Identification of cuticular lipids eliciting interspecific courtship in the German cockroach, Blattella germanica

The cuticular surface of sexually mature females of the German cockroach contains a sex pheromone that, upon contact with the male's antennae, elicits a characteristic species-specific courtship behavior. This female-specific pheromone is a blend of several long-chain methyl ketones, alcohols and aldehydes, all derived from prominent cuticular hydrocarbons found in all life stages of this cockroach. We found that contact with the antennae of 5 out of 20 assayed cockroach species elicited courtship behavior in German cockroach males. The heterospecific courtship-eliciting compounds were isolated by behaviorally guided fractionation of the active crude extracts and compared to the native sex pheromone components. We identified two active compounds from the cuticular extract of the Oriental cockroach, Blatta orientalis -- 11-methylheptacosan-2-one and 27-oxo-11-methylheptacosan-2-one; the former compound was confirmed by synthesis and proved to independently stimulate courtship in German cockroach males. These compounds share common features with, but are distinct from, any of the known contact sex pheromone components. This suggests that sex pheromone reception in the male German cockroach is unusually promiscuous, accepting a wide range of compounds that share certain features with its native pheromone, thus resulting in a broad spectrum of behavioral response to other species. We propose that several characteristics of their mating system -- chiefly, absence of closely related species in the anthropogenic environment, resulting in relaxation of selection on sexual communication, and a highly male-biased operational sex ratio -- have driven males to respond with extremely low thresholds to a wide spectrum of related compounds.     

Sex Pheromone of the Pine Sawfly Macrodiprion nemoralis (Hymenoptera : Diprionidae): Identification of (2S,3R,7R,9S)-3,7,9-Trimethyl-2-tridecanol as the Precursor for the Active Pheromone Acetate

 The main component of the sex pheromone precursor in females of Macrodiprion nemoralis was identified as a threo-3,7,9-trimethyl-2-tridecanol isomer, approximately 800 pg per female, by gas chromatography–mass spectrometry. Comparison of mass spectrometric ion chromatograms showed that the natural compound in the female extract has the same retention time and mass spectrum as one of the two synthetic threo peaks. The acetate of the synthetic 16-isomer mixture caught a large number of males in the field, confirming the structure of the active pheromone. Comparison of gas chromatograms of the natural female extract with the eight synthetic threo stereoisomers showed that the pheromone is the (2S,3R,7R,9S)-stereoisomer of 3,7,9-trimethyl-2-tridecyl acetate. Received: 12 May 1999 / Accepted in revised form: 16 September 1999     

(<Emphasis Type="Italic">Z</Emphasis>)-9-tricosene identified in rectal gland extracts of <Emphasis Type="Italic">Bactrocera oleae</Emphasis> males: first evidence of a male-produced female attractant in olive fruit fly

It is well-known that Bactrocera oleae (olive fruit fly) females attract conspecific males by using 1,7-dioxaspiro[5,5]undecane (1) as the main component of their sex pheromone, and that 1 is produced in the female rectal gland. Although some authors have claimed that B. oleae males also attract females, to date no male-produced female attractants have been found in this species. In this paper, we report the first identification of a substance unique to males and able to attract females. The findings of the study include the following: (1) females responded in a bioassay to hexane extracts obtained from rectal glands of 15-day-old B. oleae males, (2) the presence of (Z)-9-tricosene (2) was consistently and unambiguously identified in these extracts using gas chromatography (GC) and GC-mass spectrometry methods, (3) in preliminary bioactivity tests, low doses (equivalent to a few males) of chemically and stereoisomerically pure synthetic (Z)-9-tricosene (2) attracted olive fruit fly females. Interestingly, compound 2, commonly called muscalure, is also a well-known component of the house fly (Musca domestica) sex pheromone.     

