overwintering second - instar c. fumiferana larvae were obtained from the canadian forest service s insect production services ( ips ) in sault ste . marie , on , canada ( dedes 2014 ) , and were raised on artificial diet ( mcmorran 1965 ) , also produced at ips . adults were kept on a 16:8 ( l : d ) h cycle , and were 13-d - old for the study . mating trials were conducted in a wind tunnel ( 2325c , 6070% relative humidity ) flowing at 0.2  km / h ( 0.056  m / s ) that was housed in a darkened room illuminated by several red incandescent lights . treatments were compared using cages ( 20 by 17 by 14  cm ) consisting of a cardboard box with plastic window - screen coverings on all four sides and the roof . in addition to a control group containing unmanipulated males and females , treatments consisted of ( 1 ) males with hair pencils removed plus normal females ; ( 2 ) males with hair pencils washed with 5  l of 99% hexane plus normal females ; and ( 3 ) normal males plus antennectomized females . one cage was used consistently for each treatment with five males and five females in the cage at one time . as males in treatments 1 and 2 and females in treatment 3 required anesthetization in order to remove or wash body parts , all insects were anesthetized with co2 prior to placement into cages to control for any potential artifacts of co2 exposure on insect behavior . hair pencils of males in treatment 1 were removed using fine forceps . in treatment 2 , male hair pencils were washed with hexane ( 99% ) applied with a 5-l micropipette ( drummond wiretrol , broomall , pa ) . in treatment 3 , female antennae were removed close to the head using microscissors . to determine if co2 anesthetization affected mating success , we also conducted under identical conditions a separate evaluation of mating success of normal mating pair groups ( n  =  15 ) that were not anesthetized with co2 . for the experiment , cages were placed in a wind - tunnel environment during the second hour of scotophase when pheromone calling peaks ( silk and kuenen 1986 ) . females were introduced to the cages first , followed by males an hour later . as normal duration of copulation has been reported to be 3  h ( outram 1971 , delisle and hardy 1997 , delisle and simard 2002 ) , cages were checked 3  h after the introduction of males , and the number of pairs in copula were counted so that any possible copulation events within the cages would be observed . to confirm that any possible reductions in proportions of mating pairs were attributable to the presence or absence of hair pencils or female antenna and not handling , sham - operated treatments containing mating pairs that simulated the effects of handling on the insects were also observed . these included ( 1 ) males with scales removed from the dorsum of the abdomen  +  normal females ; ( 2 ) males with 3  l of hexane ( 99% ) applied to the dorsum of the abdomen  +  normal females ; and ( 3 ) normal males  +  females with their left hind leg amputated between the femur and tarsus . these were compared with hair pencil / female antennectomization treatments 13 to ensure that any possible effects on copulation success were not attributable to trauma sustained during handling . as with hair pencil / female antennectomization treatments 13 , 15 replicates of each treatment were completed ( n  =  15 ) with five males and five females being used for each replicate . to assess differences in mating success among hair pencil / female antennectomized treatments , we used a generalized linear model ( proc genmod , dist  =  binomial , link  =  logit ; sas institute 1999 ) , followed by contrast statements ( wald tests ; sas institute 1999 ) to compare all possible pairs of hair pencil / female antennectomization treatments . to determine if trauma associated with handling influenced copulation success , student s t - tests comparing the mean proportion of moths in copula of hair pencil / female antennectomized treatments versus their respective sham - operated treatments were also completed . a one - way analysis of variance ( anova ) that compared the mean proportions of moths in copula for sham - operated treatments and the control was also completed . the level of statistical significance was set at   =  0.05 for all analyses . there was a significant difference in the mean proportion of moths in copula across hair pencil / female antennectomized treatments ( =  30.85 , df  =  3 , p  <  0.0001 ) . the proportion of moths in copula ( mean    se ) in the control group ( 0.447    0.065 ) was significantly higher than that of treatment 1 ( males with hair pencils washed with 5  l of 99% hexane plus normal females ) ( 0.107    0.047 ) and treatment 2 ( males with hair pencils ablated plus normal females ) ( 0.133    0.042 ) , and treatment 3 ( normal males plus antennectomized females ) ( 0.247    0.064 ) ( wald test , p  <  0.05 ; fig . 1 ) . there was no significant difference in mating success between treatments 1 and 2 , however , mating success was significantly lower in treatments 1 and 2 than in treatment 3 ( wald test , p  <  0.05 ; fig . 