Pheromone-mediated copulatory responses of the screwworm fly,Cochliomyia hominivorax

A bioassay based on male copulatory responses occurring on contact with dead decoy insects was used to confirm the existence of a sex pheromone in the screwworm fly,Cochliomyia hominivorax. Males responded to female but not male decoys. Mated and virgin females were equally stimulatory. Activity was abolished when females were washed with hexane but partially restored by treatment with crude hexane extract of females. Responses decreased when extracts were diluted and when the number of females extracted per milliliter of hexane was decreased from 20 to 1 in the preparation of extracts concentrated to 0.4 female/l. Sexually mature female decoys of the 009 strain, the most laboratory-adapted of three strains examined in intrastrain tests, produced few copulatory attempts compared with those of Aricruz or DE-9 strains. However, newly emerged 009 as well as Aricruz females elicited responses from about 80% of sexually mature males. Those of the DE-9 strain stimulated fewer than 1%. The observation that 009 females were maximally stimulatory before becoming receptive to mating suggests that these strain differences resulted from laboratory colonization.     

Evidence for a short-range sex pheromone in female Maladera matrida beetle

Laboratory studies with live and frozen Maladera matrida female and male beetles showed that males were attracted to chemical substances emanating from the females at dusk. Beetles exhibited sexual activity (including mating) at dusk towards frozen females but not towards frozen males. Frozen females that had been extracted with methanol together with either hexane or dichloromethane or with a mixture of all three solvents did not elicit male sexual activity. Activity was fully restored when a concentrate of the extract was applied to the previously extracted frozen females. Males also responded with vigorous sexual activity to frozen males to which female extract had been applied. Deterrent chemicals appear to be absent from the male body. Males exposed to females that had been frozen during the morning displayed weak sexual activity, indicating that females lack active semiochemicals. Differences between dusk and morning extracts were found with respect to more than 20 compounds, some of which were present in much higher concentrations at dusk than in the morning, while others were not detected in the morning extract. The active component(s) of the short-range sex pheromone of female M. matrida is (are) presumably to be found among these compounds.     

Characterization of a Female-Produced Courtship Pheromone in the Parasitoid Nasonia vitripennis

Males of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) show a characteristic courtship behavior. We demonstrate that male arrestment and key behavioral elements of the courtship sequence are mediated by a female-derived contact sex pheromone. Males were arrested on paper disks treated with female extracts but not on those treated with male extracts. Male responsiveness was influenced by the surface to which female extracts were applied. Extracts applied to an extracted beetle elytron arrested males more strongly than those applied to filter paper of comparable size. However, more complex behavioral elements, such as head nodding and copulation attempts, were shown only when extracts were applied to extracted male cadavers, suggesting that tactile or visual cues synergize the male response. The chemicals involved are stable, of low volatility, and nonpolar. Dead females arrested males and elicited courtship behavior for at least 8 d. Males showed no sign of attraction to live females at a distance of 3 cm in an olfactometer. Fractionation of female extracts demonstrated that the activity was exclusively located in the nonpolar fraction. Analysis of the active fraction by gas chromatography–mass spectrometry revealed that cuticular hydrocarbons with chain lengths between 25 and 37 carbon units were present. Comparison of hydrocarbon profiles from males and females showed qualitative and quantitative differences. These results suggest that sex-specific cuticular hydrocarbons are the key signals mediating the male courtship behavior in N. vitripennis.     

Evidence for a Substrate-Borne Sex Pheromone in the Parasitoid Wasp Trichogramma brassicae

While the use of chemical cues in host location is well known in parasitoid insects, little is known about their role in sexual communication. Sex pheromones were investigated in the egg parasitoid Trichogramma brassicae, and the evidence for a substrate-borne pheromone was demonstrated. Mated and unmated males concentrated their walking in an area where virgin females (<1 or 5 days old) had walked. Males were tested 15 min after females left the area. Male response decreased over time but was always significant during the 8 min of tracking. This suggests that females deposit chemicals on the substrate for mate finding. The pheromone is soluble in hexane, as shown by two kinds of extracts: female body rinsing and rinsing of the flask where females had walked. Hexane extracts resulted in a positive response by males. The response to different doses showed a saturation above a threshold of 4–8 female equivalents. The results do not exclude the possibility that a volatile part of the pheromone attracts males over long distances. Substrate-borne pheromones may increase mating chances for virgin females searching for oviposition sites, and may also increase mating chances on emergence sites by reducing male dispersion.     

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.     

