J Chem Ecol (2016) 42:517–522 DOI 10.1007/s10886-016-0714-y

Single-Component Pheromone Consisting of Bombykal in a Diurnal Hawk , Neogurelca himachala sangaica

Takuya Uehara1,2 & Hiroshi Kitahara2 & Hideshi Naka4 & Shigeru Matsuyama3 & Tetsu Ando 5 & Hiroshi Honda3

Received: 15 February 2016 /Revised: 5 May 2016 /Accepted: 24 May 2016 /Published online: 14 June 2016 # Springer Science+Business Media New York 2016

Abstract Recent work has suggested that hawk share 16:Ald positively contributed to attractiveness, whereas E10, pheromone components but are sexually separated by quali- E12–16:Ald did so negatively, and it was concluded that the tative and quantitative differences in their pheromone blends. sex pheromone of N. himachala sangaica consists solely of During field assays on the sex pheromones of other species, a E10,Z12–16:Ald, bombykal. The negative effect of E10,E12– diurnal hawk moth, Neogurelca himachala sangaica 16:Ald on attractiveness could promote the species-specificity (: ), was frequently captured, but the of this single-component pheromone system. composition of the sex pheromone of this species was not known. Analysis of hexane extracts of the pheromone glands Keywords Lepidoptera . Sphingidae . Single component . of calling female by gas chromatography (GC) using an Pheromone electroantennographic detector (EAD) revealed two compo- nents that elicited EAD responses from male moth antennae. These components were identified by their mass spectra and Introduction retention indices on two GC columns as (10E,12Z)-10,12- hexadecadienal (E10,Z12–16:Ald) and a trace of its (10E, The Sphingidae, commonly known as hawk moths, is one of 12E)-isomer (E10,E12–16:Ald) in 98:2 ratio. In field experi- the largest families in Bombycoidea, including approximately ments, E10,Z12–16:Ald alone attracted male moths, and ad- 1450 species worldwide (Nieukerken et al. 2011). It is classi- dition of E10,E12–16:Ald significantly reduced the attractive- fied into three subfamilies, Sphinginae, , and ness, even at the naturally-occurring ratio. Analysis of the data Macroglossinae (Kawahara et al. 2009). To date, female sex using a generalized linear mixed model showed that E10,Z12– pheromones have been described for six species in the family (Tumlinson et al. 1994; Uehara et al. 2012, 2013, 2015; Wakamura et al. 1996), and a few sex attractants have been * Takuya Uehara reported (Bestmann et al. 1992; Landolt et al. 1989; Reed et al. [email protected] 1987). Sex pheromones of most of these species are composed of unsaturated aliphatic aldehydes, such as 11-hexadecenal 1 National Institute of Agrobiological Sciences (NIAS), Ohwashi 1-2, and 10,12-hexadecadienal. These components, most notably Tsukuba, Ibaraki 305-8634, Japan (10E,12Z)-10,12-hexadecadienal (E10,Z12–16:Ald), known 2 Graduate School of Life and Environmental Sciences, University of as bombykal, have been reported as sex pheromone compo- Tsukuba, Tsukuba, Ibaraki 305-8572, Japan nents of many moths (e.g., Daimon et al. 2012a;Klunetal. 3 Faculty of Life and Environmental Sciences, University of Tsukuba, 1986; McElfresh and Millar 1999; McElfresh et al. 2001; Tsukuba, Ibaraki 305-8572, Japan Raina et al. 1986). Use of different blends of these components 4 Faculty of Agriculture, Tottori University, Koyama Minami, is probably essential for establishing the species specificity of Tottori 680-8553, Japan the pheromone system in hawk moths (Uehara et al. 2015). 5 Graduate School of Bio-Applications and Systems Engineering, In previous studies, males of a diurnal hawk moth, Tokyo University of Agriculture and Technology, Neogurelca himachala sangaica (Lepidoptera: Sphingidae), Koganei 184-8588, Japan often were captured in traps baited with bombykal 518 J Chem Ecol (2016) 42:517–522

