Identification of Male-Produced Aggregation Pheromone of the Curculionid Beetle Sternechus Subsignatus

J Chem Ecol DOI 10.1007/s10886-012-0080-3 Identification of Male-Produced Aggregation Pheromone of the Curculionid Beetle Sternechus subsignatus Bianca G. Ambrogi & Angela M. Palacio Cortés & Paulo H. G. Zarbin Received: 16 November 2011 /Revised: 22 December 2011 /Accepted: 12 February 2012 # Springer Science+Business Media, LLC 2012 Abstract Analyses of the headspace volatiles produced weevils prefer legumes, such as Glycine max (L.) Merrill, as by males and females of Sternechus subsignatus hosts. The adult weevils girdle the main stem cutting the Boheman (Coleoptera: Curculionidae) revealed seven epidermis and cortex of soybean. Larvae penetrate the stem male-specific compounds. The major component was and feed on the medulla forming a gall where the plant was (E)-2-(3,3-dimethylcyclohexylidene)-ethanol, and the girdled. During early developmental plant stages, the pres- minor components were 1-(2′-hydroxyethyl)-1-methyl-2- ence of these insects can cause death or eventual decrease in isopropenylcyclobutane (grandisol), 7-methyl-3-methyleneoct- production (Hoffmann-Campo et al., 1990, 1991). Tradi- 6-en-1-ol, (Z)-2-(3,3-dimethylcyclohexylidene)-ethanol, (Z)- tional methods of control using contact insecticides are not and (E)-2-(3,3-dimethylcyclohexylidene)-acetaldehyde, and effective because adults are usually underneath leaves, in the (E)-2-(3,3-dimethylcyclohexylidene) acetic acid. The latter soil or under crop residues. In addition, immature stages compound is described for the first time as a natural product. develop inside stems limiting the action of the insecticides Only four of the seven identified compounds showed electro- (Silva et al., 1998). physiological activity. Enantioselective gas chromatography Pheromones of weevils offer a promising method of showed that the natural grandisol is the (1R,2S)-stereoisomer. direct control through mass trapping (Francke and Dettner, The major component, (E)-2-(3,3-dimethylcyclohexylidene)- 2005; Tinzaara et al., 2005). Male-produced aggregation pher- ethanol, attracted S. subsignatus in olfactometer bioassays. omones have been reported for many weevils, and generally Studies are in progress to evaluate the biological activity of are used for both host-finding and bringing the sexes together the major component and the EAD-active mixture under field (Bartelt, 1999). Previous research with S. subsignatus showed conditions. males produce volatiles attractive to both sexes (Ambrogi and Zarbin, 2008); however, the responsible compounds were Keywords Semiochemicals . Grandlure . Grandisol . unknown. Here, we report the isolation, identification, and GC-EAD . Coleoptera . Curculionidae laboratory bioassay of the male-produced aggregation pheromone of S. subsignatus. Introduction Methods and Materials The soybean stalk weevil, Sternechus subsignatus Boheman (Coleoptera: Curculionidae: Molytinae), is native to Brazil Source of Insects The colony was established from Sterne- where it is broadly distributed (Rosado Neto, 1987). These chus subsignatus adults of unknown age and mating status collected from soybean fields located in Fazenda Rio Electronic supplementary material The online version of this article Grande, Parana, Brazil, in January 2010. Sexes were (doi:10.1007/s10886-012-0080-3) contains supplementary material, separated based on the sexually dimorphic shape of which is available to authorized users. : : the fore and mid-tibia (Rosado Neto, 1987), and kept B. G. Ambrogi A. M. P. Cortés P. H. G. Zarbin (*) separately in plastic boxes (20×20×20 cm) under a Laboratório de Semioquímicos, Departamento de Química, L12:D12 photoperiod at 25±2°C and 60±5% relative Universidade Federal do Paraná, CP 19081, CEP 81531-990 Curitiba, PR, Brazil humidity. Adults were provided with fresh soybean- e-mail: [email protected] stem cuttings. J Chem Ecol Collection of Volatiles Groups of 10 males and 10 females Chemicals 7-Methyl-3-methyleneoct-6-en-1-ol (γ-isogera- were maintained separately in all-glass aeration chambers niol), (E)-2-(3,3-dimethylcyclohexylidene)-ethanol, (Z)-2- (33 cm high×3.5 cm outside diam) with two cuttings of (3,3-dimethylcyclohexylidene)-ethanol, grandisoic acid, and a fresh soybean stems (8 cm). Emitted volatiles were collected 1:1 mixture of (Z)- and (E)-2-(3,3-dimethylcyclohexylidene)- every 24 hr for 30 d (N030), and trapped on glass columns acetaldehyde were obtained from ChemTica Int., Costa Rica. A (8 cm high×1 cm diam) with 0.8 g of Super Q (Alltech, small amount of (E)-2-(3,3-dimethylcyclohexylidene)-acetic USA) (Zarbin et al., 1998). Charcoal-filtered, humidified air acid was obtained by oxidation of the corresponding aldehyde was pushed though the aeration system (1.0 L/min). using silver oxide in situ in aqueous sodium hydroxide (NaOH, Adsorbed aeration volatiles were eluted