Evidence for occurrence of mounting sex pheromone on body surface of femaleDermacentor variabilis (Say) AndDermacentor andersoni (Stiles) (Acari: Ixodidae)

MaleDermacentor variabilis andD. andersoni respond to an unknown chemical or chemicals present on the body surfaces of partially engorged conspecific female ticks. Following contact, the males mount the females and apply their mouthparts and legs against the female dorsal body surface. Then, the males turn with these appendages still in close contact and crawl to the female's venter, whereupon they locate the gonopore, probe the vulva, and copulate. Similar responses are elicited by heterospecific as well as conspecific females. However, the response is lost when the female cuticle is cleaned (delipidized) with organic solvents. It can be restored by applying hexane extracts prepared from female cuticle to the previously cleaned females. Males do not use surface texture as the primary stimulus for mate recognition. Male ticks also respond to hexane extracts applied to spherical inanimate objects, (dummy female), suggesting that a chemical or chemicals soluble in organic solvents has been transferred to these objects. These findings suggest the existence of a previously undescribed pheromone, the mounting sex pheromone (MSP). This contact sex pheromone enables males excited and attracted by 2,6-dichlorophenol to identify the female as a potential mating partner. The MSP is the second in the series of three sex pheromones guiding the hierarchy of behavioral responses which constitute tick courtship behavior.Supported by grant AI 10,986 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service, Department of Health and Human Services, Bethesda, Maryland 20205.     

Sex attraction in paper wasp,Polistes exclamans viereck (Hymenoptera: Vespidae), in a wind tunnel

A wind-tunnel bioassay was developed to test for the presence of sex attractants in the paper wasp,Polistes exclamans Viereck (Hymenoptera: Vespidae). Males exhibited significant upwind flight and attraction (chemoanemotaxis) in response to airflow passed over unmated females, and to hexane extracts of whole bodies and thoraces of unmated females. Unmated females were attracted to hexane extracts of males and to hexane extracts of each body tagma of males, suggesting distribution of the pheromone over the cuticle by grooming. The ectal mandibular and seventh sternal glands are the likely sources of the male-produced sex attractant(s) since extracts of each elicited female attraction in the flight tunnel. These glands are associated with gastral and mandibular rubbing of perch sites by territorial males ofPolistes species.     

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.     

The Role of Cuticular Hydrocarbons in Male Mating Behavior of the Mustard Leaf Beetle, <Emphasis Type="Italic">Phaedon cochleariae</Emphasis> (F.)

We investigated the role that cuticular hydrocarbons (CHC) play in sexual communication by the mustard leaf beetle, Phaedon cochleariae (Coleoptera: Chrysomelidae). In laboratory bioassays, male P. cochleariae attempted to copulate with living or freeze-killed females as often as with males. However, the duration of copulation with females was longer than with males. To elucidate the impact of CHC on this behavior, cuticular compounds of adults of both sexes were extracted with dichloromethane. Male mating attempts with glass beads treated with the dichloromethane extract were nearly as frequent as with living beetles. The dichloromethane extract was fractionated by silica gel chromatography, and the biological activity of the fractions was tested by applying them to glass beads. A non-polar hexane fraction significantly elicited mating behavior, whereas the polar methanol fraction did not, likely because it contained defensive compounds from exocrine glands located in the elytra and pronota. Interestingly, a mixture of both the non-polar and polar fraction tended to elicit more mating attempts than did the non-polar hexane fraction alone. Further fractionation of the significantly active hexane fraction by silver nitrate column chromatography revealed that saturated CHC elicited mating behavior, but the olefins did not. GC-MS analyses of dichloromethane cuticular extracts showed that the male and female CHC profiles were qualitatively identical, but differed in their relative composition. Canonical discriminant analysis showed that CHC profiles of males and females formed separate clusters. Nevertheless, the results of our bioassays demonstrated that male and female CHC did not elicit sex discriminative male behavior, but induced mating by males regardless of the sex of the partner.     

Chemical basis for asymmetric mating isolation between strains of screwworm fly,Cochliomyia hominivorax

Laboratory mating tests and bioassays for a contact, mating-stimulant pheromone were conducted within and between two strains of the screwworm fly,Cochliomyia hominivorax. No strain or sex difference in pheromone activity occurred at emergence. However, the pheromone activity of females increased with age in one strain but declined in the other. Activity also declined in males of both strains but more steeply than in the females. Thus, sexual dimorphism in pheromone activity developed with age in both strains but to differing degrees. The decline in female pheromone activity was partly compensated for within that strain by a higher male responsiveness to pheromone. Female pheromone activity and mating success were positively correlated. Virgin females and those inseminated 24–48 hr previously were equally stimulatory. It appears that the strain differences arose from selection for reduced pheromone activity during laboratory colonization.     

