The First Findings Recently, (results from 1998 season) we reported our findings about the chemical communication in C. ohridella (Svatoš et al., 1999a). Using field experiments (Table 1), wind-tunnel and electroantenno- graphic (EAG) measurements it was indubitable shown that the females are producing and releasing (Fig. 1) a very potent sex pheromone at early photophase (LD 14:10). Table 1. Catches of C. ohridella males in Delta traps, Royal Garden of the Prague castle (1988) Lure 3 Females 3 Males Control Date Moths caught: C. ohridella males (other species) 30.VII.a 113 (27) -nd 5 (1) 10.-12.IX.b 56 (14) 1 (0) 1 (0) 21.-28.IX.b 18 (3) 0 (0) 5 (1) a3rd generation; b4th generation; ndnot determined Fig. 1: Calling behaviour of one-day-old C. ohridella females (n = 1) emerged from infested leaflets. The onset of photophase was set to 9 am (stippled insert). During this active period females are exposing theirs ovipositor ventrally in typical "calling" position. Screening of the hexane washes of excised ovipositors (Fig. 2) of calling females in wind-tunnel and using EAG proved a strong activity of the extracts. Fig. 2: SEM microphotography of excised C. ohridella ovipositor The Pheromone Story The preliminary examination of hexane extracts of the calling females using GC-EAD (Struble and Arn, 1984), where male antennae were used as a biological detector, showed pronounced antennal activity on the EAD trace, but no corresponding GC peak was detected by FID detector (Fig. 3A). Furthermore, when ~ 100 female equivalents (FE) were injected on a GC/MS (ion trap) no reliable mass spectrum was obtained from the EAD-active area. Clearly, the only analytical tools available were: retention behavior of EAD peak on different GC phases examination of antennal specificity to libraries of pheromone-like synthetic compounds (EAG response profiles) micro-derivatizations of gland extracts combined with EAG Fig. 3: Sections of GC-FID-EAD traces A: a hexane extract of C. ohridella calling females (~ 5 FE) B: a synthetic mixture of 8,10-tetradecadienal isomers (100 ng) [on DB-1 phase; both co-injected with 5 ng of hydrocarbon standards] Kovats' indices (KIs) of the EAD peak (Fig. 3A) were determined using several GC phases of increasing polarity and the measured values were compared with KIs of straight-chain aldehydes, alcohols and acetates (Table 2). Table 2. A comparison of Kovats' indices (based on saturated hydrocarbons) of EAD peak in C. ohridella female extracts with some synthetic compounds GC phasea Female extracts (ca 5 FE)b 12:Ac 14:Ald 14:OH EZ-8,10-14:Ald DB-1c 1623.3 - - - 1623.9 DB-5c 1674.4 1605.9 1610.8 1675.4 1674.3 DB-WAXd 2031.2 - - - 2031.8 aJ & W Sci, 30m×0.25 mm, f.t. 0.25 µm; bfor EAD trace; c170 °C; d140-240°C @ 5°C / min Based on these measurements, a series of all geometric isomers of dodecen-1-yl acetates, tetradecen-1-ols and tetradecenals and their saturated congeners (1 mg) were tested on the EAG preparation. Both KIs and the EAG profiles obtained clearly showed that the pheromone should bear the aldehyde functionality and that unsaturation must be situated near the C-9 atom. The aldehydic nature of the pheromone was confirmed by micro-derivatization experiments where the hexane extracts, treated with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride in methanol solution, were tested both on EAG and in a wind tunnel. The pheromonal activity diminished after this derivatization. Unfortunately (E)-tetradec-9-enal which had the highest EAG potential (from EAG profiles) showed low behavioral activity (wind tunnel bioassay) and a different KI (1601.8 on DB-5) in comparison to the natural extract. Therefore, it was not considered to be the sex pheromone. From comparison of KIs on DB-1 and DB-5 phases it seems reasonable to speculate that the pheromone should have more double bonds. Its KI on the DB-5 phase is much higher than the respective KI on a DB-1 phase, which usually points