Selectivity and neuroendocrine regulation of the precursor uptake by pheromone glands from hemolymph in geometrid female moths, which secrete epoxyalkenyl sex pheromones

Macrolepidopteran female moths in families such as Geometridae produce epoxyalkenyl sex pheromones, which are biosynthesized via epoxidation of polyunsaturated hydrocarbons in their pheromone glands. The precursors, however, are expected to be produced outside of the pheromone glands, probably in oenocytes or in the fat body, and transported to the glands via hemolymph. Based on these facts, the selectivity of the epoxidation substrates and of the precursor uptake by pheromone glands was examined with two geometrid species, Hemerophila artilineata and Ascotis selenaria cretacea, using binary mixtures of deuterated precursors and their analogs, which were topically applied to the pheromone glands or injected into the abdomen. GC-MS measurements of pheromone extracts showed equal epoxidation of two polyenes, indicating a low selectivity for both processes, while the epoxidation proceeded at only one double bond specific to each species. This result makes it possible to conclude that the formation of species-specific epoxyalkenyl pheromones results from the rigid formation of polyunsaturated precursors and their epoxidation at a fixed position. Next, the neuroendocrine regulation of these processes was studied with in vivo and in vitro experiments using decapitated females. The epoxy pheromones disappeared completely within 36 h of decapitation, and epoxidation of the injected precursors was not detected in the decapitated females, which restarted the reaction by treatment with a pheromone biosynthesis-activating neuropeptide (PBAN). The precursors topically applied to glands of the decapitated females, however, were converted into epoxy pheromones without PBAN, indicating that this neuropeptide hormone accelerated the precursor uptake by pheromone glands but not the epoxidation already underway in the glands.     

Discovery of a disused desaturase gene from the pheromone gland of the moth Ascotis selenaria, which secretes an epoxyalkenyl sex pheromone

Female Ascotis selenaria (Geometridae) moths use 3,4-epoxy-(Z,Z)-6,9-nonadecadiene, which is synthesized from linolenic acid, as the main component of their sex pheromone. While the use of dietary linolenic or linoleic fatty acid derivatives as sex pheromone components has been observed in moth species belonging to a few families including Geometridae, the majority of moths use derivatives of a common saturated fatty acid, palmitic acid, as their sex pheromone components. We attempted to gain insight into the differentiation of pheromone biosynthetic pathways in geometrids by analyzing the desaturase genes expressed in the pheromone gland of A. selenaria. We demonstrated that a Δ11-desaturase-like gene (Asdesat1) was specifically expressed in the pheromone gland of A. selenaria in spite of the absence of a desaturation step in the pheromone biosynthetic pathway in this species. Further analysis revealed that the presumed transmembrane domains were degenerated in Asdesat1. Phylogenetic analysis demonstrated that Asdesat1 anciently diverged from the lineage of Δ11-desaturases, which are currently widely used in the biosynthesis of sex pheromones by moths. These results suggest that an ancestral Δ11-desaturase became dysfunctional in A. selenaria after a shift in pheromone biosynthetic pathways.     

Identification of a unique pheromonotropic neuropeptide including double FXPRL motifs from a geometrid species, Ascotis selenaria cretacea, which produces an epoxyalkenyl sex pheromone

Virgin females of the Japanese giant looper (Ascotis selenaria cretacea, Assc) in the family of Geometridae secrete an epoxyalkenyl sex pheromone to attract males. To regulate its biosynthesis in the pheromone gland, Assc females produce a pheromone biosynthesis-activating neuropeptide (PBAN) in the suboesophageal ganglion (SG), as do females in many lepidopteran species. We have isolated Assc-PBAN cDNA, which encodes 181 amino acids, including a PBAN homologue and four other putative peptides: a diapause hormone (DH) homologue, alpha-SG neuropeptide (SGNP), beta-SGNP, and gamma-SGNP, all of which shared an FXPR(K)L motif on their C-termini. Although PBANs with 30-35 amino acids have been characterized from 15 other species, the Assc-PBAN homologue consisted of 28 amino acids and showed low homology (<46%) compared with the others. Assc-beta-SGNP with eight amino acids was also shorter than the other beta-SGNPs (16-22 amino acids). Furthermore, all of the known PBAN cDNAs have a GRR sequence between beta-SGNP and PBAN as a cleavage site, but the Assc-PBAN cDNA showed an unusual GR sequence at the corresponding position, indicating the possibility of non-cleavage between the beta-SGNP and PBAN. When the GR sequence was a cleavage site, the question arose of whether or not the glutamine residue at the N-terminus of the Assc-PBAN homologue was cyclized. To identify the sequence of the Assc-PBAN, the brain-SG extract was fractionated by HPLC referring to three synthetic peptides with the predicted sequences. The chromatographic behavior of the natural pheromonotropic peptide revealed the unique structure of Assc-PBAN including beta-SGNP, i.e., SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH(2), as the first determination of PBAN from the insects producing an epoxyalkenyl sex pheromone.     

