attractive whereas (Z)-7-dodecenyl acetate alone was completely inactive. C. circumflexa is the first reported Plusiinae species which utilizes (Z)-7-dodecenyl alcohol as the major pheromone component.     

Effects of different sex pheromone compositions and host plants on the mating behavior of two Grapholita species

The two congener species Grapholita molesta and Grapholita dimorpha share two major sex pheromone components: cis-8-dodecenyl acetate (Z8-12Ac) and trans-8-dodecenyl acetate (E8-12Ac). In fact, commercial sex pheromone lures composed of only these two major components attract the males of both species. In this study, we aimed to determine the reproductive isolation components of these two species by analyzing the effects of the minor sex pheromone components and host plants. First, different ratios of the two major sex pheromone components were greatly favored by either male species. Sex pheromone gland extracts of G. dimorpha contained a lesser proportion of Z8-12Ac than that of G. molesta. In the three (apple, pear, and peach) orchards investigated in this study, a larger number of G. molesta males were attracted to the 95:5 pheromone mixture (Z8-12Ac and E8-12Ac, respectively), while a larger number of G. dimorpha males were attracted to the 85:15 mixture. Second, there was a significant variation in male attractions in different host plants. G. molesta males were more attracted to the sex pheromone lure in the apple orchards than that in the pear and peach orchards. In contrast, G. dimorpha males were more attracted to the lures in the pear and peach orchards than that in the apple orchard. Third, the minor sex pheromone components were important for reproductive isolation. Among the four minor components tested, addition of (Z)-8-dodecenol (Z8-12OH) to the major sex pheromone components significantly suppressed male attraction in G. dimorpha and slightly elevated male attraction in G. molesta. The discriminating effect of Z8-12OH was further validated using male electroantennogram analysis. These results suggest that reproductive isolation between two congeners can be achieved by variations in the minor sex pheromone components and in the host plants, as well as by changes in the ratio of the two major components.     

Identification and Characterization of Pheromone Receptors and Interplay between Receptors and Pheromone Binding Proteins in the Diamondback Moth,Plutella xyllostella

Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic of pheromone receptors. In the Xenopus-based functional study and in situ hybridization, PxylOR4 is defined as another pheromone receptor in addition to the previously characterized PxylOR1. In the study of interaction between PRs and PBPs, PxylPBPs could increase the sensitivity of the complex expressing oocyte cells to the ligand pheromone component while decreasing the sensitivity to pheromone analogs. We deduce that activating pheromone receptors in olfactory receptor neurons requires some role of PBPs to pheromone/PBP complex. If the chemical signal is not the pheromone component, but instead, a pheromone analog with a similar structure, the complex would have a decreased ability to activate downstream pheromone receptors.     

Neofunctionalization in an ancestral insect desaturase lineage led to rare Δ6 pheromone signals in the Chinese tussah silkworm

The Chinese tussah silkworm, Antheraea pernyi (Lepidoptera: Saturniidae) produces a rare dienoic sex pheromone composed of (E,Z)-6,11-hexadecadienal, (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-4,9-tetradecadienyl acetate, and for which the biosynthetic routes are yet unresolved. By means of gland composition analyses and in vivo labeling we evidenced that pheromone biosynthesis towards the immediate dienoic gland precursor, the (E,Z)-6,11-hexadecadienoic acid, involves desaturation steps with Δ⁶ and Δ¹¹ regioselectivity. cDNA cloning of pheromone gland desaturases and heterologous expression in yeast demonstrated that the 6,11-dienoic pheromone is generated from two biosynthetic routes implicating a Δ⁶ and Δ¹¹ desaturase duo albeit with an inverted reaction order. The two desaturases first catalyze the formation of the (E)-6-hexadecenoic acid or (Z)-11-hexadecenoic acid, key mono-unsaturated biosynthetic intermediates. Subsequently, each enzyme is able to produce the (E,Z)-6,11-hexadecadienoic acid by accommodating its non-respective mono-unsaturated product. Besides elucidating an unusually flexible pheromone biosynthetic pathway, our data provide the first identification of a biosynthetic Δ⁶ desaturase involved in insect mate communication. The occurrence of this novel Δ⁶ desaturase function is consistent with an evolutionary scenario involving neo-functionalization of an ancestral desaturase belonging to a gene lineage different from the Δ¹¹ desaturases commonly involved in moth pheromone biosynthesis.