Molecular characterization and evolution of pheromone binding protein genes in Agrotis moths

Pheromone-binding proteins (PBPs) are soluble transporter proteins that increase the capture and the solubilization of pheromone molecules in the lymph surrounding the olfactory receptors. A polymerase chain reaction-based method was used to identify PBP genes in Agrotis species for an evolutionary genomic study of noctuid moth PBPs. From genomic DNA we determined the structure of different PBP genes in the two closely related species, Agrotis ipsilon and A. segetum. In all, we clearly identified four genes (Aips-1, Aips-2, Aseg-1 and Aseg-2) that represent two distinct PBP orthology groups. We found that the four genes have the same exon-intron structure and that they comprise three exons and two introns but differ in length mainly in the second intron. The three exons of Aseg-2 and Aips-2 have the same lengths but both intron 1 and intron 2 differ in length between the genes. In contrast, Aips-1 and Aseg-1 show dissimilarity only in the length of intron 2. Interestingly, introns 1 and 2 are inserted in the same positions in the Aips-1, Aips-2, Aseg-1 and Aseg-2 genes. These findings show that the Agrotis PBP genes have common ancestry and probably originate from gene duplication before the speciation of ipsilon and segetum. We found that expression of Aips-1/Aseg-1 and Aips-2/Aseg-2 is antennal-specific, but expression is not restricted to the male antennae.     

Species-specific pheromonal compounds induce distinct conformational changes of pheromone binding protein subtypes from Antheraea polyphemus

We have investigated the structural features of three pheromone binding protein (PBP) subtypes from Antheraea polyphemus and monitored possible changes induced upon interaction with the Antheraea pheromonal compounds 4E,9Z-14:Ac [(E4,Z9)-tetradecadienyl-1-acetate], 6E,11Z-16:Ac [(E6,Z11)-hexadecadienyl-1-acetate], and 6E,11Z-16:Al [(E6,Z11)-hexadecadienal]. Circular dichroism and second derivative UV-difference spectroscopy data demonstrate that the structure of subtype PBP1 significantly changes upon binding of 4E,9Z-14:Ac. The related 6E,11Z-16:Ac was less effective and 6E,11Z-16:Al showed only a small effect. In contrast, in subtype PBP2 pronounced structural changes were only induced by the 6E,11Z-16:Al, and the subtype PBP3 did not show any considerable changes in response to the pheromonal compounds. The UV-spectroscopic data suggest that histidine residues are likely to be involved in the ligand-induced structural changes of the proteins, and this notion was confirmed by site-directed mutagenesis experiments. These results demonstrate that appropriate ligands induce structural changes in PBPs and provide evidence for ligand specificity of these proteins. Electronic Publication     

Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi.

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K. , Krieger, J. & Breer, H. (1989) FEBS Lett. 256, 2215-2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta 1088, 277-284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6, 11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.     

Binding properties of pheromone-binding protein 1 from the common cutworm Spodoptera litura

Pheromone-binding proteins (PBPs) were formerly thought to act as passive pheromone carriers. However, recent studies, particularly in Drosophila melanogaster, suggest that PBPs are involved in the recognition of semiochemicals, thus making ligand-binding studies more meaningful. Previously, we cloned three PBPs from Spodoptera litura (Slit), and showed that SlitPBP1 is much more abundant than the other two, particularly in male antennae. To investigate the ligand specificity of SlitPBP1, we expressed the protein in a bacterial system and performed binding experiments with the three components of the specific sex pheromones (Z9-14:Ac, Z9,E11-14:Ac and Z9,E12-14:Ac), as well as with 26 volatile ligands. The results indicated that SlitPBP1 bound all three sex pheromone components with dissociation constants between 0.6 and 1.1 μM. The same protein also bound with comparable affinities several pheromone analogs, but not plant volatiles. The presence of a double bond was the most important element for a strong binding, while its position and configuration also affected the affinity. Finally, the binding of pheromone components is strongly affected by pH, showing a critical pH value corresponding to isoelectric point of the protein. This suggests that a pH-dependent conformational mechanism might exist in SlitPBP1 for pheromone binding and release.     

Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus

The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.     