Transport of hydrocarbons by the lipophorin of insect hemolymph

When [I-¹⁴C]acetate was injected into the American cockroach, the labeled acetate was incorporated preferentially into the hydrocarbon fraction and, subsequently, the labeled hydrocarbon was released into the hemolymph where it was associated with the lipophorin (formerly called diacylglycerol-carrying lipoprotein). The label was traced to the three hydrocarbons, n-pentacosane, 3-methylpentacosane and 6,9-heptacosadiene that had been shown previously to be associated with the lipophorin. The specific capacity of lipophorin to accept hydrocarbons from oenocytes, which are believed to be the site of hydrocarbon synthesis, was demonstrated in vitro, and the uptake of hydrocarbon by lipophorin was retarded by respiratory poisoning. When lipophorin containing ¹⁴C-labeled hydrocarbon was injected into the hemocoele of cockroach, the labeled hydrocarbon soon appeared at the cuticular surface where it was deposited specifically. The above observations and our previous data support the postulate that insect lipophorin serves as the true carrier molecule for the transport of hydrocarbons from the site of synthesis (oenocyte) to the site of deposition (cuticle), in addition to its function of transporting diacylglycerol and cholesterol from the fat body and intestine.     

Identification of cuticular hydrocarbons and the alkene precursor to the pheromone in hemolymph of the female gypsy moth, Lymantria dispar

Hydrocarbons were extracted from the surface of the cuticle and from the hemolymph of adult female gypsy moths. GC and GC/MS analysis indicated that the cuticular hydrocarbons with chain lengths >21 carbons were the same as those found in the hemolymph. These consisted of mostly saturated straight chain hydrocarbons with heptacosane the major component. Methyl branched hydrocarbons were also identified including a series of tetramethylalkanes with chain lengths of 30, 32, and 34 carbons. In addition to those found on the cuticle surface, the hemolymph contained the alkene pheromone precursor, 2-methyl-Z7-octadecene and two saturated analogues, 2-methyl-octadecane and 2-methyl-hexadecane. No evidence was obtained for the presence of the pheromone 2-methyl-7, 8-epoxy-octadecane in the hemolymph. Pheromone gland extracts indicated that small amounts (<1 ng) of the alkene precursor were also present in the gland. Relatively larger amounts of the alkene precursor were found in the hemolymph at the time when pheromone titers were higher on the gland. The presence of the hydrocarbon pheromone precursor in the hemolymph is discussed in relation to possible biosynthetic pathways for producing the gypsy moth pheromone.     

Cloning and functional characterization of a fatty acid transport protein (FATP) from the pheromone gland of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone

Sex pheromones of moths are largely classified into two types based on the presence (Type I) or absence (Type II) of a terminal functional group. While Type-I sex pheromones are synthesized from common fatty acids in the pheromone gland (PG), Type-II sex pheromones are derived from hydrocarbons produced presumably in the oenocytes and transported to the PG via the hemolymph. Recently, a fatty acid transport protein (BmFATP) was identified from the PG of the silkworm Bombyx mori, which produces a Type-I sex pheromone (bombykol). BmFATP was shown to facilitate the uptake of extracellular fatty acids into PG cells for the synthesis of bombykol. To elucidate the presence and function of FATP in the PG of moths that produce Type-II sex pheromones, we explored fatp homologues expressed in the PG of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone (Type II). A fatp homologue cloned from E. japonica (Ejfatp) was predominantly expressed in the PG, and its expression is upregulated shortly after eclosion. Functional expression of EjFATP in Escherichia coli enhanced the uptake of long chain fatty acids (C₁₈ and C₂₀), but not pheromone precursor hydrocarbons. To the best of our knowledge, this is the first report of the cloning and functional characterization of a FATP in the PG of a moth producing a Type-II sex pheromone. Although EjFATP is not likely to be involved in the uptake of pheromone precursors in E. japonica, the expression pattern of Ejfatp suggests a role for EjFATP in the PG not directly linked to pheromone biosynthesis.     