1 ) . mean proportions of moths in copula for all three hair pencil / antennectomized treatments were also significantly less than their respective sham - operated treatments ( student s t - tests , p  <  0.01 , fig . 1 ) . there were no significant differences in the mean proportion of moths in copula between the control and sham - operated treatments ( one - way anova , f3 , 56  =  0.618 , p  =  0.606 ) . mean proportion of moths in copula in mating pairs not anesthetized with co2 was 0.747    0.041 ( n  =  15 ) . 1.mean (  se ) proportions of c. fumiferana mating pairs in copula in cages ( n  =  15 cages per treatment group ) . treatments include ( 1 ) five males with hair pencils ablated  +  five unmanipulated females per cage ; ( 2 ) five males with hair pencils washed with 5  l of 99% hexane  +  five unmanipulated females per cage ; and ( 3 ) five unmanipulated males  +  five antennectomized females per cage . sham - operated treatments include ( 1 ) mating pairs containing five males with scales removed from the dorsum of the abdomen  +  five normal females ; ( 2 ) mating pairs containing five males with 3  l of hexane ( 99% ) applied to the dorsum of the abdomen  +  five normal females ; and ( 3 ) mating pairs with five normal males  +  five females with their left hind leg amputated between the femur and tarsus .  *  represents significant differences between mean proportions of mating pairs in copula of treatment and sham - operated mating pairs ( student s t - test , p  <  0.01 ) . different uppercase letters represent significant differences between mean proportions of mating pairs in copula for control and hair pencil / female antennectomization treatments ( wald test , p  <  0.05 ) . different lowercase letters represent significant differences between mean proportions of mating pairs in copula for control and sham - operated treatments ( one - way anova , p  <  0.05 ) . mean (  se ) proportions of c. fumiferana mating pairs in copula in cages ( n  =  15 cages per treatment group ) . treatments include ( 1 ) five males with hair pencils ablated  +  five unmanipulated females per cage ; ( 2 ) five males with hair pencils washed with 5  l of 99% hexane  +  five unmanipulated females per cage ; and ( 3 ) five unmanipulated males  +  five antennectomized females per cage . sham - operated treatments include ( 1 ) mating pairs containing five males with scales removed from the dorsum of the abdomen  +  five normal females ; ( 2 ) mating pairs containing five males with 3  l of hexane ( 99% ) applied to the dorsum of the abdomen  +  five normal females ; and ( 3 ) mating pairs with five normal males  +  five females with their left hind leg amputated between the femur and tarsus . *  represents significant differences between mean proportions of mating pairs in copula of treatment and sham - operated mating pairs ( student s t - test , p  <  0.01 ) . different uppercase letters represent significant differences between mean proportions of mating pairs in copula for control and hair pencil / female antennectomization treatments ( wald test , p  <  0.05 ) . different lowercase letters represent significant differences between mean proportions of mating pairs in copula for control and sham - operated treatments ( one - way anova , p  <  0.05 ) . our data point strongly to the conclusion that mating success is related to the presence of hair pencil volatiles in males . in treatments where hair pencils had either been ablated or chemically washed , the mean proportion of moths in copula was much lower than in the control . significantly higher proportions of moths in copula in sham - operated treatments confirm that decreases in mating success are not related to insect handling or trauma . these results suggest that male hair pencils and their associated volatiles serve an important role in the mating success of c. fumiferana . decreases in mating success caused by the ablation of hair pencils have been observed in other species , including pseudaletia unipuncta [ haworth ] ( lepidoptera : noctuidae ) ( fitzpatrick and mcneil 1988 ) and ostrinia nubilalis ( hbner ) ( lepidoptera : pyralidae ) ( royer and mcneil 1992 , lassance and lfstedt 2009 ) . although mating was significantly lower in our hair pencil treatments , some mating instances were observed . these may be because hair pencil ablation may not have been 100% complete , with volatiles remaining after ablation or washing , a conclusion also discussed by royer and mcneil ( 1992 ) . hair pencils are also found on the wing folds of males in several tortricids ( grant 1978 ) , including c. fumiferana ( palaniswamy et  al . 