(Daimon et al. 2012b; Uehara et al. 2015). Here, we describe were digitally converted by IDAC-2 (Syntech) and visualized the identification of a sex pheromone of N. himachala and analyzed using Syntech GcEad Ver. 1.2.2. sangaica, and we provide evidence from field bioassays and electroantennography indicating that females of this species use Gas Chromatography The E/Z configuration and composi- a single compound, bombykal, as sex pheromone. tion of the pheromone candidates were determined by GC analysis. Isomers were examined by comparing their Kováts retention indices (RI) between EAD-active compounds and Methods and Materials synthetic compounds on two columns with different polarities (Kováts 1958; van Den Dool and Kratz 1963), and isomeric Late-instars of N. himachala sangaica were collected compositions were calculated from the ratio of GC peak areas. from skunkvine, Paederia scandens (), on the cam- The analyses were conducted with a GC-17 A (Shimadzu Co., pus of the University of Tsukuba (N 36°6′, E 140°5′). Larvae Ltd., Kyoto, Japan) equipped with an HP-5MS non-polar col- were reared on cut P. scandens in a rearing cage umn as above, and with an Agilent 6890 GC (Agilent (22.8 × 15.2 × 7.7 cm) under a long-day photoperiod Technologies) with a DB-23 (30 m × 0.25 mm diam, (16L:8D) at 25 ± 2 °C. After larvae pupated, pupae were 0.25-μm film thickness; Agilent Technologies) polar column. separated by sex and kept in different cages under the same The oven temperature for the non-polar column was main- conditions. Newly eclosed adults were put into a mesh cage tained at 100 °C for 2 min, raised to 250 °C at 5 °C/min, (23.5 × 30 × 33.5 cm). and then held for 10 min. The polar column was held at 130 °C for 2 min, then increased to 250 °C at 3 °C/min, and Pheromone Gland Extraction Extracts of sex pheromone held for 10 min. In both analyses, temperatures of the injector glands were made from 2-d-old virgin females in the first port and detector (FID) were 250 °C, and all samples were 2.5 to 10 h of the light period, when pheromone glands ap- injected in splitless mode. peared on the tip of female moth abdomens. After anesthetiz- ing with carbon dioxide, female abdominal tips including the Gas Chromatography–Mass Spectrometry (GC-MS) GC- sex pheromone gland were excised with ophthalmic scissors MS analysis was conducted with a JEOL MS-600H (JEOL, and extracted with approximately 50 μl of re-distilled hexane Ltd., Tokyo, Japan) at 70 eV coupled with an Agilent 6890 N per gland for 15 min under ambient temperature. Butylated GC (Agilent Technologies). The GC instrument was equipped hydroxytoluene (BHT) was added to the extract as an antiox- with a DB-5MS capillary column (25 m long × 0.25 mm diam, idant. The extracts were pooled into three stocks (20 individ- 0.25-μm film thickness; Agilent Technologies). Carrier gas uals each) and stored at −20 °C before use. was helium at a flow rate of 1 ml/min in constant flow mode. Interface and injector temperatures of the GC were 280 °C, Chemicals The four geometric isomers of 10,12- and the oven temperature was held at 100 °C for 1 min and hexadecadienyl aldehydes, alcohols, and acetates were avail- then increased by 10 °C/min to 320 °C and held for 7 min. able from a stock library in our laboratory. The purity of all compounds was confirmed to be higher than 99.5 % by gas MTAD Derivatization To determine double-bond positions, chromatography (GC; column: HP-5MS). pheromone candidates with conjugated dienes in the extracts were reacted with 4-methyl-1,2,4-triazoline-3,5-dione Gas Chromatography–Electroantennographic Detection (MTAD; Sigma-Aldrich