Environmental Entomology, 50(3), 2021, 599–604 doi: 10.1093/ee/nvab022 Advance Access Publication Date: 16 March 2021 Research Downloaded from https://academic.oup.com/ee/article/50/3/599/6174092 by Mathematics Library, University of Illinois at Urbana-Champaign user on 24 June 2021 Chemical Ecology 2-Nonanone is a Critical Pheromone Component for Cerambycid Beetle Species Native to North and South America Weliton D. Silva,1,4, Lawrence M. Hanks,2, Judith A. Mongold-Diers,2 Anna C. Grommes,2 José Maurício S. Bento,1, and Jocelyn G. Millar3 1Department of Entomology and Acarology, University of São Paulo, Piracicaba, SP 13418900, Brazil, 2Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, 3Departments of Entomology and Chemistry, University of California, Riverside, CA 92521, and 4Corresponding author, e-mail: [email protected] Subject Editor: Dong H. Cha Received 5 October 2020; Editorial decision 9 February 2021 AADate Abstract AAMonth An increasing body of evidence indicates that cerambycid beetles native to different continents may share pheromone components, suggesting that these compounds arose as pheromone components early in the evolution AAYear of the family. Here, we describe the identification and field testing of the pheromone blends of two species in the subfamily Cerambycinae that share 2-nonanone as an important component of their male-produced aggregation- sex pheromones, the South American Stizocera consobrina Gounelle (tribe Elaphidiini) and the North American Heterachthes quadrimaculatus Haldeman (tribe Neoibidionini). Along with 2-nonanone, males of S. consobrina also produce 1-(1H-pyrrol-2-yl)-1,2-propanedione, whereas males of H. quadrimaculatus produce 10-methyldodecanol. Field bioassays conducted in Brazil (targeting S. consobrina) and Illinois (targeting H. quadrimaculatus) demonstrated that adults of both species were attracted only by the blends of both their pheromone components, and not to the individual components. The use of the pyrrole as a critical component for the former species is further evidence that this compound is a common pheromone structure among cerambycines in different biogeographical regions of the world. Key words: Cerambycidae, semiochemical, management, monitoring Research over the last 15 yr has revealed that reproduction in cer- Here, we report the identification of pheromone blends for two ambycid beetles of the large subfamily Cerambycinae is mediated cerambycine species, Stizocera consobrina Gounelle (tribe Elaphidiini) by volatile aggregation-sex pheromones produced by males, which native to South America (Monné 2020) and Heterachthes quadrimac- attract both sexes (reviewed in Millar and Hanks 2017). Some ulatus Haldeman (tribe Neoibidionini) from eastern North America pheromone structures appear to be broadly shared among closely (Monné and Nearns 2020). To our knowledge, there is no published related species (e.g., congeners), and even among species in different information about the host species of S. consobrina. Hickories (Carya tribes (Hanks and Millar 2016). There also is increasing evidence species) are the most common hosts of H. quadrimaculatus, and there that pheromone structures are shared by species native to different is some evidence that larvae develop in dead hosts (Linsley 1963a, continents. For example, 3-hydroxyalkan-2-ones and the related MacRae and Rice 2007). Both species have 2-nonanone as a phero- 2,3-alkanediols are pheromone components of cerambycine species mone component, but each has a second, entirely different pheromone native to North and South America, Eurasia, and Africa (Hanks and component that is essential for strong attraction: 1-(1H-pyrrol-2-yl)- Millar 2016), suggesting these structures evolved early in the evolu- 1,2-propanedione (henceforth semanopyrrole) for S. consobrina, and tion of the subfamily. Conversely, pheromones of some cerambycine 10-methyldodecanol for H. quadrimaculatus. Attraction of adults of species appear to be less common and shared only among congeners, S. consobrina to semanopyrrole alone in an earlier study (Silva et al. whereas others may be species-specific (Ray et al. 2011; Zou et al. 2017) was the first evidence of its being an important pheromone com- 2015; Silva et al. 2016a,b; Millar et al. 2017). ponent for any South American cerambycid species. The data reported © The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: [email protected]. 