Evidence for contact sex recognition pheromone of the Asian longhorned beetle,<Emphasis Type="Italic"> Anoplophora glabripennis</Emphasis> (Coleoptera: Cerambycidae)

Field observations of the Asian longhorned beetle (Anoplophora glabripennis) mating behavior in China suggested that a female-produced contact pheromone was almost certainly involved in sex recognition. Gas chromatography–mass spectrometry (GC-MS) analysis of A. glabripennis adults' whole body cuticular extracts indicates that a series of long-chain hydrocarbons comprise the cuticular waxes of both sexes. Although for the most part the GC profiles are similar for the two sexes, five monounsaturated compounds were consistently more abundant in samples from females than in those from males. These compounds were identified as (Z)-9-tricosene, (Z)-9-pentacosene, (Z)-7-pentacosene, (Z)-9-heptacosene, and (Z)-7-heptacosene in the approximate ratio of 1:2:2:8:1, respectively. Antennal and palpi contact to a polypropylene micro-centrifuge tube coated with a synthetic mixture of the five compounds stimulated copulatory behavior in males.Electronic Supplementary Material  Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

A new class of mealybug pheromones: a hemiterpene ester in the sex pheromone of Crisicoccus matsumotoi

Mealybugs, which include several agricultural pests, are small sap feeders covered with a powdery wax. They exhibit clear sexual dimorphism; males are winged but fragile and short lived, whereas females are windless and less mobile. Thus, sex pheromones emitted by females facilitate copulation and reproduction by serving as a key navigation tool for males. Although the structures of the hitherto known mealybug pheromones vary among species, they have a common structural motif; they are carboxylic esters of monoterpene alcohols with irregular non-head-to-tail linkages. However, in the present study, we isolated from the Matsumoto mealybug, Crisicoccus matsumotoi (Siraiwa), a pheromone with a completely different structure. Using gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy, we identified the pheromone as 3-methyl-3-butenyl 5-methylhexanoate. Its attractiveness to males was confirmed in a series of field trapping experiments involving comparison between the isolated natural product and a synthetic sample. This is the first report of a hemiterpene mealybug pheromone. In addition, the acid moiety (5-methylhexanoate) appears to be rare in insect pheromones.     

Unusual pheromone chemistry in the navel orangeworm: novel sex attractants and a behavioral antagonist

Using molecular- and sensory physiology-based approaches, three novel natural products, a simple ester, and a behavioral antagonist have been identified from the pheromone gland of the navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae). In addition to the previously identified (Z,Z)-11,13-hexadecadienal, the pheromone blend is composed of (Z,Z,Z,Z,Z)-3,6,9,12,15-tricosapentaene, (Z,Z,Z,Z,Z)-3,6,9,12,15-pentacosapentaene, ethyl palmitate, ethyl-(Z,Z)-11,13-hexadecadienoate, and (Z,Z)-11,13-hexadecadien-1-yl acetate. The C₂₃ and C₂₅ pentaenes are not only novel sex pheromones, but also new natural products. In field tests, catches of A. transitella males in traps baited with the full mixture of pheromones were as high as those in traps with virgin females, whereas control and traps baited only with the previously known constituent did not capture any moths at all. The navel orangeworm sex pheromone is also an attractant for the meal moth, Pyralis farinalis L. (Pyralidae), but (Z,Z)-11,13-hexadecadien-1-yl acetate is a behavioral antagonist. The new pheromone blend may be highly effective in mating disruption and monitoring programs.     

The effects of mating status and time since mating on female sex pheromone levels in the rice leaf bug,Trigonotylus caelestialium

Although mating status affects future mating opportunities, the biochemical changes that occur in response to mating are not well understood. This study investigated the effects of mating status on the quantities of sex pheromone components found in whole-body extracts and volatile emissions of females of the rice leaf bug, Trigonotylus caelestialium. When sampled at one of four time points within a 4-day postmating period, females that had copulated with a male had greater whole-body quantities of sex pheromone components than those of virgin females sampled at the same times. The quantities of sex pheromone components emitted by virgin females over a 24-h period were initially high but then steadily decreased, whereas 24-h emissions were persistently low among mated females when measured at three time points within the 4 days after mating. As a result, soon after mating, the mated females emitted less sex pheromones than virgin females, but there were no significant differences between mated and virgin females at the end of the experiment. Thus, postmating reduction in the rate of emission of sex pheromones could explain previously observed changes in female attractiveness to male T. caelestialium.     

