Notes 657

A Fungal Metabolite as the pheromones may be due to different feeding tech­ Male Wing Gland Pheromone of the Bumble-Bee niques, and an unified biogenetic scheme for long- Wax Moth, Aphomia sociella L. chain aliphatic pheromones, which constitute the Gerhard Kunesch and Pierre Zagatti most prevalent class of compounds, may exist. We wish to report evidence for a totally different INRA-CNRS, Laboratoire des Mediateurs Chimiques, 78470 Saint Remy-les-Chevreuse, France source of pheromones in Lepidoptera. In the course Andre Pouvreau of our study of several Galleriinae species [4—6], we INRA-CNRS, Station de Recherche de Physiologie Sen­ investigated the behaviour and chemical communica­ sorielle et Comportementale des Invertebres, 91440 Bures- tion of Aphomia sociella L ., which is a major parasite sur-Yvette, France of useful, pollinating insects such as bumble-bees. Renee Cassini The predatory effect is exerted by the caterpillar INRA-SRIV, 78000 Versailles, France which not only destroys the waxy material of the Z. Naturforsch. 42c, 657—659 (1987); nests but also feeds on eggs, larvae, and pupae of the received October 14, 1986/January 2, 1987 host [7]. Aphomia sociella, Galleriinae, Male Pheromone, Fungal Female-emitted pheromones are well-known in Metabolite, Mellein Pyralid subfamilies such as the Phycitinae, Pyrausti- 3,4-Dihydro-9-hydroxy-3-methylisocoumarin [^(-)- nae or Crambinae, but they do not occur in Gal­ mellein] was identified as the major component of the male leriinae. In this subfamily, male pheromones emitted wing gland pheromone of the bumble-bee wax moth, Aphomia sociella L. (Pyralidae, Galleriinae). The presence by wing glands [ 8 ] evoke searching behaviour by of Aspergillus ochraceus Wilhelm, a mellein producing females [4—11]. microorganism, was detected in the intestines of last-instar Olfactometric tests indicated that the wing gland larvae and in bumble-bee nests. Arguments for a new type of insect-fungus relationship are presented. extracts of male A. sociella were active on females. A GC/MS analysis of the hexane extract of 120 wing glands revealed the presence of only two volatile Most sex pheromones identified in female moths compounds. The first one, a minor constituent until now are straight-chain alcohols, acetates or (< 5%), showed a molecular peak at mlz = 152 and aldehydes with 12—18 carbon atoms and one or two fragments at mlz = 84 as well as a base peak at mlz = double bonds. A general biosynthetic pathway in­ 69. This compound could be identified, by compari­ son with an authentic sample, as (Z)-2,6-nonadien- volving A 11 desaturation and chain-shortening of hexadecanoic acid as key steps, which explains most 4-olide (1) (C 9 H 1 2 0 2) previously isolated from Aphomia gularis Zeller by Y. Kuwahara [12]. structural features, has been proposed [ 1 ]. Slightly differing results have been obtained in the The identity of the major constituent with ( R)- mellein (ochracin) ( 2 ) was established by comparing study of the biosynthesis of (£)-l 1 -tetradecenal in the Eastern spruce budworm Choristoneura fumifera- its physical constants (GC-retention time, mass na [2]. Labelling studies suggest a de novo formation spectrum, and circular dichroism) with those of an of long-chain aliphatic compounds from acetate via authentic sample [13 — 14], an alcoholic precursor. On the other hand, a similar Pure (jR)-mellein, tested (5 jj,g) alone on 2 days-old investigation of the biosynthesis of (Z)-ll-hexade- virgin females, evoked the same searching behaviour cenal, a pheromone component of the rice stem as the crude extract, thus confirming its role as the borer, Chilo suppressalis, provided evidence for a major component of the wing-gland pheromone of pathway beginning with the reduction of hexadeca­ A. sociella. noic acid into the corresponding aldehyde followed Mellein (2) has previously been found in insect by introduction of the double bond in position 11 [3]. secretions on several occasions. For instance, a blend Some of the above mentioned differences in the of volatile compounds including mellein ( 2 ) is re­ biosynthetic pathways of Lepidopterous sex leased by the hair-pencil organs of the male Oriental fruit moth, Grapholita molesta Busck, which exerts a close-range attraction on calling females [15]. Mel­