with 1 ml of dis- 10% m/v) (Pepperman, 1981). Then, aqueous NaOH was tilled hexane, and adsorbent traps were changed after 10 added to an aqueous solution of silver nitrate (10% w/v), collections. The daily extracts were concentrated to 10 μl agitated until achieving complete precipitation of the silver (1 insect per 1 μl) under an argon stream. oxide, filtered, and washed with distilled water. A suspension of silver oxide (20 mg) and NaOH (10% w/v, 30 ml) in 1 ml of Analytical Procedures Gas chromatographic (GC) analyses water was vigorously stirred with a magnetic stirrer. The mix- were conducted to detect sex-specific compounds using a ture of 2-(3,3-dimethylcyclohexylidene)-acetaldehyde (100 μl) Shimadzu GC-2010. The instrument was equipped with a isomers was added to the silver oxide suspension in small DB-5 column (30 m×0.25 mm i.d. and 0.25 μm film thick- portions over 30 mins, and then stirred for 3 hr. The colloidal ness; J&W Scientific Inc., USA), and 1 μl of extract was silver was filtered and washed with water. The filtrate was injected in the splitless mode at an injector temperature of acidified with concentrated HCl, the precipitate was collected, 250°C. The column oven was held at 50°C for 1 min, raised and washed with water. The filtrate was extracted with diethyl to 250°C at 7°C/min and kept constant for 10 min. Helium ether, and the organic phase was extracted with NaHCO3 was used as the carrier gas at a column head pressure of solution to remove the carboxylic acid as its salt. The aqueous 170 kPa. The Kovats indexes (KI) of the sex-specific com- solution was acidified with HCl solution until it reached pH 3, ponents were calculated using a series of saturated C10-C26 and then extracted with diethyl ether. The ether solutions were hydrocarbons co-injected with the samples in the same dried over anhydrous Na2SO4. These procedures were all per- DB-5 GC column (Kovats, 1965). The ratio of the seven formed at room temperature. Pure (1R,2S)-grandisol was male-specific compounds was calculated based on the area kindly provided by Prof. Kenji Mori, Tokyo University, Japan of the GC peaks of twenty extracts. (Mori and Fukamatsu, 1992). Chiral GC of grandisol was performed using an HP- Chiral 20B (30×0.25 mm i.d. and 0.25 μm film thickness) Microderivatizations The latter extract (100 μl) diluted in at a constant temperature of 150°C for 170 min with a gas 200 μl of hexane was reduced over Pd/C (10%, 1 mg) at pressure of 160 kPa. room temperature under a hydrogen (25 psi) in a Parr Extracts were analyzed by GC-electroantennographic apparatus for 2 hr. After filtration, the filtrate was concen- detection (GC-EAD) using a Shimadzu GC-2010, and a trated and analyzed by GC (Zarbin et al., 2000). Syntech electroantennography system (Hilversum, The For esterification, crude extract (100 μl) was diluted in Netherlands). The same column as described previously was hexane (100 μl), and an ethereal solution of diazomethane used, and 1 μl of extract was injected in the splitless mode at was added at 0°C until the solution turned yellow. The an injector temperature of 250°C. Temperature was main- mixture was stirred at room temperature for 30 min, and tained at 70°C for 1 min, raised to 120°C at 4°C/min, elevated directly analyzed by GC (Zarbin et al., 2000) to 250°C at 10°C/min, and held for 10 min. The column effluent was split 1:1, with one part going to the FID at a Laboratory Bioassays Bioassay were performed to deter- temperature of 270°C and the other through a heated transfer mine the attractiveness of the major S. subsignatus male- line into a humidified airstream (280 ml/min) directed to specific component in a glass Y-tube olfactometer 4 cm in antennal preparation of a female or male. Antennae of 30 diam, 40 cm long with 20-cm long arms operated at 4 L/min males and females were excised. The base and distal part of flow of humidified and charcoal filtered air (Zarbin et al., the antenna was fixed between two stainless steel electrodes 2007a). The behavior of 43 males and 38 females was using electrically conductive gel (Signa gel, Parker Labs., NJ). observed individually for 20 min. The insect was considered GC-EAD recordings were analyzed with Syntech GC-EAD32 to have a chosen a particular arm when the weevil traveled software (version 4.6 Hilversum, The Netherlands). 5 cm past the division of the basal tube. Age and mating GC-mass spectrometry (GC-MS) analyses of extracts from status of bioassayed beetles were not controlled. The main males were conducted using a Shimadzu QP5050A MS component present in the extracts was tested, along with a system equipped with a DB-5 capillary column as described host plant (HP) extract (Ambrogi and Zarbin, 2008). For above was used in the electron impact mode at 70 eV. each replication, we used a piece of filter paper (2×2 cm) J Chem Ecol Fig.

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