J Chem Ecol (2016) 42:517–522 DOI 10.1007/s10886-016-0714-y Single-Component Pheromone Consisting of Bombykal in a Diurnal Hawk Moth, 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 moths 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 (Lepidoptera: Sphingidae), 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, Smerinthinae, 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 Insects Late-instars of N. himachala sangaica were collected compositions were calculated from the ratio of GC peak areas. from skunkvine, Paederia scandens (Rubiaceae), 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.