Male-Produced Sex Pheromone of the Carrion Beetles, Oxelytrum Discicolle and Its Attraction to Food Sources

J Chem Ecol (2013) 39:1056–1065 DOI 10.1007/s10886-013-0329-5 Male-Produced Sex Pheromone of the Carrion Beetles, Oxelytrum discicolle and its Attraction to Food Sources Douglas H. Fockink & Kleber M. Mise & Paulo H. G. Zarbin Received: 3 May 2013 /Revised: 4 July 2013 /Accepted: 8 July 2013 /Published online: 6 August 2013 # Springer Science+Business Media New York 2013 Abstract Carrion beetles are part of the great diversity of Introduction insects collected on cadavers. In Brazil, beetles of the genus Oxelytrum have great forensic importance in post mortem inter- Species of Silphidae are known as carrion beetles because val (PMI) estimation. We investigated the system of chemical they are commonly encountered in vertebrate carcasses (Peck communication in the attraction of these necrophagous beetles. and Anderson 1985). As they seem strictly necrophagous Gas chromatographic analysis (GC) of female and male aeration during larval stages, Silphidae are recognized as useful in extracts revealed the presence of two male-specific compounds, forensic investigations because they may be indicators of the produced in a ratio of 94:6. Bioassays showed that the combina- post mortem interval (PMI) when they are found in corpses tion of male produced volatiles and the odor of a food source (Payne 1965;Smith1986) The family is recognized as an (carcass volatiles) were attractive to females. Mass and infrared important forensic indicator; in fact, Oxelytrum species al- spectral analyses of the male-specific compounds suggested that ready are being used to determine the PMI of corpses in they were both unsaturated hydrocarbons. Several micro- Argentina (Oliva and Di Iorio 2008). Moreover, there are derivatizations were carried out with the natural products, and records of occurrences in the initial stages of decomposition the target structures were identified as (Z)-1,8-heptadecadiene of carcasses in Brazil (Mise et al. 2007; Ururahy-Rodrigues (major) and 1-heptadecene (minor). The structure of the minor et al. 2010). Despite their forensic importance, no prior studies component was assigned by co-injection with a commercial have addressed the chemical ecology of Oxelytrum species. standard. A seven-step synthesis was developed to synthesize Silphid species of the genus Nicrophorus are known to (Z)-1,8-heptadiene, which co-eluted with the major natural prod- feed and reproduce on small vertebrate carcasses. Haberer uct on three different GC stationary phases. Y-tube olfactometer et al. (2008) verified that males of Nicrophorus vespilloides assays showed that the mixture of synthetic standards in the release sex pheromones that attract females. However, males naturally occurring proportion was slightly attractive to females. of Dermestes maculatus (Coleoptera: Dermestidae) use a The results contribute both to the understanding of the chemical combination of sex pheromones and decomposing odors that ecology of O. discicolle and to its potential to improve the attract females (von Hoermann et al. 2011, 2012). accuracy of PMI estimation. The aim of this paper was to test whether Oxelytrum discicolle shows a similar behavior and to investigate the Keywords Coleoptera . Silphidae . Forensic entomology . system of carcass invading as well as chemical communica- Micro-derivatization . (Z)-1,8-heptadecadiene . GC/FT-IR . tion among conspecifics. The study focused primarily on the Post mortem interval identification and syntheses of sex pheromones. : : D. H. Fockink K. M. Mise P. H. G. Zarbin Methods and Materials Laboratório de Semioquímicos, Departamento de Química, Universidade Federal do Paraná, CP 19081, CEP 81531-990 Curitiba, PR, Brazil Insects * P. H. G. Zarbin ( ) A colony of Oxelytrum discicolle was started with insects Departamento de Química – UFPR, CP 19081, CEP 81531-990 Curitiba, PR, Brazil collected from an araucaria forest within the experimental e-mail: [email protected] area of UFPR, Paraná, Brazil (25º25′S; 49º14′W). Newly J Chem Ecol (2013) 39:1056–1065 1057 emerged adults were separated by sex and kept in cages 50 MHz, respectively) as CDCl3 solutions. Chemical shifts (40×20×20 cm) at 25±1 °C, with 65±15 % relative humid- were expressed in ppm relative to TMS. ity and a 12/12 h light/dark photoperiod. Insects were fed with ground meat. Microderivatization Collection of Volatiles Catalytic Hydrogenation with Palladium on Charcoal (Pd/C) Volatiles were collected by the aeration method (Zarbin et al. 1999, 2009). Seven insects of each sex were placed in aeration Extracts of aerations from males in hexane were added to chambers (33×3.5 cm ID.) containing daily moistened cottons ~0.5 mg of Pd/C (5 % Pd) in a glass vial. A balloon filled and a continuous 1 L/min flow of humidified