Co., Ltd., St. Louis, MO, USA). (GC-EAD) GC-EAD analysis was performed on an HP5890 MTAD (1 %) in dichloromethane was added to one female Series II GC (Hewlett Packard, Palo Alto, CA, USA) equivalent extract in dichloromethane until a slight pink color equipped with an HP-5MS column (30 m in length × persisted (Young et al. 1990). The reaction mixture was ana- 0.32 mm in diam, 0.25-μm film thickness; Agilent lyzed by GC-MS as described above. Technologies, Santa Clara, CA, USA). The oven temperature was held at 130 °C for 2 min and then increased to 250 °C at Field Bioassays The attractiveness of candidate pheromone 5 °C/min. The temperatures of the detector and injector were components was tested in field bioassays conducted at three 250 °C, and the outlet for the EAD was maintained at 300 °C. locations on the campus of the University of Tsukuba (N Injection was splitless, and carrier gas was helium. The efflu- 36°6′, E 140°5′) September in 2013 and 2014. Sticky traps ent from the GC column was split at a 1:1 ratio between the (SE-traps, 30 × 27-cm bottom plate with a roof; Sankei flame ionization detector (FID) and the EAD. The effluent Chemical Co., Ltd., Kagoshima, Japan) were baited with a was delivered in humidified air (23 °C) to the antennal prep- gray rubber septum (West Company, Singapore) containing aration connected to an electrode (EAG Type PRG-2 probe; 0.5 mg of the test chemicals. Test chemicals were dissolved Syntech, Kirchzarten, Germany) via electrically conductive in hexane with 0.5 % BHT then added to the septum. The traps gel (Spectra 360, Fairfield, NJ, USA). The antennal responses were set in a randomized design. Once a week, catches were J Chem Ecol (2016) 42:517–522 519

Hexane BHT recorded, sticky traps renewed, and the positions of the treat- ments were rotated to avoid positional effects. Daily counts A B for each treatment were pooled to eliminate the day factor, 0.1 mV which was irrelevant for our purposes. To examine the effects of lures, the number of male catches (count data) was analyzed using a generalized linear mixed model (GLMM) with a Poisson distribution and a log link function. Subsequently, significant explanatory variables were compared by Tukey’s test. A GLMM with a Poisson distribu- tion and a log link function also was used to estimate how components contribute to male catches. The number of male catches was used as the response variable. Relative amounts of E10,Z12–16:Ald and (10E,12E)-10,12-hexadecadienal (E10,E12–16:Ald) were treated as explanatory variables. 0481216 Retention time (min) Model comparison was conducted using Akaike’sinformation criterion (AIC) (Crawley 2005). The lowest AIC value was Fig 1 Gas chromatography–electroantennographic detection analysis of pheromone gland extract from Neogurelca himachala sangaica on an used to select the model that was most suitable for describing HP-5MS column [upper trace, antennal response; lower trace, flame the contribution of components to the male catches. These ionization detector (FID)] analyses were performed using R version 3.1.0 (R Core Team 2015)andthelme4(Batesetal.2015)andmultcomp In GC-EAD analyses of the four geometric isomers of 10, (Hothorn et al. 2008) packages. 