Identification of Three Sex Pheromone Components of the Female Saturniid Moth Antheraea pernyi (Lepidoptera: Saturniidae)* Hans-Jürgen Bestmann, Athula B. Attygalle, Thorolf Brosche, Joachim Erler, Hans Platz, Jürgen Schwarz, Otto Vostrowsky, and Wu Cai-Hong Institut für Organische Chemie, Universität Erlangen-Nürnberg, Henkestraße 42, D-8520 Erlangen, Bundesrepublik Deutschland Karl Ernst Kaissling Max Planck Institut für Verhaltensphysiologie, D-8131 Seewiesen, Bundesrepublik Deutschland Chen Te-Ming Department of Biology, Beijing University, Beijing, PR China Z. Naturforsch. 42c, 631 — 636 (1987); received December 12, 1986 Sex Pheromone, Antheraea pernyi, Saturniidae, Lepidoptera By means of electroantennography and single cell recordings, GC and GCMS analyses and GC analysis with EAG detection ( 6 £ ',llZ )6 - ,ll-hexadecadienal, (6£,11 Z)- 6 ,ll-hexadecadienyl ace­ tate and (4£,9Z)-4,9-tetradecadienyl acetate were identified as the primary components of the sex pheromone of female Antheraea pernyi (Lepidoptera: Saturniidae). The composition of two female saturniid moth sex Material and Methods pheromones has been reported to date, (Z)-5- Insect material decenyl isovalerate as the attractant of the pine em­ peror moth, Nudaurelia cytheraea (F.) [2], and a mix­ Pupae of Antheraea pernyi were provided by the Institute of Silkworm Science of Liaoning Province, ture of (6£,11 Z)-6 ,ll-hexadecadienyl acetate and PR China. The insects were raised in the laboratory ( 6 £",11 Z)-6 ,ll-hexadecadienal as the pheromone of the wild silkworm moth, Antheraea polyphemus on wild oak foliage, two generations each year. The (Cramer) [3], first generation pupae, raised between March and In the course of electrophysiological studies on in­ July, dit not diapause. The second generation co­ terspecific relationships of pheromone perception in coons were maintained in underground holes, at 5 °C saturniid moths [4], we were interested in the com­ during the winter time. After diapause, raising the position of the pheromone blend of another silk­ temperature to 20—25 °C caused emergence three to worm moth, Antheraea pernyi (Saturniidae). This four weeks later. species appears in the north-eastern parts of China. The pupae were sexed and kept at 22 °C in plastic boxes lined with moistened filter paper. A reversed * Pheromones 59 [1]. 16:8 h light: dark cycle was maintained throughout Abbreviations: LC, liquid chromatography; GC, gas chro­ the study. Eclosed adults were collected daily. To matography; FSCC, fused silica capillary column; GCMS, observe the calling behaviour, the insects were ex­ GC combined mass spectrometry; EAG, electro- amined under a red light during the scotophase. antennogram; EAD, electroantennographic detector; FID, flame ionisation detector; E6Z11-16:A1, (6£\11Z)-6,11- hexadecadienal; E6Zll-16:Ac, ( 6 £,llZ)-hexadecadienyl acetate; E4Z9-14:Ac, (4£,9Z)-4,9-tetradecadienyl ace­ Pheromone extraction tate; E6-16:Ac, (£)-6-hexadecenyl acetate; Z 11-16:Ac, (Z)-ll-hexadecenyl acetate; 16:Ac, hexadecyl acetate; Abdominal tips of individual virgin females were TCD, thermal conductivity detector. excised and stored in 1 ml hexane at —20 °C. The Reprint requests to Prof. Dr. H.-J. Bestmann. hexane extract was filtered through glass wool and Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen concentrated by blowing a fine stream of nitrogen 0341 - 0382/87/0500 - 0631 $01.30/0 over it. Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 632 H.-J. Bestmann et al. ■ Pheromones 59 LC fractionation compounds present in the column effluent were de­ termined (electroantennographic detection, EAD). LC fractionation was performed on a 30 x 1.5 cm silica gel column (0.06—0.2 mm) by an eluent system of increasing polarity [5]. Hexane extracts of abdomi­ GC peak trapping of the tetradecadienyl acetate nal tips were separated into 15 fractions [5] and the The glandular tissues of 5 calling females were ex­ fractions were reconcentrated before use. tracted into 10 |a1 of hexane. The extract was reduced to 5 |xl and injected onto a 4% OV-17 GC column GC fractionation (glass, 80—100 mesh Gas Chrom Q, 2 m x 4 mm, A 2 m x 0.25" steel GC column, packed with 10% 210 °C isothermal, Hewlett-Packard 5750 G gas SE30 on Chromosorb W, 100/120 mesh, mounted in chromatograph). The effluent was split 99:1 (trap: a HP 5750 gas chromatograph equipped with a ther­ FID, all glass splitter). The effluent volume corre­ mal conductivity detector was used. sponding to that of C14-acetates was trapped in a glass tube cooled in dry ice. Five |a1 each of crude hexane extracts were injected (inj. temp. 250 °C, oven 180 °C, det. 250 °C) and the effluent, after passing the TCD, was condensed in Microozonolysis cooled (—78 °C) pasteur pipettes. The pipettes were Dry ozone was passed through the glass tube con­ exchanged at two-minute intervals and each conden­ taining the sample to be ozonised, via a fused silica sate was dissolved in 150 |xl hexane each. The solu­ tube (0.4 mm OD) for 20 sec, until 0 3 eluted from tions were subsequently used for electrophysiological the other end and turned a piece wet starch-KI paper testing. blue. The tube was immediately sealed and chromatographed by a solid sample injection tech­ Encapsulation of pheromone glands nique. The abdominal tips of 3-day-old female moths were excised during the period of maximum calling. Results and Discussion The intersegmental membrane between the seg­ Electrophysiological studies ments VIII and IX containing the pheromone com­ ponents was excised under a binocular microscope Single sensillum recording performed with sensory according to Attygalle et al. [6 ]. The samples were hairs of male antennae of A. pernyi resulted in the sealed in soda glass capillaries ( 2 cm x 2 mm) and detection of three types of receptor cells. Two of used for immediate analysis or stored at —20 °C. these cells were most effectively excited by the two compounds, ( 6 £ ',llZ )-6 ,ll-hexadecadienal and Gas chromatography with solid sample injection (6 £ ,llZ )-6 ,ll-hexadecadienyl acetate, already known as the pheromone components of the satur- Capillary gas chromatography with flame ioniza­ niid moth A. polyphemus [3]. This result indicated tion detection was performed with a Packard United- that these two substances can also be regarded as Technologies 438 A instrument equipped with a split- candidates for pheromone components of A. pernyi. less injector and a Shimadzu Chromatopac C-R3A This was supported by the fact that male A. pernyi data system. FSCC (25 m x 0.22 mm) SP-2340, antennae showed a complete electroantennogram 0.21 fx film, 60 °C for 2 min, 60—195 °C at 4 °C/min. reaction to gland volatiles of female Antheraea Samples sealed in glass capillaries were chromato­ polyphemus [8 ] and have two types of receptor cells graphed by a solid sample injection technique [ 6 ]. responding very sensitively to these two compounds [9]- Gas chromatography with EAG detection Furthermore, morphological investigations The effluent of a CPSil 19 FSCC (25 m x 0.22 mm, showed that the sensory hairs of male antennae of mounted in a Packard United-Technologies gas A. pernyi have rarely a third receptor cell [10]. In chromatograph) was split and one part of it directed A. polyphemus, nerve impulses of a third cell have over a male insect antenna [5, 7], By means of im­ been recorded [9] suggesting the existence of a third planted capillary electrodes on the isolated antenna pheromone component. One (or more) pheromone the retention times of electrophysiologically active components other than E6Z11-16:A1 and E6Z11- H.-J. Bestmann etal. • Pheromones 59 633 16: Ac were predicted for A. pernyi based on com­ decadienyl acetate (type B). Since A. polyphemus parisons with other saturniid species [9]. antennae produce larger response amplitudes to the From abdominal tips of 15 female moths a hexane acetate, and those of A. pernyi are more responsive extract was prepared. The extract was gas to the aldehyde, the respective antennae can be em­ chromatographically fractionated, and each fraction ployed as biological detectors for these substances. was tested by the electroantennogram technique With the aid of these detectors, the presence of (EAG) as well as with single cell recording of single E 6 Z 11-16:Al as well as E 6 Z 11-16:Ac in certain gas sensory hairs of male Antheraea antennae. EAG re­ chromatographic fractions of hexane extracts of A. cordings performed with A. pernyi male antennae pernyi glands was established. These fractions had gave significantly high response amplitudes to those the same retention times as those of authentic sam­ fractions having the retention times corresponding to ples of E6Z11-16:A1 and E6Zll-16:Ac, respec­ those of E6Z11-16:A1 and E6Zll-16:Ac, respec­ tively. tively. Similarly, single cell recordings of the GC A quantification of the relative amounts of these fractions were performed with sensory hairs of males two pheromone components could be made by com­ of the related saturniid A. polyphemus. The sensilla paring the impulse rates of sensory cells of male trichodea of this species possess two specialized types A. polyphemus antennae given by the respective of receptor cells, one maximally responding to fractions of female A. pernyi and A. polyphemus ex­ ( 6 11 Z)-6 ,ll-hexadecadienal (cell type A in tracts, with those from known quantities of E6Z11- Fig. 1) and the other one to ( 6 £ ',llZ )-6 ,ll-hexa- 16:A1 and E6,Zll-16:A c (Table I).