Attraction of two lacewing species to volatiles produced by host plants and aphid prey

It is well documented that host-related odors enable many species of parasitoids and predatory insects to locate their prey and prey habitats. This study reports the first characterization of prey and prey host odor reception in two species of lacewings, Chrysoperla carnea (Say) and Chrysopa oculata L. 2-Phenylethanol, one of the volatiles emitted from their preys host plants (alfalfa and corn) evoked a significant EAG response from antennae of C. carnea. Traps baited with this compound attracted high numbers of adult C. carnea, which were predominantly females. One of the sex pheromone components (1R,4aS,7S,7aR)-nepetalactol of an aphid species, Acyrthosiphon pisum (Harris) attracted only C. oculata adults. Single sensillum recordings showed that the olfactory neurons of C. carnea responded to both 2-phenylethanol and aphid sex pheromone components, but those of C. oculata only responded to the latter.     

No discrimination against previous mates in a sexually cannibalistic spider

In several animal species, females discriminate against previous mates in subsequent mating decisions, increasing the potential for multiple paternity. In spiders, female choice may take the form of selective sexual cannibalism, which has been shown to bias paternity in favor of particular males. If cannibalistic attacks function to restrict a male's paternity, females may have little interest to remate with males having survived such an attack. We therefore studied the possibility of female discrimination against previous mates in sexually cannibalistic Argiope bruennichi, where females almost always attack their mate at the onset of copulation. We compared mating latency and copulation duration of males having experienced a previous copulation either with the same or with a different female, but found no evidence for discrimination against previous mates. However, males copulated significantly shorter when inserting into a used, compared to a previously unused, genital pore of the female.     

The response of Trissolcus basalis to footprint contact kairomones from Nezara viridula females is mediated by leaf epicuticular waxes

Chemical footprints left behind by true bugs are perceived as contact kairomones by scelionid egg parasitoids. Female wasps encountering a contaminated artificial substrate display a characteristic arrestment posture, holding the body motionless and antennating the surface. In the system Nezara viridula (L.) and its egg parasitoid Trissolcus basalis (Wollaston), previous studies have shown that the kairomone mediating such behavior is part of N. viridula’s cuticular hydrocarbons (CHC) and furthermore that the wasp’s ability to discriminate host male and female footprints is mainly based on the presence/absence of nonadecane (nC₁₉). In this study, the effect of epicuticular waxes of leaves of broad bean, Vicia faba, on wasp responses to footprints of N. viridula females were investigated. Approximately 20% of T. basalis females displayed an arrestment posture when released on the adaxial leaf surfaces of broad bean plants with intact wax layer and without host chemical contamination; whereas ∼70% of wasps displayed the arrestment posture when intact leaves were contaminated by host female footprints. Adaxial leaf surfaces of broad bean plants dewaxed with an aqueous solution of gum arabic and afterwards contaminated by N. viridula females induced arrestment responses in about 10% of female wasps; the same percentage of arrestment (10%) was observed when the wasps were released on leaves contaminated by host females and subsequently dewaxed. The side of the polymer film that was appressed to the leaf surface, peeled from the contaminated leaves, induced an arrestment posture in about 95% of observed wasps. Scanning electron microscopy (SEM) revealed that the epicuticular waxes occurred as a film densely crystallized as irregularly shaped platelets with spherical granules randomly distributed. These findings demonstrated that epicuticular waxes of broad bean leaves can mediate the foraging behavior of T. basalis females by absorbing contact kairomones of the host.     

网湖沉积物正构烷烃分布特征及其记录的环境变化

通过对网湖沉积岩芯中正构烷烃含量和组成特征的分析,探讨了网湖近百年来的湖泊环境变化.结果表明,网湖沉积岩芯中正构烷烃的碳数范围在n-C14~n-C33之间,其中以高碳数组分为主,并具有明显的奇偶优势,反映了沉积物有机质以大型水生植物和陆生植物贡献为主,较低的2n-C31/(n-C27+n-C29)比值指示陆源输入中以木本植物输入为主.根据正构烷烃参数指示的沉积物有机质来源变化特征,近百年来网湖水体环境变化具有如下3个阶段:20世纪50年代以前,网湖与长江水体交换频繁,湖泊水体处于低营养环境状态,沉积物正构烷烃高/低分子量正构烷烃比值(H/L)和陆/水生类脂物比值(TAR)较高,沉积物有机质主要来源于陆生植物和大型水生植物,湖泊浮游藻类贡献少;20世纪50~80年代,H/L和TAR值明显下降,中、短链正构烷烃的比例略有升高,表明陆源植被对沉积物有机质的贡献降低,水生植物和浮游藻类贡献的有机质增加,但较低的2n-C17/(n-C23+n-C25)值表明浮游藻类有机质较低,此时湖泊水体较为稳定,湖泊受长江水位影响减小,湖泊营养水平有所升高;1980s以来,总体上湖泊受流域人类活动影响明显,湖泊水体营养水平升高,沉积物正构烷烃表现为H/L和TAR值升高后下降,正构烷烃总量和2n-C17/(n-C23+n-C25)比值显著升高,2000年后尤其明显,表明湖泊沉积物有机质输入增加,其中湖泊浮游藻类贡献明显增加.     

