Courtship Behavior in Relation to the Female Sex Pheromone in the Parasitoid, <Emphasis Type="Italic">Aphidius ervi</Emphasis> (Hymenoptera: Braconidae)

Mating in the aphid parasitoid, Aphidius ervi, is mediated by sex pheromones. Virgin females produce pheromones that stimulate both upwind flight and elicit close-range courtship behavior by males. Field studies and laboratory bioassays demonstrated that time of day and adult age affect both the emission of, and receptivity to, the sex pheromones. In contrast, mating affected female pheromone production, but not male responsiveness.     

Male-produced aggregation pheromone of the maize weevil,Sitophilus zeamais,and interspecific attraction between threeSitophilus species

A dual-choice pitfall bioassay was used to demonstrate the existence of a male-produced aggregation pheromone in the maize weevil. Both males and females showed a highly significant preference for extracts of disks exposed to wheat-feeding males over extracts of disks exposed to wheat kernels only. Neither sex responded significantly to extracts from females. Mating did not reduce pheromone release by males. Response by virgin females to pheromone was significantly higher than response by mated females, but males of either mating status responded equally well. There was no apparent daily periodicity in the responsiveness to pheromone. Rice and maize weevils showed a strong interspecific cross-attraction. Granary weevils of both sexes responded well to maize weevil extracts, but only females showed a significant response to rice weevil extracts. Neither maize nor rice weevils responded significantly to granary weevil extracts.     

Pheromone-mediated mating in the aphid parasitoid,Aphidius nigripes (hymenoptera: Aphididae)

Mating in the aphid parasitoid,Aphidius nigripes, is mediated by a female sex pheromone that stimulates both upwind flight and courtship behavior by males. There is no evidence of any overt behavior associated with the release of the pheromone, but bioassay results, using female extracts, suggest that the pheromone is produced in the abdomen, although it may be actively released from the entire body surface. Age does not appear to have a major influence on either pheromone production by the female or on male responsiveness. However, field data suggest the presence of a strong diel periodicity in the emission of and/or receptivity to the pheromone. Laboratory bioassay results indicated diel changes in the male response but more refined techniques are needed to determine if such periodicity exists in pheromone production by females. Following mating, female pheromone production is significantly reduced. The potential use of the parasitoid sex pheromone in integrated management of aphids is discussed.     

Effects of Age and Mating on Female Sex Attractant Pheromone Levels in the Sorghum Plant Bug, <Emphasis Type="Italic">Stenotus rubrovittatus</Emphasis> (Matsumura)

Previous work has shown that male sorghum plant bugs, Stenotus rubrovittatus (Matsumura) (Heteroptera: Miridae), are more attracted to young virgin females than to old virgin females and are not attracted to mated females. Therefore, we examined the effects of age and mating status on female sex pheromone levels. The pheromone components, hexyl butyrate, (E)-hex-2-en-1-yl butyrate, and (E)-4-oxohex-2-enal, were collected from females in two different ways. First, the compounds were extracted from whole bodies of each female and quantified by gas chromatography-mass spectrometry (GC-MS). Pheromone levels extracted from virgin female bodies decreased with age. In females that had just mated, pheromone levels did not differ from those of virgin females, and pheromone levels in mated females remained relatively constant up to 15 d after mating. This absence of change in pheromone levels extracted from whole bodies of mated females is not congruent with previous reports of lack of male attraction to these females. In a second method for pheromone recovery (adsorption on a PDMS-coated stir bar), GC-MS analysis showed that mated females released lower amounts of pheromone components compared to those emitted by virgin females of the same age (4 d). As was the case with whole body extracts, young virgin females (3 d) released higher amounts of pheromone components than did old virgin females (8 d). The results suggest that male response in S. rubrovittatus is dictated more by the quantities of pheromone components released into the volatile headspace by females than by the quantities present in the body.     

First Contact Pheromone Identified for a Longhorned Beetle (Coleoptera: Cerambycidae) in the Subfamily Prioninae