Field evaluation of female sex pheromone of the artichoke moth, Gortyna xanthenes

Field responses of Gortyna xanthenes (Germar) males to traps baited with different mixtures of the female sex pheromone components were evaluated in an artichoke field. Catches were compared to those obtained by virgin females and light traps. The best results were achieved by utilizing a mixture of 5 mg Z11-16:Ald + 0.15 mg Z9-16:Ald + 0.15 mg 16:Ald + 0.12 mg Z11-16:OH, which captured only G. xanthenes males 3–5 times more than light traps and ca two times more than one virgin female. Starting from the basic mixture Z11-16:Ald (89–92%) + Z9-16:Ald (2–4%), the addition of 16:Ald (2–4%) and of Z11-16:OH (2–3%) produced an increase of G. xanthenes and a decrease of H, armigera (Hb.) catches. The inhibitory action of Z11-16:OH towards H. armigera males was confirmed.

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Evaluation sur le terrain des constituants de la phéromone sexuelle de la noctuelle de l'artichaut (Gortyna xanthenes)

Résumé Dans un champ d'artichaut de la province de Salerno on a evalué la réponse des mâles de G. xanthenes à mélanges divers des constituants de la phéromone sexuelle produite par la femelle. Les meilleurs résultats ont été obtenus en employant un mélange de 5 mg Z11-16:Ald + 0.15 mg Z9-16:Ald + 0.15 mg 16:Ald + 0.12 mg Z11-16:OH, qui a capturé seulement les mâles de G. xanthenes en mesure 3–5 fois plus élevée que les pièges lumineux et ca. deux fois plus élevée que la femelle vierge. En partant du mélange base Z11-16:Ald (89–92%) + Z9-16:Ald (2–4%), l'addition de 16:Ald (2–4%) et de Z11-16:OH (2–3%) a causé un accroissement des captures de G. xanthenes et une diminution des captures de Heliothis armigera. Cela confirme l'action inhibitoire du Z11-16:OH à l'égard des males de H. armigera.

     

Pre-exposure modulates attraction to sex pheromone in a moth

In behavioural experiments we investigated the influence of previous short exposure to sex pheromone on subsequent response of male Spodoptera littoralis moths to sex pheromone. We found that pre-exposed males showed increased sensitivity to female sex pheromone after a single exposure to a pheromone plume compared to that found in na?ve males. The increased responsiveness lasted for at least 27 h after the exposure, showing that it was not just a short-term sensitization of the males. Exposure to the odour source without upwind movement towards the source was enough to increase the responsiveness. Physical activation without exposure to odour did not affect responsiveness. The increase in responsiveness after exposure was higher when the males were pre-exposed to natural female pheromone gland extract than when they were exposed to a higher dose of the main component, even though both odour sources elicited similar upwind attraction in na?ve males. Thus, the quality of the pheromone mixture to which males were exposed influenced the subsequent response.     

Reidentification of the female sex pheromone of the Indian meal moth, Plodia interpunctella: evidence for a four-component pheromone blend