黄河河南段水体中正构烷烃的分布特征与来源解析

本研究于2010年8月采集黄河河南段26个表层水及悬浮颗粒物样品,采用气相色谱-质谱联用仪(GC-MS)测定22种正构烷烃(C_(14)~C_(36))的含量,分析其组成特征,并利用特征参数解析其来源.结果表明,黄河河南段水相中正构烷烃浓度为521~5 843 ng·L~(-1),平均浓度为1 409 ng·L~(-1),组成特征以C_(25)为主峰碳的高碳单峰型.悬浮颗粒相中正构烷烃浓度范围为463~11 142 ng·L~(-1),平均浓度为1 951 ng·L~(-1),组成特征表现为双峰型,C_(25)为主峰碳的高碳烃占优势,同时存在低碳峰.多特征参数OEP、CPI、%Wax C_n以及TAR表明,黄河河南段水相及悬浮颗粒物中正构烷烃主要来源于化石燃料的燃烧,同时存在陆生植物来源.     

Sex pheromone chemistry and field trapping studies of the elm spanworm Ennomos subsignaria (Hübner) (Lepidoptera:Geometridae)

The elm spanworm, Ennomos subsignaria (Hübner), occurs throughout Canada and the eastern United States and can be a destructive forest pest on a wide range of deciduous trees. Gas chromatography/mass spectrometry (GC/MS) and coupled gas chromatographic–electroantennographic detection (GC/EAD) analysis of pheromone gland extracts, in combination with chemical synthesis and field trapping studies have identified (2S, 3R)-2-((Z)-oct-2′-enyl)-3-nonyl oxirane (hereafter Z6-9S, 10R-epoxy-19:H) as the female-produced sex pheromone. Significantly more male moths were captured between 1–100 μg loadings of this compound on red rubber septa in sticky traps compared to blank (unbaited) traps; catches then declined at higher dosages (500–1000 μg). The other isomeric enantiomer, (2R, 3S)-2-[(Z)-oct-2′-enyl]-3-nonyl oxirane (Z6-9R, 10S-epoxy-19:H), at a 10-μg dosage did not elicit trap capture. The likely precursor to the active epoxide, (Z, Z)-6, 9-nonadecadiene ((Z, Z)-6, 9-19:H), identified in virgin female sex pheromone glands, did not elicit trap capture either, and inhibited trap capture when combined with the active epoxide. Racemic 2-((Z)-oct-2′-enyl)-3-nonyl oxirane showed no significant difference in trap capture compared with Z6-9S, 10R-epoxy-19:H, indicating that the opposite enantiomer was not antagonistic. The addition of the EAD-active diene epoxide enantiomers (2S, 3R)-2-[(Z, Z)-octa-2′, 5′-dienyl]-3-nonyl oxirane or (2R, 3S)-2-[(Z, Z)-octa-2′, 5′-dienyl]-3-nonyl oxirane in admixture with Z6-9S, 10R-epoxy-19:H (at 10% of the latter) did not enhance or decrease trap capture compared to Z6-9S, 10R-epoxy-19:H oxirane alone, so they are not likely pheromone components. This pheromone, impregnated in rubber septa at less than 100-μg dosage, can now be used as a trap bait to develop detection and monitoring strategies for this insect.     

Insecticide resistance may enhance the response to a host-plant volatile kairomone for the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis> (L.)