Little is known of the reproductive behavior of longhorned beetles (Coleoptera: Cerambycidae) in the subfamily Prioninae. Mallodon dasystomus (Say), the hardwood stump borer, is a widely distributed prionine that is native to the southern U.S. Here, we explored the chemically-mediated mating behavior of M. dasystomus, and tested the hypothesis that males recognize females by a contact pheromone. In mating bioassays, all males tested attempted to mate with females only after contacting females with their antennae. Moreover, all males attempted to mate with solvent-washed dead females treated with as little as 0.15 ± 0.03 female equivalents of conspecific cuticular extracts, confirming that compounds on the cuticle of females are essential for mate recognition. Cuticular hydrocarbon profiles of females contained 13 compounds that were not present in profiles of males. Among the female-specific compounds, two co-dominant methyl-branched alkanes, 2-methylhexacosane (2Me-C₂₆) and 2-methyloctacosane (2Me-C₂₈), accounted for 17% of the total hydrocarbons. Our strategy for identifying the contact pheromone was to synthesize and test the bioactivity of female specific compounds, starting with the most abundant. In bioassays, males displayed mating behavior in response to synthetic 2Me-C₂₆ and 2Me-C₂₈ when tested individually. Furthermore, when these compounds were tested in combination, they elicited the full progression of mating behaviors, suggesting that 2Me-C₂₆ and 2Me-C₂₈ make up the contact pheromone. These findings are further evidence of the critical role of contact pheromones in mating systems of longhorned beetles.     

In the Nick of Time: Males of the Parasitoid Wasp <Emphasis Type="Italic">Pimpla disparis</Emphasis> Respond to Semiochemicals from Emerging Mates

Males of the parasitoid wasp Pimpla disparis Viereck (Hymenoptera: Ichneumonidae) aggregate on parasitized gypsy moth, Lymantria dispar, host pupae when the emergence of a prospective mate is imminent or under way. We tested the hypotheses that the developing parasitoid (“DePa”) inside the host pupal case produces a pheromone that attracts and arrests mate-seeking males, and that the pheromone is most effective during the emergence of the parasitoid from the host. Results obtained in two-choice laboratory experiments, with 4–7-d-old virgin males, indicate that (1) DePa-derived semiochemicals arrest males, (2) the opening of a host pupal case strongly arrests males, and (3) the arrestment cue emanates from oral fluid secreted by both female and male parasitoids while they chew their way out of a host pupal case. This phenomenon implies that emerging females, which are haplodiploid and can reproduce without mating, do not engage in active pheromone signaling to attract males, and that mate-seeking males co-opt chemicals involved in eclosion as a mate-finding cue, taking a 50% chance that the prospective mate is a female.     

Particular Levels of Odors Released by Virgin Females Attract Conspecific Males of the Funnel-Web Spider <Emphasis Type="Italic">Allagelena difficilis</Emphasis>

Female-released chemical signals are crucial clues for mate-searching males to locate and gain sexual receptivity of conspecific females. Abundant behavioral evidence indicates that female spiders release sex pheromones to guide mate-searching behavior of conspecific mature males. However, the chemical nature of spider pheromones is poorly understood. Females of the funnel-web spider, Allagelena difficilis, employ sit-and-wait tactics for mating. Field observations indicate that males leave their retreats to search for potential mates during the breeding season. Therefore, we investigated whether virgin females release a sex attractant to conspecific males and then explored the chemical nature of the female pheromone. Four fatty acids extracted from the female bodies (palmitic acid, linoleic acid, cis-vaccenic acid and stearic acid) constitute a multiple-component sex attractant to conspecific males in A. difficilis. Unexpectedly, mated females also produce the same fatty acids, but at trace levels. Two-choice experiments showed that males were significantly attracted by the blend of the four fatty acids in appropriate concentrations while avoiding the blend consisting of the same acids at very low concentrations, suggesting that mate-searching males are able to discriminate virgin females from mated females by the quantities of female-specific fatty acids in the funnel-web spider A. difficilis.     

Sexual communication and associative learning in the parasitic waspItoplectis conquisitor (Say)

Extracts of newly emerged virgin females and newly emerged virgin males of the parasitic waspItoplectis conquisitor were highly active in eliciting sexual behavior in older males. Newly emerged males did not respond to the extracts. A reliable biological assay was devised in which the responses of males to extract could be evaluated under varying conditions. Optimum male responses to female extract were obtained at 27 °C and at 1.0 female equivalents (the amount of pheromone extractable from the body surface of a female). A decrease in sexual response was observed when males were repeatedly exposed to pheromone. Males also learned to associate bioassay conditions with the presence of pheromone.     

Airborne Chemical Communication in the Wolf Spider Pardosa milvina

Most studies involving chemical communication in spiders focus on contact pheromones attached to spider silk. Here we tested if males of the wolf spider Pardosa milvina use airborne pheromones to identify, locate, and follow females. Using a two-choice olfactometer, we tested the response of adult male P. milvina to a number of potential chemical cues while controlling for concomitant visual and vibratory stimuli. An airborne chemical cue from adult virgin female P. milvina elicited a positive taxis response from the male. We also tested adult male responses to penultimate instar female P. milvina, one adult male P. milvina, and two adult males together. In each case, test males showed no attraction to the stimuli. Additional experiments were run with pitfall traps baited with adult virgin female P. milvina as attractants. Again, we controlled for visual and vibratory cues from females. Pitfall traps containing virgin females captured significantly more males than control traps. Collectively, these experiments demonstrate evidence of an airborne sex pheromone in P. milvina.     