Pheromone gland extracts from calling female Plodia interpunctella contained at least seven compounds that consistently elicited electroantennographic responses from male antennae upon gas chromatographic analysis. Three of these compounds were found to be the previously identified gland constituents, i.e., (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), (Z,E)-9,12-tetradecadienal (Z9,E12-14:Ald) and (Z,E)-9,12-tetradecadienol (Z9,E12-14:OH). A fourth EAD-active compound was identified as (Z)-9-tetradecenyl acetate (Z9-14:OAc). The homologue (Z)-11-hexadecenyl acetate (Z11-16:OAc) was also identified in the extracts, but showed no EAD activity. The identity of all five compounds was confirmed by comparison of GC retention times and mass spectra with those of synthetic standards. In flight tunnel tests there were no significant differences in response of male P. interpunctella to the bait containing all four EAD-active compounds and the responses to female gland extacts. A behavioural assay of different two-compound blends in the flight tunnel showed that only addition of the corresponding aldehyde to the major pheromone component Z9,E12-14:OAc raised the male response. A subtractive assay, however, revealed that the exclusion of any of the compounds from the complete four-compound blend reduced its activity significantly. We thus conclude that the female-produced sex pheromone of P. interpunctella consists of at least four components, i.e., Z9,E12-14:OAc, Z9,E12-14:Ald, Z9,E12-14:OH and Z9-14:OAc.In a field trapping test performed in a storage facility, the four-component blend attracted significantly more males of P. interpunctella than traps baited with Z9,E12-14:OAc alone. In contrast, the highest number of Ephestia kuehniella males was found in the traps baited with this major component, suggesting that the secondary pheromone components contribute to the species specificity of the blend.     

Hormonal control of female sex pheromone biosynthesis in the redbanded leafroller moth,Argyrotaenia velutinana

Pheromone biosynthesis in female redbanded leafroller moths (RBLR) is under control of a neuropeptide produced in the brain. A bioassay consisting of isolated abdomens was developed to test the mode of action of the pheromone biosynthesis activating neuropetide (PBAN). Pheromone titer and incorporation of radiolabeled acetate into pheromone could be monitored with this bioassay. Synthetic PBAN with sequences identical to PBAN isolated from Heliothis zea and Bombyx mori were active in inducing synthesis of pheromone in RBLR. Removal of the ventral nerve cord in isolated abdomens did not inhibit the action of PBAN. Small amounts of PBAN-like activity was found in hemolymph collected from normal females but not from decapitated females. Severing the VNC in vivo in normal females did not lower the pheromone titer. These data indicate that PBAN is released into the hemolymph and then travels to its site of action. A two-fold increase in both pheromone titer and radiolabeled acetate incorporation upon incubation with PBAN was shown with isolated pheromone glands. However, the differences between control and PBAN-induced values were smaller than those obtained with the isolated abdomen culture bioassay where a seven-fold increase was observed. A decrease in pheromone titer was seen upon the in vivo removal of the corpus bursae from normal females. Removal of the corpus bursae in the isolated abdomen cultures also abolished the activity of PBAN. However, cutting the cervix bursae and leaving the corpus bursae in the abdomen culture increased both titer and radiolabeled acetate incorporation into pheromone without the presence of PBAN. An aqueous extract made from the corpus bursae of 5-day-old females was also active by itself in inducing pheromone biosynthesis in the isolated abdomen cultures. Experiments performed using newly emerged females confirmed that the corpus bursae extracts will induce pheromone biosynthesis. These results indicate that both PBAN and the corpus bursae are involved in controlling pheromone biosynthesis in RBLR.     

Polyunsaturated hydrocarbons in the hemolymph: biosynthetic precursors of epoxy pheromones of geometrid and arctiid moths

Female moths of many species in Geometridae, Arctiidae and some other macrolepidopteran families produce epoxy pheromones, which are probably derived from polyunsaturated hydrocarbons. In order to understand a biosynthetic site, hemolymph from both sexes of two geometrid species, Ascotis selenaria cretacea and Hemerophila artilineata, and one arctiid species, Spilosoma imparilis, was shaken with n-hexane and the solvent extracts were analyzed by GC-MS. Each extract of the female hemolymph sex-specifically included polyunsaturated hydrocarbons corresponding to the pheromonal epoxy components in addition to many saturated hydrocarbons, but no epoxy compounds were detected in it. Based on this analysis, deuterated polyunsaturated hydrocarbons were injected into the abdomens of two geometrid females, and the labeled epoxy components were successfully yielded from the pheromone glands. This result indicated that the polyunsaturated hydrocarbons occurring in the female hemolymph were direct pheromone precursors, which might be produced outside the pheromone gland, probably in oenocytes associated with abdominal epidermal cells or in the fat body, and transported to the pheromone gland via the hemolymph for their epoxydation and emission into the atmosphere.     