12-hexadecadienyl aldehydes, alcohols, and acetates, most of the compounds elicited antennal responses from males, but the strongest responses were to the aldehydes (Fig. 2). Results Field Bioassays Because two previous reports showed that Pheromone Identification Analysis of pheromone gland ex- E10,Z12–16:Ald could attract male moths (see tracts by GC-EAD showed that two components, A and B, Introduction), E10,Z12–16:Ald alone, the natural mixture of elicited responses from the antennae of male moths (Fig. 1). In E10,Z12–16:Ald and E10,E12–16:Ald in 98:2 ratio, and a GC-MS analyses, components A and B showed ion peaks at solvent control were first compared. E10,Z12–16:Ald and m/z 236 (M+, 37 %), 67 ([C H ]+, base peak), 81, 95, 96 5 7 the natural mixture attracted male moths, but the solvent con- ([C H ]+, 42 %), and 109 ([C H ]+, 29 %). The molecular 7 12 8 13 trol did not (Fig. 3). In GLMM analysis, E10,Z12–16:Ald ion (M+)atm/z 236, and fragment ions at m/z 67 and separated alone and the natural mixture showed significant explanatory by 14 mass units indicated a conjugated diunsaturated aliphat- power for male attraction. Tukey’s test showed that the single ic with a possible molecular formula of C H O. The 16 28 component was more attractive than the natural mixture dehydrated ion of a conjugated dienyl aldehyde is difficult to (P <0.01). detect (Uehara et al. 2012); thus, the spectrum is consistent To clarify the effect of E10,E12–16:Ald, the amount rela- with hexadecadienal. Additionally, M+ and two conspicuous tive to that of E10,Z12–16:Ald was increased (Fig. 4). E10, diagnostic ions at m/z 96 and 109 suggested conjugated dou- Z12–16:Ald alone was significantly more attractive than the ble bonds at the 10 and 12 positions (ω4andω6positions)in blend containing 5 % E10,E12–16:Ald (P < 0.05) and highly a straight carbon chain (Ando et al. 1988;Andoand Yamakawa 2011). The positions of the double bonds in these components Table 1 Kováts retention indices (RI) of candidate pheromone were confirmed by derivatization with MTAD, which reacts components A , B and authentic hexadecadienals chromatographed on specifically with conjugated dienes. The mass spectrum of the a non-polar (HP-5MS) and polar (DB-23) GC column MTAD reaction product with components A and B exhibited Compounds RI (HP-5MS) RI (DB-23) diagnostic ions at m/z 349 (M+), 208 (base peak, 100 %, [M- + + C9H17O] ), and 306 ([M-C3H7] ). RI values of components A Component A 1863 2386 and B on both the HP-5MS and DB-23 columns matched Component B 1879 2395 those of E10,Z12–16:Ald and E10,E12–16:Ald (Table 1). Z10E12–16:Ald 1854 2372 These results strongly suggest that components A and B are E10Z12–16:Ald 1863 2386 – – E10,Z12 16:Ald and E10,E12 16:Ald, respectively. The ratio Z10Z12–16:Ald 1873 2392 was determined based on the peak area and RIs from the GC E10E12–16:Ald 1879 2396 analyses as 98:2 respectively. 520 J Chem Ecol (2016) 42:517–522

Ald OH OAc *** 20 yad rep rep yad * 0.1 mV 15 *** de r u tpac s tpac 10 h tom tom 5 ela

ZE EZ ZZ EE M 0 EZ 0900 95 100 EE 010100 50 Ratio of EZ and EE isomer 14 16 18 20 Fig 4 Numbers of male Neogurelca himachala sangaica attracted to Retention time (min) traps baited with mixtures of E10,Z12–16:Ald and E10,E12–16:Ald. Fig 2 Gas chromatography–electroantennographic detection analysis of Box plots represent the minimum observation, 25th, 50th (median), and four geometric isomers of 10,12-hexadecadienyl aldehydes, alcohols, and 75th percentiles, and the maximum observation; circles are outliers acetates (5 ng each and Z10,Z12–16:Ald 2 ng) on an HP-5MS column (September 4–12, 2013; total number of moths trapped =153; number (upper trace, antennal response; lower trace, FID) of replications =13) significantly more attractive than the blend containing 10 % 16:Ald was positive, whereas that of E10,E12–16:Ald was E10,E12–16:Ald. The latter was not significantly more attrac- negative, i.e., E10,Z12–16:Ald contributed positively to male tive than the unbaited solvent control (P > 0.05) (Fig. 4). attraction, but E10,E12–16:Ald contributed negatively. GLMM analysis of these results showed that the regression models including one (E10,Z12–16:Ald) and two (E10,Z12– 16:Ald and