Risk of Egg Parasitoid Attraction Depends on Anti-aphrodisiac Titre in the Large Cabbage White Butterfly <Emphasis Type="Italic">Pieris brassicae</Emphasis>

Males of a variety of insects transfer an anti-aphrodisiac pheromone to females during mating that renders them less attractive to conspecific males. In cabbage white butterflies, the transfer of an anti-aphrodisiac can result in the unwanted attraction of tiny egg parasitoid wasps of the genus Trichogramma that hitch-hike with mated female butterflies to a host plant where they parasitize the freshly laid butterfly eggs. Here, we show that the anti-aphrodisiac benzyl cyanide (BC) of the large cabbage white Pieris brassicae is depleted by frequent display of the mate-refusal posture that signals a female’s unreceptivity to mating. This depletion of BC is ecologically important because it results in a reduced risk of attracting the hitch-hiking egg parasitoid Trichogramma brassicae to mated female butterflies over time since mating. Our results indicate for the first time that a reduction in anti-aphrodisiac titre in mated females due to frequent adoption of the mate-refusal posture is beneficial to both mated females and males particularly when parasitoid pressure is high.     

Age and Mating Status Do Not Affect Transcript Levels of Odorant Receptor Genes in Male Antennae of Heliothis virescens and Heliothis subflexa

In biological systems, it is expected that gene expression levels generally will correlate with temporally varying physiological and biological needs, and that gene expression levels could regulate biological capabilities. In moth species, male response to female sex pheromones often is affected by moth age and mating status. Odorant receptors (ORs) expressed in neurons within male antennae are critical for detecting the female pheromones. Therefore, we hypothesized that the expression level of these receptor proteins would be affected by age and mating status of male moths. We examined expression levels of two OR genes that are preferentially expressed in the male antennae of Heliothis virescens (HvOR13 and HvOR15) and Heliothis subflexa (HsOR13 and HsOR15). Antennae were dissected from virgin males at 2 h, 1 d, 2 d, 4 d, and 8 d. We also dissected antennae from 4-d-old mated males. We found that age had no effect on expression levels of either OR in either species, except for a small difference in HsOR15 expression between 2 h and 8-d-old virgin males. Furthermore, we found no effect of mating status on expression level of these ORs in either species. We discuss these findings in relationship to studies of age and mating status effects on male electrophysiological and behavioral response to female pheromones, and contrast our results to studies on the effects of age and mating status on gene expression of pheromone receptor proteins and pheromone binding proteins in other moths.     

Multiple Roles of a Male-Specific Compound in the Sexual Behavior of the Dried Bean Beetle, <Emphasis Type="Italic">Acanthoscelides Obtectus</Emphasis>

Males of Acanthoscelides obtectus (Coleoptera: Chrysomelidae, Bruchinae) emit methyl (E,R)-2,4,5-tetradecatrienoate that attracts females for mating. This study identified further roles for this compound in the sexual behavior of A. obtectus. Earlier observations revealed that males touched females with their antennae while tandem-running with them and initiated mounting and copulation, whereas they showed no such behavior toward other males. A series of subsequent laboratory choice tests were set up to establish if certain cuticular compounds aid contact sex recognition in A. obtectus. Males chose virgin females over other males. The activity toward females could be eliminated by rinsing with hexane, but was regained by application of female extract onto previously rinsed females. Gas chromatographic (GC) comparison of hexane extracts revealed the presence of two male-specific compounds, methyl (E,R)-2,4,5-tetradecatrienoate and octadecanal, which were absent from the behaviorally active female samples. Of the two compounds, methyl (E,R)-2,4,5-tetradecatrienoate was found to be responsible for the inhibition of male sexual behavior, similar to that observed with crude male extracts applied to virgin females. Furthermore, males preferred virgin over mated females. GC analyses revealed the presence of methyl (E,R)-2,4,5-tetradecatrienoate in mated females in amounts sufficient to curtail mating attempts. It appears that methyl (E,R)-2,4,5-tetradecatrienoate, besides being a male-produced sex pheromone, acts as a male-recognition signal in A. obtectus. Males also transfer it onto females during mating, resulting in mated females being avoided by courting males.     