The behavioral and electroantennographic responses of Cydia pomonella (L.) to the ripe pear volatile ethyl (2E,4Z)-2,4-decadienoate (Et-E,Z-DD), were compared in insecticide-susceptible and -resistant populations originating from southern France. A dose–response relationship to this kairomonal attractant was established for antennal activity and did not reveal differences between susceptible and resistant strains. Conversely, males of the laboratory strains expressing metabolic [cytochrome P450-dependent mixed-function oxidases (mfo)] or physiological (kdr-type mutation of the sodium-channel gene) resistance mechanisms exhibited a significantly higher response to Et-E,Z-DD than those of the susceptible strain in a wind tunnel experiment. No response of the females to this kairomone could be obtained in our wind-tunnel conditions. In apple orchards, mfo-resistant male moths were captured at significantly higher rates in kairomone-baited traps than in traps baited with the sex pheromone of C. pomonella. Such a differential phenomenon was not verified for the kdr-resistant insects, which exhibited a similar response to both the sex pheromone and the kairomonal attractant in apple orchards. Considering the widespread distribution of metabolic resistance in European populations of C. pomonella and the enhanced behavioral response to Et-E,Z-DD in resistant moths, the development of control measures based on this kairomonal compound would be of great interest for the management of insecticide resistance in this species.     

Diel periodicity of pheromone release by females of Planococcus citri and Planococcus ficus and the temporal flight activity of their conspecific males

The diel periodicity of sex pheromone release was monitored in two mealybug species, Planococcus citri and Planococcus ficus (Hemiptera; Pseudococcidae), using sequential SPME/GCMS analysis. A maximal release of 2 ng/h pheromone by 9–12-day-old P. citri females occurred 1–2 h before the beginning of photophase. The highest release of pheromone by P. ficus females was 1–2 ng/2 h of 10–20-day-old females, approximately 2 h after the beginning of photophase. Mating resulted in termination of the pheromone release in both mealybug species. The temporal flight activity of the males was monitored in rearing chambers using pheromone baited delta traps. Males of both P. citri and P. ficus displayed the same flight pattern and began flying at 06:00 hours when the light was turned on, reaching a peak during the first and second hour of the photophase. Our results suggest that other biparental mealybug species display also diel periodicities of maximal pheromone release and response. Direct evaluation of the diel periodicity of the pheromone release by the automatic sequential analysis is convenient and will be very helpful in optimizing the airborne collection and identification of other unknown mealybug pheromones and to study the calling behavior of females. Considering this behavior pattern may help to develop more effective pheromone-based management strategies against mealybugs.     

Territorial meadow pipit males (<Emphasis Type="Italic">Anthus pratensis</Emphasis>; Passeriformes) become more aggressive in female presence

Although mate guarding as prevention of extra-pair copulation is common among birds, evidence for aggressive behaviour involving physical contact related to mate guarding in passerines is scarce and cases of the presence of one partner directly influencing the aggressiveness of the other are lacking. We investigated the intra-specific territorial behaviour of male meadow pipits (Anthus pratensis; Passeriformes: Motacillidae) at the beginning of the breeding season by placing a pipit model accompanied by an intra-specific song playback in the territory of socially paired males and compared the responses of males whose mates were physically present during trials with those whose females were out of sight. The level of aggression of males was significantly higher in the presence of the female; half of the males in this group physically attacked the model (the most intense and risky aggressive behaviour). Physical attacks did not occur among males whose female was absent during the trial; response to the playback by most of these males was only weak. This pattern may be related to the prevention of extra-pair copulation; if the risks involved in the conflict are outweighed by potential loss of paternity, such aggressive mate guarding may pay off. The apparently overlooked effect on the territorial behaviour of a partner’s passive physical presence during conflict should be further evaluated because it may be important for the design and interpretation of results of behavioural experiments.     

Sexual behavior,cannibalism, and mating plugs as sticky traps in the orb weaver spider <Emphasis Type="BoldItalic">Leucauge argyra</Emphasis> (Tetragnathidae)

Unpublished field observations in Leucauge argyra, a tropical orb weaver spider, suggest the occurrence of conspicuous mating plugs that could reduce or prevent remating attempts. Otherwise, the sexual behavior of this species remains unknown. The aims of this study were to describe the courtship behavior and copulation in L. argyra and investigate mating plug formation in this species. Fourteen virgin females and 12 plugged females were exposed to up to three males and checked for mating plug formation. Of the 12 virgins that copulated, nine produced plugs (five immediately after copulation), and the five plugged females that copulated produced another mating plug immediately after copulation. We did not detect the transfer of any male substance during copulation but observed a whitish liquid emerging from female genital ducts. Plug formation was positively associated with male twanging during courtship. One virgin and four plugged females cannibalized males. In seven trials with virgins and in three trials with plugged females, the male’s palp adhered to a substance that emerged from female genital ducts and spread on her genital plate. The male had to struggle energetically to free his glued palp; two of these males were cannibalized while trying to release their palps. Females seem to determine copulation duration by altering the timing of mating plug formation and through sexual cannibalism. This is the first case reported of a mating plug as a sticky trap for males.     