E10,E12–16:Ald) explanatory variables were sig- Discussion nificant (Table 2). The AIC value indicated that the model including two variables was a better fit than the model includ- Analyses of extracts of pheromone glands of female ing one variable. The Poisson regression formula of the best N. himachala sangaica by GC-EAD, GC/MS, and GC lead model was determined to be: ln N = a (0.010 ± 0.002) + b to identification of two potential pheromone components, (−0.026 ± 0.004), where N is the number of males attracted, E10,Z12–16:Ald and E10,E12–16:Ald, at a ratio of 98:2. and a and b are the relative amounts of E10,Z12–16:Ald and Field trapping bioassays and GLMM analysis showed that E10,E12–16:Ald, respectively. The coefficient of E10,Z12– the E10,Z12–16:Ald contributed to attracting males, but E10,E12–16:Ald inhibited this attractiveness. These results ** suggest that the sex pheromone of N. himachala sangaica consists of E10,Z12–16:Ald, bombykal, alone. Although a yad rep de rep yad 15 trace of E10,E12–16:Ald was detected in extracts of phero- mone glands of females, addition of even 2 % of this isomer to E10,Z12–16:Ald significantly reduced its attractiveness in rutpac shtom elaM shtom rutpac 10 field tests, and addition of 10 % completely removed the

Table 2 Comparisons of models explaining the number of male 5 catches to the binary blend

Modela AICb ΔAICc

0 EZ* +EE* 281.4 0 98 EZ 0 100 EZ* 287.7 6.3 EE 002 EE 523.1 241.7 Ratio of EZ and EE isomer Fig 3 Numbers of male Neogurelca himachala sangaica attracted to a EZ: E10,Z12–16:Ald, EE: E10,E12–16:Ald traps baited with 10,12–16:aldehydes and mixtures. Box plots represent b The bold value indicates the lowest AIC (GLMM, coefficient estimate) the minimum observation, 25th, 50th (median), and 75th percentiles, and c Δ the maximum observation; circles are outliers (September 3–11, 2014; The AIC column shows the difference in AIC between the model with total number of moths trapped =132; numbers of replications 15, 19, and the lowest AIC value and each other model 17 for treatments from left) *Statistical significant explanatory variables (P <0.001) J Chem Ecol (2016) 42:517–522 521 attractiveness. The composition of the volatile blend actually pheromone components have similarly-sized glomeruli, emitted by the virgin female moth was not determined. whereas species with a single pheromone have glomeruli of The sex pheromone systems of most hawk moths re- the same type, but with one glomerulus extensively enlarged. ported thus far consist of geometrical isomers of 11- It is proposed that recognition of a mixture of two components hexadecenal and bombykal, and their mixtures. requires two similarly sized glomeruli. When a moth evolves Furthermore, since the first identification of bombykal to using just one of the two components, the volume of either from a pheromone gland of the silk moth Bombyx mori glomerulus may be enlarged to increase the sensitivity to this by Kaissling et al. (1978), it has been reported to be a single component, while at the same time, the other glomeru- pheromone component in many moths. It is likely that lus may change its role to an inhibitory one (Daimon 2014; species-specificity in N. himachala sangaica is main- Namiki et al. 2014). A similar scenario may explain the evo- tained through antagonism of heterospecific pheromone lution of the single-component pheromone of N. himachala components to conspecific components. In our experi- sangaica. However, further investigations of the responsive- ments, addition of 10 % E10,E12–16:Ald to E10,Z12– ness of male antennae and the antennal lobe organization of 16:Ald completely removed its attractiveness to male N. himachala sangaica are required to address