An Oral Male Courtship Pheromone Terminates the Response of Nasonia vitripennis Females to the Male-Produced Sex Attractant

Sex pheromones are crucial for mate finding in many animals. Long-range attraction, mate recognition, and the elicitation of sexual receptiveness during courtship are typically mediated by different compounds. It is widely unknown, however, how the different components of a species’ pheromone system influence each other. Here, we demonstrated in the parasitoid wasp Nasonia vitripennis that females quickly cease to respond to the male sex attractant after they contact a male’s oral secretion during courtship. We used this behavioral switch to monitor the fractionation of head extracts from male wasps for identification of the bioactive compounds as a blend of ethyl oleate, ethyl linoleate, and ethyl α-linolenate. This is the first identification of a cephalic courtship pheromone in parasitic Hymenoptera. Plasticity in pheromone-mediated sexual behavior of female insects has hitherto been attributed to the transfer of bioactive proteinaceous molecules with the male ejaculate. The pheromone interaction reported here sheds new light on the sexual communication of insects by showing that the sex pheromone response of females can be terminated by males independent of sperm transfer.     

Primer and Short-Range Releaser Pheromone Properties of Premolt Female Urine from the Shore Crab <Emphasis Type="Italic">Carcinus maenas</Emphasis>

The European shore crab Carcinus maenas is considered to rely on a female pheromone when mating. Evidence, however, is scarce on how the urine pheromone in itself affects males. We investigated male primer and releaser responses to female pheromones with methods that minimized effects from females, delivering female urine either as a pump-generated plume or deposited on a polyurethane sponge. We delivered the pheromone at different concentrations in far, near, and close/contact range to get a picture of how distance affects behavioral response. Our results show that substances in premolt female urine (PMU) function as primer and potent short-range releaser pheromones. Based on the olfactometer and sponge tests, we conclude that PMU stimulus in itself is sufficient to elicit increased search and mating-specific behaviors such as posing, posing search, cradle carrying, and stroking. Pheromone concentrations do not seem to be important for attenuating search and posing as long as the level is above a certain threshold concentration. Instead, pheromone levels seem to play a role in male acceptance of females, recruiting more males to respond, and generating better responses with increasing concentration.     

High Chemical Diversity in a Wasp Pheromone: a Blend of Methyl 6-Methylsalicylate,Fatty Alcohol Acetates and Cuticular Hydrocarbons Releases Courtship Behavior in the Drosophila Parasitoid Asobara tabida

Wasps of genus Asobara, a larval parasitoid of Drosophila, have become model organisms for the study of host-parasite interactions. However, little is known about the role of pheromones in locating mates and courtship behavior in this genus. In the present study, we aimed to identify the female courtship pheromone in Asobara tabida. The chemical compositions of solvent extracts from male and female wasps were analyzed by GC/MS. These extracts, fractions thereof, and synthetic pheromone candidates were tested for their activity in behavioral bioassays. The results demonstrate that the courtship pheromone of A. tabida is characterized by a remarkable chemical diversity. A multi-component blend of female-specific compounds including methyl 6-methylsalicylate (M6M), fatty alcohol acetates (FAAs), and cuticular hydrocarbons (CHCs) released male courtship behavior. Using a combinatory approach that included both purified natural products and synthetic analogs, it was shown that none of the three chemical classes alone was sufficient to release a full behavioral response in males. However, a blend of M6M and FAAs or combinations of one or both of these with female-derived CHCs resulted in wing-fanning responses by males comparable to those elicited by the crude extract of females. Thus, components from all three chemical classes contribute to the bioactivity of the pheromone, but none of the elements plays a key role or is irreplaceable. The fact that one of the FAAs, vaccenyl acetate, is also used as a kairomone by Asobara females to locate Drosophila hosts suggests that a pre-existing sensory responsiveness to vaccenyl acetate might have been involved in the evolution of the female sex pheromone in Asobara.     

Female Sex Pheromone in Trails of the Minute Pirate Bug, <Emphasis Type="Italic">Orius minutus</Emphasis> (L)

Orius minutus (L.) (Heteroptera: Anthocoridae) is a natural enemy of agricultural pests such as thrips, aphids, and various newly hatched insect juveniles. In this study, we conducted 1) behavioral assays for evidence of contact sex pheromone activity in trails of O. minutus, and 2) chemical analysis to identify the essential chemical components of the trails. Males showed arrestment to trails of mature virgin females but not to trails from either conspecific nymphs or immature females. Females also showed arrestment to trails from conspecific males, although the response was weaker than that exhibited by males. The activity of female trails lasted for at least 46 h after deposition. Males showed a response irrespective of mating experience. Following confirmation that a contact sex pheromone was present in the trails of female O. minutus, we used a bioassay-driven approach to isolate the active chemicals. After fractionation on silica gel, the n-hexane fraction was found to be biologically active to males. A major compound in the active fraction was (Z)-9-nonacosene; this compound was found only in trail extracts of mature virgin females. Synthetic (Z)-9-nonacosene arrested O. minutus males, indicating that it is the major active component of the contact sex pheromone in the trails of female O. minutus.     