towards conjugation (Attygale and Morgan, 1988). Based on this consideration, and on the measured EAG profiles, we prepared mixtures of all geometric isomers of 7,9-, 8,10-, and 9,11-tetradecadienals using a general strategy shown for the tetradeca-8,10-dienal (7) isomers: (a) lithiumacetylide.EDA complex / DMSO (b) 1. DIBAH / hexane, 2. iodine / THF (c) 1-pentyne, terakis(triphenylphosphine)palladium, n-butylamine, CuI / benzene (d) 1. dicyclohexylborane, 2. AcOH , 3. pH 10 (NaOH) / hydrogenperoxide (e) ferric chloride / acetanhydride (f) 1. NaOH / MeOH, 2. PDC / dichloromethane (g) PhSH / benzene, 80°C The EAG examination of the mixtures of geometric isomers showed that only an isomeric mixture of 7 displays the highest antennal activity. Geometric isomers of the aldehyde 7 were reasonably separated on GC capillary columns and we were able to obtain GC-EAD of the individual isomers. Although males' antennae were, to some extent, sensitive to more than one geometric isomer in the mixture we could clearly eliminate the ZE-7 and EE-7 isomers. The EZ-7 isomer showed higher EAD activity than ZZ-7 isomer (Fig. 2B). When EZ-7 was measured on GC-EAD using several GC phases the corresponding EAG activity showed identical retention behavior (at sub-ng amounts) to hexane- extracted female abdomens (Table 1). In wind tunnel behavior assay 1 - 0.1 pg of the EZ-7 isomer displayed high attractiveness, which was comparable to 3 FE of gland extract (100% of all tested males were activated, took off and 80% of them localized the odor source and tried to copulate with it). In contrast, the pure ZZ-7 displayed a different KI to the natural extract and its behavioral activity was negligible. In preliminary field experiments sticky traps baited with 5 ng of EZ-7 isomer (loaded on BBL Taxo paper disc, 1/2 inch dia) were, similarly to virgin females, highly attractive for C. ohridella males (Fig. 4). All the presented data confirm that EZ-7 isomer is the main component of C. ohridella sex pheromone. Fig. 4: WOW! Result of the first field test of synthetic pheromone (EZ-7) (420 C.ohridella males were caught overnight) Insect antenna is shown here as a powerful analytical tool when spectroscopic techniques reach their sensitivity limits. It is extremely sensitive to pheromone components via s pecialized pheromone receptors, however, at elevated concentrations it, in certain extend, responds to pheromone analogs. Consequently, by screening of libraries of the analogs at different concentrations we can direct our structural information towards the pheromone structure. (8E,10Z)-Tetradeca-8,10-dienal (EZ-7) (8E,10Z)-Tetradeca-8,10-dienal (EZ-7) is, to the best of our knowledge (The Pherolist), a newly discovered sex pheromone and the first identified sex pheromone in genus Cameraria. Other isomers of 7 have been described as attractants for males of other Lepidopteran species; EE-7 for Acrocercops sp. and ZZ-7 for Phyllonorycter sp. The EZ conjugated double bond system is quite common, found for example in bombykol, the first described sex pheromone. The use and preparation of tetradeca-8,10-dienals are protected by Czech Patent: Svatoš, A., Kalinová, B., Hoskovec, M., Kindl, J. and Hrdý, I. (1999): Tetradeca-8,10-dienals and their using as specific leaf-miner sexual attractants. Czech Patent, N° PV-2156-99, 16.VI.1999 (in Czech). The Pheromone Story is a digest of our published papers: Svatoš, A., Kalinová, B., Hoskovec, M., Kindl, J. Hovorka O. and Hrdý, I.: Identification of a new lepidopteran sex pheromone in picogram quantities using an antennal biodetector: (8E,10Z)-tetradeca-8,10-dienal from Cameraria ohridella. Tetrahedron Letters 40, 7011-7014 (1999b). Hoskovec, M., Saman, D., and Svatoš, A.: Synthesis of (8E,14Z)-tetradeca-8,10-dienal, sex pheromone of horse chestnut leafminer (Cameraria ohridella) and all its geometrical isomers. Collect. Czech. Chem. Commun. 65, 511-523 (2000). to Cameraria main page MH © 10.IX.2002