this issue. N. himachala sangaica. In previous studies, we reported that two sympatric hawk moth species use E10,Z12– Acknowledgments We thank the two anonymous reviewers for their 16:Ald as a pheromone component, valuable comments on the manuscript. This project was supported by oldenlandiae F. (Lepidoptera: Sphingidae) (Uehara et al. Grants-in-Aid for JSPS Fellows and for Young Scientists (Start-up), 2012)andHemaris affinis (Butler) (Lepidoptera: JSPS KAKENHI Grant Number 15H06854. This research was in partial Sphingidae) (Uehara et al. 2015). Although it was noted fulfillment of a Ph.D. degree (TU) from the University of Tsukuba. that males of N. himachala sangaica were captured in traps baited with E10,Z12–16:Ald alone (Uehara et al. 2015), none were caught in blends containing more than References 30 % of E10,E12–16:Ald (Uehara et al. 2012, 2015). Furthermore, in previous studies in which N. himachala Ando T, Yamakawa R (2011) Analyses of lepidopteran sex pheromones – sangaica naturally occurred, no male moths were attracted by mass spectrometry. Trends Anal Chem 30:990 1002 – Ando T, Ogura Y, Uchiyama M (1988) Mass spectra of lepidopterous sex to a binary mixture of (Z)-11-hexadecenal (Z11 16:Ald) pheromones with a conjugated diene system. Agric Biol Chem 52: and E10,Z12–16:Ald (Uehara et al. 2015), suggesting that 1415–1423 Z11–16:Ald could also inhibit the attractiveness of E10, Baker TC (2008) Balanced olfactory antagonism as a concept for under- Z12–16:Ald. standing evolutionary shifts in moth sex pheromone blend. J Chem – Baker (2008) proposed a theory of olfactory antago- Ecol 34:971 981 Bates D, Maechler M, Bolker B, Walker S (2015) lme4: Linear mixed- nism balance, and introduced a multi-dimensional inter- effects models using Eigen and S4_. R package version 1.1–9, URL pretation to pheromone mixture recognition based on ol- https://CRAN.R-project.org/package=lme4 factory receptors. The theory stressed that even a single- Bestmann HJ, Erler J, Garbe W, Kern F, Martischonok V, Schäfer D, component pheromone must be considered a mixture at Vostrowsky O, Wasserthal LT (1992) Pheromone components of the neurophysiological level. The sex pheromone system the female elephant hawk-moth, elpenor,andthesilver- striped hawk-moth, Hippotion celerio. Experientia 48:610–613 of N. himachala sangaica would be a good example of Crawley MJ (2005) Statistics, an introduction using R. John Wiley, New this in that females produce just bombykal, but males York,p.208 may be able to detect not only the conspecific phero- Daimon T (2014) Title in Japanese. Sanshi-Konchu Biotec 83:105–114 mone but also heterospecific pheromone components in Daimon T, Fujii T, Fujii T, Yokoyama T, Katsuma S, Shinoda T, Shimada mixtures. T, Ishikawa Y (2012a) Reinvestigation of the sex pheromone of the wild silkmoth Bombyx mandarina: the effects of bombykal and It is possible that the pheromone system of N. himachala bombykyl acetate. J Chem Ecol 38:1031–1035 sangaica arose from an ancestral pheromone that consisted of Daimon T, Fujii T, Yago M, Hsu YF, Nakajima Y, Fujii T, Katsuma S, multiple components, like other hawk moth pheromones. IshikawaY ST (2012b) Female sex pheromone and male behavioral According to the molecular phylogenetic tree of hawk moths responses of the bombycid moth Trilocha varians: comparison with those of the domesticated silkmoth Bombyx mori. proposed by Kawahara et al. (2009), the multi-component Naturwissenschaften 99:207–215 pheromone appears to be the most common ancestral trait. Dool HD, Kratz PD (1963) A generalization of the retention index system In Bombycidae, in which single-component pheromone sys- including linear temperature programmed gas–liquid partition