Sulfhydryl-reagent inhibition of olfaction in a moth and effects of exposure to pheromone compounds or a pheromone analogue

The effects on olfaction of N-ethylmaleimide (NEM), a specificreagent of free sulfhydryl groups, were studied in the mothMamestra brassicae. The antennae of male M.brassicae bear twotypes of specialist receptor neurons involved in pheromone communication.One type is tuned to (Z)-11-hexadecenyl acetate (Z11-16:Ac),the main pheromone component; the second type is tuned to (Z)-9-tetradecenylacetate (Z9-14:Ac), an interspecific inhibitor not producedby the females of this species. Vapours of NEM irreversiblyinhibited the electro-antennographic (EAG) responses to Z11-16:Acand Z9-14:Ac. When Zll-16:Ac was applied before and during NEMtreatment, the responses to Z9-14:Ac were preserved and someprotection was observed in the responses to Zll-16:Ac. In return,Z9-14:Ac partially prevented the disappearance of responsesto Zll-16:Ac but not to Z9-14:Ac. A third compound, hexadecylacetate (16:Ac), found in the pheromone gland, but not detectedby the antennal receptors, did not prevent the inhibition causedby NEM.     

草地贪夜蛾三个SfruPBPs对本种及同域种劳氏粘虫性信息素及腺体组分的亲和力

[目的]测定草地贪夜蛾Spodoptera frugiperda 3个信息素结合蛋白(pheromone binding protein,PBP)(SfruPBP)对草地贪夜蛾及同域近缘种劳氏粘虫Leucania loreyi性信息素及腺体组分的结合特性,探究草地贪夜蛾这3个SfruPBPs在两种昆虫不同性信息素组分识...     

Structure-activity studies with pheromone-binding proteins of the gypsy moth,Lymantria dispar

Pheromone olfaction in the gypsy moth, Lymantria dispar, involves accurate distinction of compounds with similar structure and polarity. The identified sex pheromone is (7R,8S)-2-methyl-7,8-epoxyoctadecane, 1a, and a known antagonist is (7Z)-2-methyloctadec-7-ene, 4a. The first step in pheromone olfaction is binding of odorants by small, soluble pheromone-binding proteins (PBPs), found in the pheromone-sensing hairs. We have studied the molecular determinants recognized by the two PBPs found in the gypsy moth, using three pheromone/PBP binding assays. Results indicate that (i) PBPs bind analogs of the pheromone with some discrimination; (ii) PBPs experience enhancement of binding when presented with 1a or its enantiomer and 4a simultaneously; and (iii) the binding enhancement is also seen at high ligand:PBP ratios. We found no evidence of allostery, so the synergistic binding effects and the concentration effect may only be explained by multimerization of PBPs with each other, which leads to more than one population of binding sites. We suggest that the enhanced ligand binding at high ligand:PBP ratios may serve to sequester excess ligand and thereby attenuate very strong signals.     

小地老虎中国种群性信息素的成分鉴定及田间试验（英文）

研究了小地老虎中国种群的性信息素组分,3日龄处女蛾单腺体性信息素提取物中性信息素的含量非常低。GC和GC-MS分析表明,小地老虎性信息素含有5种成分：顺-7-12碳乙酸酯（A）、顺-9-14碳乙酸酯（B）、顺-11-16碳乙酸酯（C）、顺-5-10碳乙酸酯（D）和顺-8-12碳乙酸酯（E）。它们的含量分别为：（0.245±0.098）、（0.080±0.031）、（0.089±0.03）、(0.085±0.031)和 (0.105±0.065)ng, 这5种物质的百分比分别为40.451±13.66、13.176±5.279、14.943±5.142、14.392±6.10和17.225±9.792,前3种物质的百分比为58.75±9.429、18.91±7.539和22.34±7.209。田间试验表明,性信息素单一组分均未诱到雄蛾,AB以3∶1的比例配成的诱芯对雄蛾有一定的引诱活性,一个诱捕器平均诱捕到2.6头。ABC组分以3∶1∶1的比例配成的诱芯对雄蛾引诱活性显著增强,一个诱捕器平均诱捕量为7.40头,是AB（3∶1）诱芯的2.8倍。诱芯中性信息素的含量对诱蛾活性有明显的影响,在剂量为200 µg时的平均诱捕量最高。     

Molecular and functional characterization of an odorant binding protein of the Asian elephant,Elephas maximus: implications for the role of lipocalins in mammalian olfaction