Multicomponent sex pheromone inMacrocentrus grandii Goidanich (Hymenoptera: Braconidae)

A multicomponent sex pheromone inMacrocentms grandii Goidanich (Hymenoptera: Braconidae) was demonstrated using wind-tunnel and field bioassays. In wind-tunnel bioassays, three Florisii fractions from female wasps (hexane, 5%, and 50% ether in hexane) and one from male wasps (50% ether in hexane) were attractive to males. The hexane and 5% ether in hexane fractions each elicited similar male behavioral responses. These included upwind anemotaxis, casting, landing on the source, wing fanning, and mating attempts between males. The 50% ether in hexane fraction, whether male- or female-derived, initiated a strong flight response by males but few landings on the source. In addition, the 50% ether in hexane fraction was found to synergize strongly the hexane fraction. All females tested in the wind tunnel gave no responses to male- or female-derived extracts. Field tests generally supported the wind-tunnel results, although no field attraction was observed for the 5% ether in hexane fraction.     

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.     

Mating stimulant of the pine weevilHylobius abietis (L.)

Factors eliciting copulatory behavior in mature maleHylobius abietis were studied in the laboratory. Dead female weevils were sexually attractive, while dead mature males and pentane-extracted female weevils were not. The sexual attractiveness of dead females declined with time after death. Pentane extracts of whole female weevils or of the anterior or posterior parts of their bodies elicited a copulatory response when applied to decoys. In contrast, extracts of hindgut or frass were inactive. Juvenile males were sexually attractive for about four weeks, after which their attractiveness gradually declined. The results indicate that the mating stimulant is present on the body surface of female and juvenile male weevils, and it can be extracted with pentane.     

Scented Males and Choosy Females: Does Male Odor Influence Female Mate Choice in the Mediterranean Fruit Fly?

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), displays a lek mating system characterized by a high level of female discrimination among potential mates. The basis of female choice is not understood, but recent studies indicate that male exposure to the aroma of certain plant structures or essential oils may increase mating success. In particular, exposure to the aroma of ginger root oil (GRO) enhances male mating frequency, and several sterile-male release programs against C. capitata have incorporated ‘aromatherapy’ (large-scale exposure of pre-release insects to GRO) to increase the effectiveness of control efforts. We investigated the mechanism underlying female preference for GRO-exposed males. Two sets of experiments were conducted. In the first, we monitored female attraction to (1) freshly killed flies, or (2) paper discs that contained hexane extracts from varying treatments. In these tests, females were sighted more often (1) near GRO-exposed than non-exposed males (even when the males were visually concealed) and (2) near extracts from GRO-exposed than non-exposed males. These findings suggest a ‘perfume effect’, whereby female mate choice is mediated by olfactory differences. In the second set, we compared (1) mate choice between intact females and females from which both antennae had been surgically removed, and (2) mating success between intact males and males from which both antennae had been surgically removed before GRO exposure. Intact females preferred GRO-exposed males, whereas females lacking both antennae rarely mated and showed no preference between GRO-exposed and non-exposed males. In the opposite treatment (intact females but surgically altered males), GRO-exposed males lacking both antennae mated as frequently as GRO-exposed intact males. These data suggest that female choice was dependent on olfactory perception of male odor but that male mating success did not depend on olfactory perception of GRO aroma, suggesting, in turn, that GRO conferred a mating advantage through an external phenomenon (possibly alteration of cuticular scent) rather than through internal processing (pheromone synthesis).     

Evidence for sex pheromones produced by males and females inBlatta orientalis (Dictyoptera,Blattidae)

Males and females ofBlatta orientalis produce a volatile sex pheromone attractive at a distance. The male initiates courtship behavior by adopting a calling posture while emitting his sex pheromone. He exposes the anterior region of his tergites by stretching his abdomen horizontally. Morphological observations show the existence of male tergal glands on the anterior region of tergites 1 to 8. Behavioral assays of females utilizing methylene chloride extracts of various male body parts reveal that extracts of the anterior region of tergites are highly attractive and induce more positive responses that any other body part. Once near the male, the attracted female opens widely her genital atrium thus exposing her atrial glands. Behavioral assays reveal that these atrial glands are certainly the site of production of the female sex pheromone.     

Close-range attraction in Lygocoris pabulinus (L.)

Males of the green capsid bug, Lygocoris pabulinus, exhibit a specific courtship behavior, i.e., a vibration of the abdomen. When both live and dead females were offered to males, this vibration behavior was elicited in most of the males tested. When females were dissected into separate body parts, heads, wings, and legs elicited equal responses, while thorax plus abdomen elicited a much lower response. When separate body parts were extracted, the leg extracts elicited significantly stronger responses than any other extract. This suggests that female L. pabulinuslegs are either the source of a close-range sex pheromone or that pheromone is accumulated on the legs due to grooming behavior. The leg extracts contained several hydrocarbons such as n-alkenes, n-alkanes, and some methylalkanes. Female extracts contained more (Z)-9-pentacosene and male extracts contained more (Z)-9-heptacosene. Substrates on which females had walked elicited similar responses as female legs, indicating that the pheromone is deposited on the substrate. This enlarges the functional range of low-volatility compounds, which are thought to function only when sexes are in close vicinity or in contact.     