Variation in sex pheromone emission does not reflect immunocompetence but affects attractiveness of male burying beetles—a combination of laboratory and field experiments

Life history theory predicts a trade-off between male sexual trait expression and immunocompetence. Using burying beetles, Nicrophorus vespilloides, as a model, we investigated the relationship between male immune function, sex pheromone emission, and attractiveness under field conditions. In the first experiment, we tested whether there is a positive correlation between immune capacity, sex pheromone characteristics (quantity, relative composition, and time invested in pheromone emission), and male attractiveness. As a measurement of immune capacity, we used an individual’s encapsulation ability against a novel antigen. In the second experiment, we specifically examined whether a trade-off between chemical trait expression and immune function existed. To this end, we challenged the immune system and measured the subsequent investment in sex pheromone emission and the attractiveness of the male under field conditions. We found that a male’s immunocompetence was neither related to the emission of the male’s sex pheromone nor to its attractiveness in the field. Furthermore, none of the immune-challenge treatments affected the subsequent investment in pheromone emission or number of females attracted. However, we showed that the same males that emitted a high quantity of their sex pheromone in the laboratory were able to attract more females in the field. Our data suggest that the chemical signal is not a reliable predictor of a male’s immunocompetence but rather is a general important fitness-related trait, with a higher emission of the sex pheromone measured in the laboratory directly affecting the attractiveness of a male under field conditions.     

Reversed sex change by widowed males in polygynous and protogynous fishes: female removal experiments in the field

Sex change, either protogyny (female to male) or protandry (male to female), is well known among fishes, but evidence of bidirectional sex change or reversed sex change in natural populations is still very limited. This is the first report on female removal experiments for polygnous and protogynous fish species to induce reversed sex change in the widowed males in the field. We removed all of the females and juveniles from the territories of dominant males in the cleaner wrasse Labroides dimidiatus (Labridae) and the rusty angelfish Centropyge ferrugata (Pomacanthidae) on the coral reefs of Okinawa. In both species, if new females or juveniles did not immigrate into the territories of the widowed males, some of them emigrated to form male–male pairs. When a male–male pair formed, the smaller, subordinate partner began to perform female sexual behaviours (n = 4 in L. dimidiatus; n = 2 in C. ferrugata) and, finally, released eggs (n = 1, respectively). Thus, the reversed sex change occurred in the widowed males according to the change of their social status. These results suggest that such female removal experiments will contribute to the discovery of reversed sex change in the field also in other polygnous and protogynous species.     

Female sex pheromone and male behavioral responses of the bombycid moth <Emphasis Type="Italic">Trilocha varians</Emphasis>: comparison with those of the domesticated silkmoth <Emphasis Type="Italic">Bombyx mori</Emphasis>

Analysis of female sex pheromone components and subsequent field trap experiments demonstrated that the bombycid moth Trilocha varians uses a mixture of (E,Z)-10,12-hexadecadienal (bombykal) and (E,Z)-10,12-hexadecadienyl acetate (bombykyl acetate) as a sex pheromone. Both of these components are derivatives of (E,Z)-10,12-hexadecadienol (bombykol), the sex pheromone of the domesticated silkmoth Bombyx mori. This finding prompted us to compare the antennal and behavioral responses of T. varians and B. mori to bombykol, bombykal, and bombykyl acetate in detail. The antennae of T. varians males responded to bombykal and bombykyl acetate but not to bombykol, and males were attracted only when lures contained both bombykal and bombykyl acetate. In contrast, the antennae of B. mori males responded to all the three components. Behavioral analysis showed that B. mori males responded to neither bombykal nor bombykyl acetate. Meanwhile, the wing fluttering response of B. mori males to bombykol was strongly inhibited by bombykal and bombykyl acetate, thereby indicating that bombykal and bombykyl acetate act as behavioral antagonists for B. mori males. T. varians would serve as a reference species for B. mori in future investigations into the molecular mechanisms underlying the evolution of sex pheromone communication systems in bombycid moths.