The sex pheromone present in the pre-ovulatory urine of female Asian elephants is the simple lipid (Z)-7-dodecen-1-yl acetate (Z7-12:Ac). Using radiolabeled probes, we have identified a pheromone binding protein that is abundant in the mucus of the trunk; this protein is homologous to a class of lipocalins known as odorant binding proteins (OBPs). To test five previously proposed roles for the OBP in chemosensory perception, we determined the equilibrium dissociation constant of the OBP-pheromone complex, as well as the association and dissociation rates. Using a mathematical model in conjunction with experimental data, we suggest that the binding and release of the pheromone by the OBP are too slow for the OBP to function in transporting the pheromone through the mucus that covers the olfactory sensory epithelium. Our data indicate that the elephant OBP only modestly increases the solubility of the pheromone in the mucus. Our results are most consistent with the notion that elephant OBP functions as a scavenger of the pheromone and possibly other ligands, including odorants. In light of these findings, and published results for other mammalian OBP-ligand complexes, a general model for the role of OBPs in mammalian olfaction is proposed. Moreover, the potential implications of these findings for interaction of Z7-12:Ac with insect antennal proteins are discussed.     

Identification of the sex pheromone of Isoceras sibirica Alpheraky (Lepidoptera, Cossidae)

We discovered that extracts of the female sex pheromone gland of the carpenterworm moth Isoceras sibirica Alpheraky, a pest of Asparagus officinalis Linn., contained (Z)-7-tetradecen-1-ol (Z7-14:OH), (Z)-9-tetradecen-1-ol (Z9-14:OH), (Z)-7-tetradecenyl acetate (Z7-14:Ac), (Z)-9-tetradecenyl acetate (Z9-14:Ac), and (Z)-9-hexadecadecenyl acetate (Z9-16:Ac). The average levels of the chemicals in a single sex pheromone gland of a calling moth were (0.71 +/- 0.24) ng, (1.42 +/- 0.44) ng, (4.36 +/- 0.32) ng, (8.71 +/- 0.26) ng, and (0.82 +/- 0.38) ng, respectively. The electroantennography (EAG) analysis of these chemicals and their analogues demonstrated that Z9-14:Ac triggered significantly the male EAG response. Traps with rubber septa lure impregnated with Z9-14:Ac (500 microg/septum), Z7-14:Ac (250 microg/septum), and Z9-16:Ac (50 microg/septum) were more effective in catching male moths than traps with other baits or virgin females. Addition of Z7-14:OH and Z9-14:OH to rubber septa did not enhance the efficiency of the trap.     

Sex pheromone of Etiella behrii, a pod borer of soybean in Indonesia: identification and field attraction

Four EAG-active components were detected in GC-EAG analyses of hexane extracts from virgin Etiella behrii (Zeller) (Lepidoptera: Pyralidae) females. These components were identified as dodecyl acetate (12:Ac), (E)-9-dodecenyl acetate (E9-12:Ac), either (Z)-9- or (E)-11-tetradecenyl acetate (Z9- or E11-14:Ac), and (Z)-11-tetradecenyl acetate (Z11-14:Ac) by comparison of retention indices on both nonpolar and polar GC columns. The available extract was insufficient for further GC-MS or other chemical analyses (<0.2 ng/female). In field tests carried out in East Java, a 10:90 mixture of E9-12:Ac and Z11-14:Ac showed attractiveness to male moths and addition of 12:Ac and/or E11-14:Ac significantly increased the trap catches while addition of Z9-14:Ac showed no significant effect. Maximum attraction was obtained with 5.35 or 10.7 g/rubber septum of a mixture of E9-12:Ac, Z11-14:Ac, 12:Ac and E11-14:Ac at the ratio of 10:90:0.7:6.3, respectively. The role of pheromone blends in species discrimination between E. behrii and the related E. zinckenella (Treitschke) is discussed.     