Identification of female sex pheromone in alfalfa blotch leafminer,Agromyza frontella (Rondani)(Diptera: Agromyzidae)

Gas chroinatography of hexane extracts of 3-day-old virgin A.frontella adults revealed a branched saturated hydrocarbon present only in females. This compound was isolated by argentation chromotography and preparative GC, identified by GC and GC-MS as 3,7-dimethylnonadecane, and subsequently synthesized. Three-day-old virgin females contained 54.1 ± 3.5 ng (± SEM;N = 48) of 3,7-dimethylnonadecane. Male cadavers do not elicit male courtship behavior, but when treated with 18 ng of 3,7-dimethylnonadecane they were as attractive as cadavers of 3-day-old virgin females. Black cotton knots were less attractive than male cadavers when treated with 3,7-dimethylnonadecane, but all bioassays indicated that this compound is an important semiochemical modulating male mating behavior.     

Sex pheromone communication in the screwworm,Cochliomyia hominivorax: Ontogenetic and strain effects

A laboratory behavioral assay was used to examine ontogenetic and strain effects on sex pheromone production and reception in the screwworm, which employs a female-produced contact pheromone to stimulate male copulatory attempts. Pheromone levels peaked between three and six days after adult emergence in females of two strains that had been colonized fewer than 50 generations. Pheromone was detectable at low levels in newly emerged flies of both sexes but disappeared from males by two days of age, when males first became responsive to females. A similar decline in pheromone with ontogenetic age was previously described for females of a strain colonized over 100 generations. Interstrain tests between the two newer strains, which differed by 35 generations in duration of colonization, demonstrated lower female pheromone activity but higher male responsiveness to pheromone in the older strain, despite the similar shape within these strains of curves relating female age and pheromone activity. The data suggest that conditions of screwworm colonization result in gradual loss of the pheromone peak that develops three to six days after emergence in newly colonized females. Higher male responsiveness associated with longer colonization involved change in both thresholds and dose dependency and may be relevant to control of the screwworm via the sterile insect technique.     

Arrestment of male twospotted spider mite caused by female sex pheromone

A glass slide bioassay was used to evaluate male twospotted spider mite,Tetranychus urticae Koch, arrestment caused by quiescent deutonymph extract. Males that were guarding quiescent deutonymphs prior to being tested were arrested by a 3-mm-diameter circle of quiescent deutonymph extract; nonguarding males and adult females were not arrested. Extracts of allT. urticae instars tested caused male arrestment, but mean arrestment duration was longest with quiescent deutonymph extract. Arrestment by volatile perception of pheromone and upwind orientation to point sources of extract were not observed. The mono- and sesquiterpene alcohols previously identified as components of the pheromone did not arrest males. HPLC separation of extract resulted in four active fractions; a subtractive bioassay showed that three were essential to elicit maximum male response.     

European corn borer: Pheromonal catabolism and behavioral response to sex pheromone

When physiologically excessive amounts of the female sex pheromone of the European corn borer (ECB) or esters analogous to the pheromone were applied to the antennae of males, their behavioral responsiveness to pheromone in a flight tunnel was significantly impaired for 2 hr. Concurrent quantitative analyses of heptane extracts of the male antennae by gasliquid chromatography showed that the compounds applied to antennae were hydrolyzed and, at 2 and 4 hr posttreatment, little or none of the compound applied or hydrolysis product was detectable in the antennal extracts. After 4 hr of in vivo incubation, male responsiveness to pheromone was restored among moths treated with the analogs but not among moths treated with pheromone. Esterase activity on the antennae was moderately inhibited in vivo by a pheromone analog that is a so-called transition-state esterase inhibitor, 1,1, 1-trifluoro-14-heptadecen-2-one. However, the analog did not inhibit male behavior when it was coevaporated with pheromone in a flight-tunnel assay. Therefore, in the presence of pheromone, the analog did not compete well for esterase or the pheromone receptor. Treating the antennae of intact males with tetrahydrofuran obliterated sex pheromone response capability in males, but the treatment did not significantly attenuate esterase and other catabolic activity of the antennae. Indications are that degradation of esters on the ECB antennae involves substrate-nonspecific esterase activity and other metabolic processes that in turn remove hydrolysis products from the antennae. Maintenance of a male's ability to respond to pheromone is linked to these processes.