Sexual behavior ofMatsucoccus josephi (Homoptera: Margarodidae)

The daily emergence patterns ofMatsucoccus josephi adults and third-instar male larvae raised on artificially infested saplings ofPinus halepensis were determined. A single peak of emergence was found for adult males between 0300 and 0500 hr. Adult females emerged throughout the day, with maximum emergence between 0500 and 0700 hr. Two daily emergence periods were observed in third-instar male larvae, one between 0300 and 0900 hr and the other between 1700 and 2100 hr. Airborne pheromone emitted by adult virgin females was collected using a flow system. No significant differences were recorded in the attraction of the male to crude pheromone collected at different times of day. A single female, virgin or half an hour after mating, was sufficient to attract the males in an olfactometer system. From minor differences in pheromone release throughout the day, and from the lesser degree of attraction by females half an hour after mating, it is assumed that there is no mechanism controlling the daily release of the female sex pheromone. Reduction of pheromone emission after mating is suggested.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 2743-E, 1989 series.     

Pheromone Communication in <Emphasis Type="Italic">Nasonia vitripennis</Emphasis>: Abdominal Sex Attractant Mediates Site Fidelity of Releasing Males

Males of the parasitic wasp Nasonia vitripennis (Hymenoptera: Pteromalidae) use a substrate-borne sex pheromone to attract virgin females. The pheromone is synthesized in the rectal vesicle and deposited via the anus by dabbing movements of the abdominal tip. The chemicals attracting the females are composed of a mixture (4R,5R- and (4R,5 S)-5-hydroxy-4-decanolides (HDL) being synergized by the trace component 4-methylquinazoline (4-MeQ) which is not attractive for females when offered alone. Here we show that male pheromone deposits are not only attractive to virgin females but also for the releasing males themselves. In an olfactometer bioassay, males were strongly attracted by their own pheromone markings but were unable to discriminate between their own markings and those deposited by other males. Polar fractions of pheromone gland extracts containing the HDLs and 4-MeQ were also highly attractive for males. Bioassays using synthetic pheromones in natural doses revealed that combinations of HDL/4-MeQ and 4-MeQ alone attracted males whereas the HDLs alone were behaviorally inactive. Furthermore, males did not discriminate between HDL/4-MeQ and 4-MeQ alone. We conclude that the trace component 4-MeQ mediates site fidelity of N. vitripennis males at sites previously marked with the abdominal sex pheromone. The use of 4-MeQ to stay at and to return to scent-marked patches rather than marking new ones might be a strategy to economize semiochemical use in N. vitripennis males.     

Identification of a Sex Pheromone From Male Yellow MealWorm Beetles, <Emphasis Type="Italic">Tenebrio molitor</Emphasis>

The sex pheromone released by the adult female Tenebrio molitor, 4-methyl-1-nonanol, is well known. In addition, there is evidence that adult males release a pheromone that attracts females. The purpose of the present study was to isolate and identify male-released pheromone(s). Emissions from virgin adult males and females were collected on filter paper and extracted with pentane. Extracts were analyzed by gas chromatography-mass spectrometry. One male-specific compound was detected and identified as (Z)-3-dodecenyl acetate (Z3-12:Ac). In arena bioassays, E3-12:Ac was attractive to females only, at 1 and 10 μg doses. E3-12:Ac was also attractive to females at a 10-μg dose. The presence of both male and female pheromones, each attracting the opposite sex, may contribute to maintaining a high-density population of both sexes.     

Evidence for a sex pheromone in the hide beetle,Dermestes maculatus (De Geer) (Coleoptera: Dermestidae)

Adults of the hide beetle,Dermestes maculatus (De Geer), were shown to have a female-produced sex pheromone which excited males. Male response was positively correlated with increasing age, but females produced a higher level of pheromone at 6–8 days of age than at younger or older ages. Extractable female pheromone and male response varied over the photophase with peak values that occurred during the latter hours. Quantitative bioassay indicated that the 50% male response level (RD₅₀) would be elicited by a pheromone exposure of 0.01 female equivalents (FE). Females extracted 24 hr after mating had a lower level of extractable pheromone than did virgin females of the same age.Mention of a proprietary product does not constitute an endorsement by the Minnesota Agricultural Experiment Station.