Ligand binding to six recombinant pheromone-binding proteins of<Emphasis Type="Italic"> Antheraea polyphemus</Emphasis> and<Emphasis Type="Italic"> Antheraea pernyi</Emphasis>

Binding properties of six heterologously expressed pheromone-binding proteins (PBPs) identified in the silkmoths Antheraea polyphemus and Antheraea pernyi were studied using tritium-labelled pheromone components, (E,Z)-6,11-hexadecadienyl acetate (³H-Ac1) and (E,Z)-6,11-hexadecadienal (³H-Ald), common to both species. In addition, a known ligand of PBP and inhibitor of pheromone receptor cells, the tritium-labelled esterase inhibitor decyl-thio-1,1,1-trifluoropropanone (³H-DTFP), was tested. The binding of ligands was measured after native gel electrophoresis and cutting gel slices. In both species, PBP1 and PBP3 showed binding of ³H-Ac1. In competition experiments with ³H-Ac1 and the third unlabelled pheromone component, (E,Z)-4,9-tetradecadienyl acetate (Ac2), the PBP1 showed preferential binding of Ac1, whereas PBP3 preferentially bound Ac2. The PBP2 of both species bound ³H-Ald only. All of the six PBPs strongly bound ³H-DTFP. Among unlabelled pheromone derivatives, alcohols were revealed to be the best competitors for ³H-Ac1 and ³H-Ald bound to PBPs. No pH influence was found for ³H-Ac1 binding to, or its release from, the PBP3 of A. polyphemus and A. pernyi between pH 4.0 and pH 7.5. The data indicate binding preference of each of the three PBP-subtypes (1–3) for a specific pheromone component and support the idea that PBPs contribute to odour discrimination, although to a smaller extent than receptor activation.Abbreviations Ac1 (E,Z)-6,11-hexadecadienyl acetate - Ac2 (E,Z)-4,9-tetradecadienyl acetate - Ald (E,Z)-6,11-hexadecadienal - AMA 1-amino-anthracene - cpm counts per min - DTFP decyl-thio-1,1,1-trifluoropropanone - ES-MS electrospray mass spectrometry - OH (E,Z)-6,11-hexadecadienol - PAGE polyacrylamide gel electrophoresis - PCR polymerase chain reaction - PBP pheromone-binding protein - SDS sodium dodecyl sulphate - Z-11 OH Z-11 hexadecenolCommunicated by G. Heldmaier     

Functional organization of the macroglomerular complex related to behaviourally expressed olfactory redundancy in male cabbage looper moths

Abstract. The neurophysiological bases for behaviourally expressed olfactory redundancy in the sex pheromone communication system of the cabbage looper moth, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), were examined by coupling the cut-sensillum extracellular recording technique with a highly specific neuronal marking method for moth peripheral receptors. In seventy-two antennal sensilla, axonal pathways of cobalt-stained neurones could be traced into the male-specific macroglomerular complex in the antennal lobe. In T. ni males this comprises five glomeruli, two of which are subdivided into morphologically, and in some instances functionally identifiable, regions. Axonal arborizations of forty-eight neurones (single stainings) showed high fidelity (98%) for containment within a specific glomerulus or glomerular subdivision, and the neuropil targeted seemed to be related to the specificity of a neurone to a particular female-emitted sex pheromone component (27-12:Ac, Z7-14:Ac, Z9-14:Ac, 12:Ac, 11–12:Ac, Z5-12:Ac), or to a behavioural antagonist (Z7-12:OH). Double (twenty-one) and multiple stainings (three) showed axons projecting into two or more glomeruli, respectively, with 100% fidelity for the component-specific glomerulus or glomerular subdivision to be targeted. We suggest that the potential for a single minor component to cross-stimulate two or more neurones within a sensillum may enable partial blends to continue to provide sensory input into all of the pheromone-processing glomeruli of the complex. Our interpretation is that redundancy occurs at the receptor level on neighbouring dendrites, and thus allows various four-component partial blends to evoke full pheromone-mediated behaviour.     

Do Pheromone Binding Proteins Converge in Amino Acid Sequence When Pheromones Converge?

Convergence in amino acid sequences between proteins can be strong evidence for selection. Here, I look for evidence of convergence in the amino acid sequences of pheromone binding protein (PBP) in response to convergence in pheromones. PBPs are involved in sex pheromone reception by the antennae of male moths. In this role PBPs may selectively bind pheromone components and experience convergent selection in response to convergence in pheromone components. However, examination of the PBPs of the taxa that have converged upon the use of (E)- or (Z)-11-tetradecenyl acetate as their major pheromone component reveals little evidence for convergence in the PBPs identified from these taxa. A few sites show a pattern consistent with convergence or parallelism; however, it cannot be ruled out that these sites share the ancestral state. Two of these sites fall within the proposed binding region of PBPs. These results suggest that PBPs either have not converged in sequence or have converged at very few sites in response to convergence on the same pheromone component. Received: 29 July 1999 / Accepted: 8 November 1999     