charcoal-filtered with hydrogen was attached to the vial, and the reaction was air. The collecting apparatus was maintained at the same stirred for approximately 2 h. The product solution was temperature and photoperiod as the colony. The volatiles were filtered and analyzed by GC/MS (Attygalle 1998). captured in glass traps (11×1 cm ID) containing 20 mg of HayeSep-D adsorbent polymer (Analytical Research systems, Partial Reduction with Diimine Inc., Gainesville, FL, USA). The adsorbed volatile compounds were eluted from the polymer once a day with doubly The extract (10 μl) containing insect volatiles was evaporat- distilled hexane (400 μl) and concentrated under argon to ed to near dryness and then diluted with 20 μl of absolute 160 μl. Extracts were stored at −20 °C for chemical analyses ethanol in a vial. Subsequently, 10 μl of hydrazine hydrate and bioassays. To determine the day cycle, collections were (10 % in ethanol) and 10 μl of hydrogen peroxide (0.6 % in made every 12 h. The carcass volatiles of a laboratory rat ethanol) were added. The vial was heated to 60 °C for (Rattus norvegicus) were collected in the bloated phase using 20 min. After cooling to room temperature, the reaction the procedures described above. was quenched with 20 μl of HCl (10 % in water) and extracted with three portions of hexane (40 μl each). The Chemical Analysis organic extract was dried over anhydrous sodium sulfate and evaporated under nitrogen to the initial volume of about Extracted volatiles were analyzed with a Shimadzu GC2010 10 μl, of which 1 μl was analyzed by GC/MS (Attygalle gas chromatograph equipped with an FID detector, a RTX-5 1998). (Restek, 30 m×0.25 m×0.25 μm film thickness) capillary column, and helium as the carrier gas. The GC was operated Thiomethylation with Dimethyl Disulfide (DMDS) in splitless mode (250 °C). The temperature began at 100 °C for 1 min, increased at 7 °C/min until reaching 250 °C, and A9-μl portion of the product of partial reduction was added maintained at this temperature for 10 min. To determine the to 10 μl of dimethyl disulfide (DMDS) and 10 μl of iodine Kovats indices (Lubeck and Sutton 1983) and to examine the solution (5 % in carbon disulfide). The reaction mixture was co-injection of the natural products with synthetic standards, maintained at 60 °C for 24 h in small-volume sealed vials. RTX-WAX (Restek, 30 m×0.25 m×0.25 μm film thickness) Excess iodine was reduced with sodium thiosulfate (10 % in and ECTM-1 (Alltech, 30 m×0.25 m×0.25 μm film thick- water) and extracted with three 20-μl portions of hexane. ness) capillary columns were employed. The organic extract was dried over anhydrous sodium sul- Gas chromatography–mass spectrometry (GC/MS) data fate, evaporated under nitrogen to the initial volume of about were acquired using a Shimadzu QP2010-Plus electron ion- 10 μl, and 1 μl was analyzed by GC/MS (Attygalle 1998; ization mass detector operating in electron impact mode Jham et al. 2005). (70 eV) with an RTX-5 (Restek, 30 m×0.25 m×0.25 μm) For microderivatization of the extract obtained from aer- capillary column. The injector mode and program tempera- ations, the reaction mixture was heated to 90 °C for 48 h. ture were the same as described above. Extraction procedures were identical to those performed with Volatiles also were analyzed by coupled gas chromatog- the partially reduced product. raphy - Fourier transform infrared spectroscopy (GC/FT-IR) with a Shimadzu GC2010 gas chromatograph linked to a Syntheses DiscovIR/GC infrared detector (4000–750 cm−1, resolution of 8 cm−1, Spectra Analysis, Marlborough, MA, USA). The 7-Bromoheptan-1-ol (4) capillary column, injector mode and program temperature were the same as those described above. To a mixture of 1,7-heptanediol (3) (4.00 g, 30.30 mmol) and 1H- and 13C-NMR spectra of the synthetic compounds toluene (40.0 ml), 48 % aqueous HBr (2.0 ml) was added. The were recorded on a Bruker ARX-200 spectrometer (200 and heterogeneous mixture was stirred and heated at reflux for 6 h 1058 J Chem Ecol (2013) 39:1056–1065 while trapping the water by using a Dean-Stark apparatus. yield (1.09 g, 0.32 mmol) (Kang and Park 1988). IR Max, TLC analyses indicated that substantial amounts of diol 3 still cm−1: 870, 1034, 1085, 1206, 1352, 1464, 2856, 2931. 1H remained. Thus, an additional quantity of HBr (2.0 ml) was NMR(200MHz,CDCl3,ppm):δ 0.84–0.91 (m, 3H); 1.24– added, and the mixture was heated at reflux for a further 12 h, 1.90 (m, 28H); 2.10–2.22 (m, 4H); 3.32–3.55 (m, 2H); 3.68– 13 at which time TLC analyses showed no diol 3 remaining. The 3.93 (m, 2H); 4.56–4.60 (m, 1H). C NMR (50 MHz, CDCl3, reaction mixture was allowed to cool to room temperature, ppm): δ 14.2; 18.4; 18.2; 19.8; 22.7; 25.6; 26.2; 28.6; 28.9; and the phases were separated.

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