chro- tems have been reported in several species, the possible evo- matography. J Chromatogr 11:463–471 lutionary scenario with respect to antennal lobe organization Hothorn T, Bretz F, Westfall P (2008) Simultaneous inference in general parametric models. Biometrical J 50:346–363 was described by Namiki et al. (2014). According to Namiki Kaissling KE, Kasang G, Bestmann HJ, Stransky W, Vostrowsky O et al. (2014), glomerular organization in the antennal lobe is (1978) A new pheromone of the silkworm moth Bombyx mori. associated with pheromone reception. Species with two Naturwissenschaften 65:382–384 522 J Chem Ecol (2016) 42:517–522

Kawahara AY, Mignault AA, Regier JC, Kitching IJ, Mitter C (2009) male responses to pheromone in a flight tunnel. J Chem Ecol Phylogeny and biogeography of hawkmoths (Lepidoptera: 12:229–237 Sphingidae): evidence from five nuclear genes. PLoS One 4:e5719 Reed DW, Underhill EW, Giblin EM (1987) Attraction of sphingid moths Klun JA, Leonhardt BA, Schwarz M, Day A, Raina AK (1986) Female (Lepidoptera: Sphingidae) to 10,12-hexadecadienyl aldehydes and sex pheromone of the pickleworm, Diaphania nitidalis acetates: evidence of pheromone components. J Chem Ecol 13:931– (Lepidoptera: Pyralidae). J Chem Ecol 12:239–249 942 Kováts E (1958) Gas-chromatographische Charakterisierung organischer Tumlinson JH, Mitchell ER, Doolittle RE, Jackson DM (1994) Field tests Verbindungen. Teil 1: Retentionsindices aliphatischer Halogenide, of synthetic Manduca sexta sex pheromone. J Chem Ecol 20:579– Alkohole, aldehyde und ketone. Helv Chim Acta 41:1915–1932 591 Landolt PJ, Tumlinson JH, Brennan MM (1989) Attraction of Amphion Uehara T, Naka H, Matsuyama S, Ando T, Honda H (2012) Identification floridensis (Lepidoptera: Sphingidae) to bombykal, (E,Z)-10,12- and field evaluation of sex pheromones in two hawk moths – hexadecadienal. Fla Entomol 72:324 327 lewisii and Theretra oldenlandiae McElfresh JS, Millar JG (1999) Geographic variation in sex pheromone (Lepidoptera; Sphingidae). Appl Entomol Zool 47:227–232 blend of Hemileuca electra from southern California. J Chem Ecol – Uehara T, Naka H, Matsuyama S, Vang LV, Ando T, Honda H (2013) 25:2505 2525 Identification of conjugated pentadecadienals as sex pheromone McElfresh JS, Hammond AM, Millar JG (2001) Sex pheromone compo- – components of the sphingid moth, tancrei. J Chem Ecol nents of the buck moth Hemileuca maia. J Chem Ecol 27:1409 1422 39:1441–1447 Namiki S, Daimon T, Iwatsuki C, Shimada T, Kanzaki R (2014) Antennal Uehara T, Naka H, Matsuyama S, Ando T, Honda H (2015) Identification lobe organization and pheromone usage in bombycid moths. Biol of the sex pheromone of the diurnal hawk moth, affinis.J Lett 10:20140096 Chem Ecol 41:9–14 Nieukerken EJ et al. (2011) Order Lepidoptera Linnaeus, 1758. In: Zhang, Z.-Q. (Ed.) biodiversity: an outline of higher-level classifica- Wakamura S, Yasuda T, Watanabe M, Kiguchi K, Shimoda M, Ando T tion and survey of taxonomic richness. Zootaxa 3148:212–221 (1996) Sex pheromone of the sweetpotato hornworm, Agrius R Core Team (2015) R: A language and environment for statistical com- convolvuli (L.) (Lepidoptera: Sphingidae): identification of a major component and its activity in a wind tunnel. Appl Entomol Zool 31: puting. R Foundation for statistical computing, Vienna, Austria. – URL https://www.R-project.org/. 171 174 – Raina AK, Klun JA, Schwarz M, Day A, Leonhardt BA, Douglass Young DC, Vouros P, Holick MF (1990) Gas chromatography mass LW (1986) Female sex pheromone of the melonworm, spectrometry of conjugated dienes by derivatization with 4-meth- – Diaphania hyalinata (Lepidoptera: Pyralidae), and analysis of yl-1,2,4-triazoline-3,5-dione. J Chromatogr 522:295 302