榆木蠹蛾雄蛾对性信息素不同组分及其不同比例和剂量混合物的风洞行为反应

【目的】本研究旨在深入了解榆木蠹蛾Holcocerus vicarius(Walker)信息素通讯系统。【方法】在风洞中观察了榆木蠹蛾雄蛾对合成性信息素不同组分及其不同比例及剂量混合物的行为反应。【结果】单组分试验结果表明,仅有Z7-14:Ac能够引起少量雄蛾完成从兴奋到接触诱芯并出现预交尾的全部行为反应,其余4个单组分(E3-14:Ac,Z3E5-14:Ac,E3-14:OH和Z3-14:OH)只能引起雄蛾兴奋,均不能引起雄蛾向性信息素源定向飞行。二元混合物(Z7-14:Ac+E3-14:Ac)明显增加雄蛾完整的性行为反应比例,三元混合物(Z7-14:Ac+E3-14:Ac+Z3E5-14:Ac)比例为10∶4∶4,剂量为1 300μg时有71.7%雄蛾发生预交尾,剂量为1 000μg时有70.3%雄蛾发生预交尾,两者差异不显著(P0.05)。【结论】榆木蠹蛾风洞行为实验为进一步研究其性信息素相关生物学特性及应用性信息素对榆木蠹蛾进行综合防治奠定了基础。     

Field trials of synthetic sex pheromone lures for the large cabbage-heart caterpillar moth, Crocidolomia pavonana (Lepidoptera: Crambidae) in Indonesia

The efficacy of synthetic female sex pheromone lures for Crocidolomia pavonana (Fabricius) (Lepidoptera: Crambidae) in the cabbage fields of Java and Bali, Indonesia, was investigated by varying the composition and dosage of the components. The lure containing a synthetic pheromone blend of (Z)-11-hexadecenyl acetate (Z11–16: Ac) and (Z)-9-tetradecenyl acetate (Z9–14: Ac) at a 10:1 ratio acquired significantly more male catches than single component lures and the control lure. Meanwhile, no attraction was observed when lures with 1:1 and 1:10 blends were tested. The composition of Z11–16: Ac and Z9–14: Ac at a ratio of 5, 10 and 20:1 attracted more males than the control lures. Dosage studies showed that 0.055 and 0.55 mg of a mixture of Z11–16: Ac and Z9–14: Ac (10:1 ratio) attracted more males than the control. These results are the first demonstration of the efficacy of synthetic pheromone for C. pavonana in field conditions. The present study suggests the feasibility of pheromone-based monitoring as a simple and low-cost technique for integrated pest management of this pest.     

Response specificity of male olfactory receptor neurones for the major and minor components of a female pheromone blend

Abstract The sex attractant of the female redbanded leafroller moth, Argyrotaenia velutinana (Walker), is a blend of seven compounds. Specialized olfactory receptor neurones had been found for only two of the compounds, (Z)-11-tetradecenyl acetate (Z11-14:Ac) and (E)-11-tetradecenyl acetate (E11–14:Ac). These receptor neurones were always found in pairs within the long trichoid sensilla, which are the most abundant multi-pored sensilla on the male antenna. A systematic survey of all regions of the male antenna with standard extracellular recording techniques was undertaken to find receptor neurones responsive to the remaining five minor components of the female pheromone. Of the 113 long trichoid sensilla sampled, all contained two receptor neurones, one specialized for Z11–14:Ac and a second specialized for Ell –14:Ac. A comparable number of recordings were then obtained from the less abundant classes of multi-pored sensilla. Two new receptor neurone types were found, responsive to the minor pheromone components (E)-9-dodecenyl acetate (E9-12:Ac) and (Z)-9-dodecenyl acetate (Z9-12:Ac). Scanning electron micrographs indicated that these recordings were obtained from shorter, narrower trichoid sensilla. The majority of these sensilla appeared to contain three neurones capable of spontaneous action potential production. In each sensillum, only one receptor neurone appeared to respond to stimulation with a minor component of the female blend. The remaining two neurones did not respond to the chemical stimuli evaluated.