Reprint requests to G. Kunesch. lein ( 2 ) has also been detected in the male mandibu­ Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen lar gland secretions of some carpenter ant species 0341-0382/87/0500-0657 $01.30/0 (e.g. Camponotus pennsylvanicus, C. herculeanus 658 N otes and C. noveboracensis [16], and in the gaster of O HO Rhytidoponera metallica [17]. In spite of the fact that mellein (2) has been found several times in various insect secretions, the pres­ ence in insects of a typical fungal metabolite [18] was intriguing, especially since an entomogenous strain of Aspergillus flavus has been isolated from pupae of the wax moth, Galleria mellonella, another Galleriinae species [19], These observations prompted us to look for a pos­ sible exogenous origin of mellein (2) in A. sociella. A systematic search for mellein-producing microorgan­ isms was undertaken at various instars and with adult males of A. sociella. (A. sociella), which feeds on infested pollen and When wing glands of adult male moths were care­ wax. The exact mechanism by which one of the fully clipped under sterile conditions and maintained metabolites of the fungus ends up in the wing glands in pure culture on Czapek’s agar medium, no fungus of male bumble-bee wax moths remains unknown. could be detected. A parallel search for fungi in the The biosynthesis of insect sex pheromones by intestines of adult males was equally unsuccessful. In microorganisms will most probably remain a rare contrast, several fungus strains could be isolated event. But the origin of mellein (2) and other fungal from the alimentary canals of last instar larvae. One metabolites like (3) [23] and (4) [24] in insect secre­ of these strains was identified as Asp. ochraceus tions (particularly of social insects) deserves certainly Wilhelm [20], reconsideration. The role of microorganisms in the In order to check its capability to produce mellein synthesis of aggregation pheromones was thoroughly (2), Asp. ochraceus was cultivated on Fries’s mod­ investigated. For example, Bacillus cereus isolated ified liquid medium for one month [21]. Mellein (2) from Ips paraconfusus guts converts a-pinene to cis- was identified by RF on TLC, its characteristic bright verbenol, one component of the aggregation phero­ fluorescence at 366 nm, GC-retention time, and its mone of this species [25]. Woods decayed by the diagnostic mass spectral fragmentation. fungus Lenzites trabea produce (Z)-3,(£,£)-6,8-

The presence of a widespread mellein producing dodecatrienol- 1 , which is also found to be used as a microorganism in the alimentary canals of last-instar trail pheromone by the termite Reticulitermes vir- larvae of A. sociella may suggest a biosynthesis of the ginicus [26]. A close examination of the habitat of wing gland pheromone by a fungus present in the many social insects may provide evidence for a new intestines. The passage of exogenous chemicals type of relationship between insects and fungi, in through the gut walls of insects is not without prece­ addition to the well explored use of “fungus gardens” dent. Male Danaus butterflies obviously use dietary for nutrition [27, 28]. alkaloids to synthesize the chief volatile component of their scent organs [ 2 2 ], Materials and Methods Laboratory-reared A. sociella feed on a non­ sterilized diet of pollen and wax. A sample of a bum- Insects ble-bee nest was maintained in pure culture on Laboratory-reared Bombus nests were exposed Czapek’s agar medium and a mellein-producing Asp. outdoor until colonization by A. sociella larvae was ochraceus was detected among several other fungal observed. The caterpillars were fed with bumble-bee strains. It should be noted that the chromatographic wax and pollen. analysis of a methylene chloride extract of a bumble­ Histological investigations revealed that the male bee nest did not provide any evidence of mellein ( 2 ) moths possess a gland located on the basal part of the in the food of larvae. Thus, it seems that a wide­ forewing. These glands were carefully clipped and spread microorganism (Asp. ochraceus), which is soaked in hexane for 2 h at room temperature. The introduced by bumble-bees into their honey-combs resulting extract was then filtered and reduced to synthesizes the sex-pheromone of a parasite about 100 |il under N 2 flow. Notes 659

Each female moth to be tested was placed inside a sis was accomplished on a Ribermag R-10-10C (Ner- small cylindrical box (5 cm x 8 cm diameter). Crude mag) equipped with a Sidar data acquisition system. extracts or synthetic compounds were blown over the The fungus cultures were separated from the female through a pasteur pipette. The female moth medium by ultrafiltration. The medium was then ex­ which responded positively to the stimulation had to tracted twice with ethyl acetate. The organic layer climb up the inner walls of the box and escape in less was dried over anhydrous MgS0 4 and evaporated than 2 min. This climbing behaviour is very similar to after filtration. The crude extract obtained was then that of Eldana saccharina [4]. purified by preparative thin-layer chromatography (hexane:ethyl acetate = 9:1). Analyses Crude extracts and pure compounds were studied Acknowledgements by gas chromatography (GC) (Fractovap 2900 Carlo We thank Professor Y. Kuwahara for a sample and Erba, capillary column 25 m, CPSil 5 CB, isother- spectral and chromatographic data of (Z)-2,6-non- mally operated at 140 °C). Micropreparative GC was adien-4-olide, C. F. Bousquet for providing us with performed on a 2 m ('/s") column packed with 3% a sample of mellein and C. Malosse for GC and SE-30 on GCQ at 150 °C (Perkin-Elmer 3920). Gas GC-MS measurements. We thank also Professor J. chromatography-mass spectrometry (GC-MS) analy- Meinwald for helpful comments on the manuscript.

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