Artificial night lighting disrupts sex pheromone in a noctuid moth

1. One major, yet poorly studied, change in the environment is the increase in nocturnal light pollution. Although this strongly alters the habitat of nocturnal species, the ecological consequences are poorly known. Moths are well known to be attracted to artificial light sources, but artificial light may affect them in other ways as well. 2. In this study, female Mamestra brassicae moths were subjected to various types of low‐intensity artificial night lighting with contrasting spectral compositions (green‐rich, red‐rich, warm white) or to a dark control treatment and the effects on their sex pheromone production and composition were tested. 3. Artificial night lighting reduced sex pheromone production and altered the chemical composition of the pheromone blend, irrespective of spectral composition. Specifically, amounts of the main pheromone component Z11‐16:Ac were reduced, while the deterring compounds Z9‐14:Ac, Z9‐16:Ac, and Z11‐16:OH were increased relative to Z11‐16:Ac when females were kept under artificial light. These changes may reduce the effectiveness of the sex pheromones, becoming less attractive for males. 4. These results show for the first time that artificial light at night affects processes that are involved in moth reproduction. The potential for mitigation through manipulation of the spectral composition of artificial light appears limited.     

Control of the biosynthetic pathway of Sesamia nonagrioides sex pheromone by the pheromone biosynthesis activating neuropeptide

(Z)-11-Hexadecenyl acetate, the main pheromone component of Sesamia nonagrioides sex pheromone, is biosynthesized from palmitic acid by Delta(11)-desaturation followed by reduction and acetylation. Production of (Z)-11-hexadecenyl acetate is regulated by the Pheromone Biosynthesis Activating Neuropeptide (PBAN). Transformation of (Z)-11-hexadecen-1-ol into the corresponding acetate is a target step for PBAN in the regulation of this biosynthetic sequence, thus being the first example of a PBAN-activated acetylation. The production of the minor component (Z)-11-hexadecenal is also stimulated by PBAN. The usefulness of pentafluorobenzyloxime-derivatives for the analysis of aldehyde pheromone constituents by gas chromatography coupled to mass spectrometry is also reported.     

Study of the genetics of female sex pheromone production and male behavioral response in a moth, Ostrinia orientalis

Inheritance patterns of female sex pheromone production and male behavioral response were studied in Ostrinia orientalis. Results showed that the production of the female sex pheromone in O. orientalis was mainly controlled by a single autosomal factor, while the male behavioral response was controlled by a sex‐linked major gene.     

Analysis of odorant-binding proteins in antennae of a geometrid species, Ascotis selenaria cretacea, which produces lepidopteran Type II sex pheromone components

Information on the olfactory system in antennae of Geometridae moths is very limited, and odorant-binding proteins (OBPs) working as transporters of lipophilic odors have not been identified. In the first investigation on this family of insects, we examined antennal OBPs of the Japanese giant looper, Ascotis selenaria cretacea. RT-PCR experiments using several pairs of degenerate primers designed from known cDNA sequences encoding lepidopteran OBPs successfully amplified partial sequences of two pheromone-binding proteins (PBPs), named AscrPBP1 and AscrPBP2 in reference to their corresponding nucleotide sequence homologies with other PBPs. Using 5′- and 3′-rapid amplification of cDNA end strategies, a cDNA clone for AscrPBP1 encoding a protein of 141 amino acids was isolated. Western blotting with the antiserum against recombinant AscrPBP1 overexpressed in Escherichia coli showed that the AscrPBP1 gene was more strongly expressed in male antennae than in female antennae. Furthermore, natural AscrPBP1was isolated by immunoprecipitation with the antiserum, and its binding ability was evaluated by using synthetic sex pheromonal compounds with a C₁₉ chain. The result indicated that AscrPBP1 bound not only the pheromone components, 3,6,9-nonadecatriene and its 3,4-epoxy derivative, but also unnatural 6,7- and 9,10-epoxy derivatives. While no general odorant-binding proteins (GOBPs) were amplified in the RT-PCR experiments, two antisera prepared from GOBP1 and GOBP2 of Bombyx mori suggested the occurrence of at least two GOBPs in the A. s. cretacea antennae.     