1979 ) . although it is possible that pheromones may also be emitted from these regions as well , detectable amounts are likely obscured based on the significant reduction in mating success in pairs where hair pencils had been manipulated . we observed that mating success in pairs containing antennectomized females was significantly lower than in the control group . this result may be due to the female not being able to detect hair pencil volatiles , causing disruptions to the mating sequence . antennectomization of females has been shown to reduce mating success in o. nubilalis ( royer and mcneil 1992 ) and heliothis virescens f. ( lepidoptera : noctuidae ) ( hillier and vickers 2004 ) . ( 1979 ) also observed eag responses in female c. fumiferana antenna that were exposed to male c. fumiferana hair pencil extracts . although these studies support our conclusion , it is possible that antennectomization may also prevent the female from detecting her own sex pheromones and thus potentially disrupt her own pheromone production . palaniswamy and seabrook ( 1978 , 1985 ) observed that c. fumiferana female pheromone production can be induced by the detection of female sex pheromone . if pheromone emittance does not occur , then male attraction and subsequent mating may be reduced due to an inability of the male to locate the female through pheromone detection . the significantly higher amount of successful matings in the antennectomized treatment compared with mating pairs where hair pencils had been manipulated is also interesting as it suggests that other olfactory organs could exist . ( 1986 ) suggested that sensilla on the labial palps of several hawkmoth ( manduca spp . [ hbner ] ( lepidoptera : sphingidae ) ) species could be secondary olfactory receptors . ablation of labial palps in male c. fumiferana did not , however , affect mating success ( sanders and lucuik 1992 ) . future experiments analyzing the structure of female labial palps may be useful in determining if such structures exist . observed mating success in the control group ( 42% ) was much lower than that observed by sanders and lucuik ( 1992 ) ( 82% ) . we attribute this to co2 anesthetization of all insects prior to observations . mating trials conducted with insects not exposed to co2 anesthetization yielded mating proportions ( 74.1    4.10% ) more similar to those of sanders and lucuik ( 1992 ) , thereby indicating that co2 anesthetization was a likely factor in the relatively low mating successes observed in the control group . insect handling also did not significantly influence mating success , a result also observed by sanders and lucuik ( 1992 ) and royer and mcneil ( 1992 ) in c. fumiferana and o. nubilalis , respectively . at present , the chemical composition of this potential volatile or blend of male volatiles is unknown . studies have shown hair pencil blends to vary across species . in g. molesta , male hair pencil volatile blends include mellein , methyl jasmonate , methyl 2-epi - jasmonate , and trans - ethyl cinnamate ( nishida et  al . 1982 ) , whereas ( e)-2-methyl-2-butenoic acid ( tiglic acid ) is the primary component in hair pencil extracts from male conogethes punctiferalis [ guene ] ( lepidoptera : pyralidae ) ( kimura and honda 1999 ) . analyses of c. fumiferana hair pencil extracts through gc - ms analyses and synthesis of compounds and bioassays are currently underway . although the specific role of these compounds in the mating sequence of c. fumiferana is also unknown , other studies have demonstrated that hair pencil volatiles could fulfil a variety of roles , including acting as female attractants ( baker et  al . 1985 , krasnoff and dussourd 1989 , krasnoff and roelofs 1989 , landolt and heath 1989 ) and as compounds that arrest female behaviors , including sex pheromone production ( hendricks and shaver 1975 ) . other studies have suggested that females may be able to assess mate quality based on hair pencil volatile composition ( baker and card 1979b ) , and others suggest that hair pencil volatiles may inhibit competing males from approaching females both pre- ( lecomte et  al . 1998 , hillier et  al . 2007 ) and post- ( andersson et  al . 2003 ) copulation . this latter possibility may be useful in interfering with the mating behavior of c. fumiferana , possibly in conjunction with female 95:5 e / z11 - 14:ald compounds . further studies on the composition and identity of male - derived hair pencil compounds in conjunction with behavioral assays are required .
male lepidoptera often possess specialized scales , called hair pencils that emit volatiles that are critical to mating success . 
 spruce budworm , choristoneura fumiferana ( clemens ) ( lepidoptera : tortricidae ) , males will display hair pencils to females before attempting copulation . 
 the importance of volatiles on these hair pencils is , however , not clear . 
 we compared the proportion of successful copulations in unmanipulated mating pairs to pairs where males had their hair pencils either removed or chemically washed , and to pairs where females were antennectomized . 
 mean proportions of successful matings were significantly lower in pairs where hair pencils had been manipulated or where females had been antennectomized compared with unmanipulated mating pairs . 
 there was no significant difference in mating success between treatments where hair pencils had been manipulated ; however , mating success was significantly lower in hair pencil treatments than in antennectomized treatments . 
 mean copulation proportions in hair pencil / antennectomized treatments were also significantly less than in respective sham - operated treatments . 
 our results suggest that volatiles are associated with hair pencils , and they may be required for mating success in c. fumiferana .