Alkenyl sex pheromone analogs in the hemolymph of an arctiid Eilema japonica and several non-arctiid moths

The majority of moth species utilize compounds derived from de novo synthesized fatty acids as their sex pheromones (type I). In contrast, species belonging to two recently diverged moth families, Arctiidae and Geometridae, utilize alkenes and their epoxides, which are derived from dietary essential fatty acids (EFAs), as their sex pheromones (type II). In the latter species, EFAs are considered to be converted into alkenes, often after chain elongation, in specialized cells called oenocytes. These alkenes are transported through the hemolymph to the pheromone gland, from which they are secreted with or without further modifications. We confirmed that the appearance of EFA-derived alkenes in the hemolymph was closely associated with the completion of pheromone gland formation in an arctiid moth Eilema japonica. Analyses of the hemolymph of several moth species utilizing type-I sex pheromones demonstrated the occurrence of (Z,Z,Z)-3,6,9-tricosatriene (T23), a typical type-II component, in the hemolymph of a noctuid Mamestra brassicae and two crambids Ostrinia furnacalis and Ostrinia scapulalis. Our results demonstrated that moths utilizing type-I pheromones have the ability to synthesize type-II sex pheromones, and suggested that recently diverged groups of moths may have secondarily exploited EFA-derived alkenes as sex pheromones.     

Synthesis of the four components of the female sex pheromone of the painted apple moth,Teia anartoides

Four pheromone components of the female painted apple moth (Teia anartoides), an Australian insect pest, were synthesized. These were (Z)-6-henicosen-11-one (1), (6Z, 8E)-6,8-henicosadien-11-one (2), (Z)-cis-9,10-epoxy-6-henicosene (3), and (Z)-cis-9,10-epoxy-6-icosene (4). 2-Dodecanone was converted to 1 and 2, and both the enantiomers of 3 and 4 were synthesized from the enantiomers of 4-tert-butyldimethylsilyloxy-cis-2,3-epoxy-1-butanol.     

Identification of components of the female sex pheromone of the potato tuber moth, Scrobipalpopsis solanivora

The sex pheromone produced by virgin female S. solanivora moths has been shown to contain (E)-3-dodecenyl acetate with approximately 2% of the Z isomer by electroantennography and gas chromatography. In the field, traps baited with (E)-3-dodecenyl acetate alone or in combination with 1% or 2% of the Z isomer caught at least as many male S. solanivora moths as those baited with virgin female moths, and there was some evidence that addition of 5% of the Z isomer reduced catches.

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Résumé S. solanivora est l'un des plus importants lépidoptères détruisant les pommes de terre en Amérique Centrale. L'analyse par chromatographie en phase gazeuse (GC) des produits obtenus par lavage de l'ovipositeur de femelles vierges, associée à des électro-anténnogrammes (EAG) a révélé un composé actif unique en EAG. Les durées de rétention pour ce constituant correspondaient à celles des acétates monoinsaturés à 12 carbones, et les analyses sur colonne GC liées avec une phase liquide cristal ont montré un pic principal correspondant à l'acétate (E)-3-dodécénul et un pic secondaire, correspondant à l'isomère Z, et au niveau de 2,5% du pic principal. L'acétate dodécyl a aussi été décelé en quantités variables, approximativement 10% du pic principal.Dans la nature, des pièges contenant de l'acétate (E)-3-dodécényl, seul ou mélangé avec 1 ou 2% de l'isomère Z, ont capturé autant des mâles de S. solanivora que des pièges contenant des femelles vierges, et certaines indications montrent que l'addition de 5% de l'isomère Z réduit les captures.

     

Discrimination of sex pheromone blends in the olfactory system of the moth

Intracellular analysis of olfactory neurons in the internallobes of several speicies of months has revealed a psysiologisallydivorse population of projection neurons connecting the pheromone-processingcenter (the male-specific macroglormerular complex) with severalarea of the protocerebrum. Some of these output elements carryinformation about only a single pheromone in the female's complexblend, which is consistent with the ‘component hypothesis’of behavioral excitation. Other output neurons, which receivemore complex synaptic input, can distinguish the complete blendfrom the individual pheromones, thereby lending support to the‘blend hypothesis’ of behavioral excitation. Theresults suggest that even within the pheromone-processing subsystermin male insects, which is largely distinct from the rest ofthe olfactory system, there exist different but parallel linesof pheromonal information coding that ultimately govern thesteteotyped mate-seeking behaviors.