599 600 Environmental Entomology, 2021, Vol. 50, No. 3 here show that the attraction of this species to semanopyrrole is syner- Collection and Analysis of Beetle-Produced gized by 2-nonanone. Compounds Adults of S. consobrina that were used for the collection of head- space volatiles were caught with sentinel traps, outfitted with dry Materials and Methods Downloaded from https://academic.oup.com/ee/article/50/3/599/6174092 by Mathematics Library, University of Illinois at Urbana-Champaign user on 24 June 2021 collection jars, deployed in Anhembi throughout October 2018. Sources of Chemicals Traps were baited with semanopyrrole because this species was at- 2-Nonanone was purchased from Aldrich Chemical Co. (Milwaukee, tracted in significant numbers to this compound during field bio- WI). Semanopyrrole was synthesized as described in Zou et al. assays that targeted other cerambycine species (Silva et al. 2017). (2016), and 10-methyldodecanol as described in Silva et al. (2016a). Traps were serviced daily, and beetles were sent on the date of cap- ture to the Laboratory of Chemical Ecology and Insect Behavior, Study Sites University of São Paulo, Piracicaba (~85 km from Anhembi). Sex of Stizocera consobrina was targeted with a field experiment conducted beetles was determined based on the characteristic punctation on the in a forest remnant of Cerrado (Brazilian savanna) located at the propleura of males (Martins 2005). Beetles were held under labora- Forest Science Experimental Station of the University of São Paulo tory conditions (25 ± 2°C, 60 ± 10% RH, 12:12 (L:D) h, and 5000 (Anhembi SP, Brazil; −22.7060, −48.1669; https://www.esalq.usp.br/ lux light intensity) for 24 h prior to collecting volatiles. Headspace svee/Anhembi/). The experimental site comprised a ~5 ha area cov- volatiles were collected from males and females held individually ered with original forest remnants and mature, reforested tree spe- in 500 ml cylindrical glass chambers with the internal surfaces of cies native to Cerrado biome, including representatives from several half the chamber lined with paper toweling to provide a surface for botanical families, primarily Fabaceae. Immediately surrounding perching and hiding. Beetles were supplied with 10% sucrose solu- the area, there were pasture lands with scattered trees of the species tion in glass vials for nourishment. Headspace volatiles were col- Senegalia polyphylla (D.C.) Britton & Rose and Eucalyptus grandis lected with glass pipettes (8.5 cm long × 0.5 cm i.d.) containing the Hill ex Maiden. adsorbent HayeSep Q (150 mg of 80/100 mesh; Supelco, Bellefonte, The field experiment targeting H. quadrimaculatus was con- PA) held in place with glass wool plugs. Collectors were connected ducted at Forest Glen Preserve in Vermilion Co., Illinois (40.0152, to the outlets of chambers with screw caps fitted with PTFE ferrules. −87.5677; Vermilion County Conservation District). The preserve is Air purified with activated charcoal was pushed with an oil-free ~730 ha in area and consists primarily of beech-maple and oak-hick- compressor pump through the chamber at constant flow of ~150 ml/ ory forest types that have been unmanaged since 1968 (http://www. min regulated by flowmeters. Individual beetles (four males and five vccd.org/). females) were continuously aerated for periods of 48 h as many as three times. System contaminants were monitored by aerating cham- bers that contained only paper towel liners and feeder vials. Volatiles General Methods of Trapping were stripped from collectors by washing with three successive ali- Attraction of beetles to synthesized candidate pheromones was tested quots of 500 μl of dichloromethane into 2-ml silanized amber glass with black cross-vane panel traps (corrugated plastic; AlphaScents, vials, which were stored at −30°C. Aeration extracts were not spiked Portland, OR in Illinois, and traps of similar design that were with an internal standard because there was no plan to quantify custom-built of similar materials in Brazil; Silva et al. 2020). Efficiency emission of pheromone. of both types of traps was improved by coating interior surfaces with Extracts of volatiles from adults of S. consobrina initially were the fluoropolymer dispersion Fluon PTFE (AGC Chemicals Americas, analyzed in Brazil by gas chromatography with flame ionization Inc., Exton, PA; Graham et al. 2010). Traps were suspended from detection (GC-FID) to identify those which contained compounds frames of polyvinylchloride irrigation pipe and positioned ~0.5 m absent in controls, specifically any sex-specific compounds. Two- above the ground. The frames were mounted on 1-m steel reinfor- microliter aliquots were injected into a GC-2010 gas chromato- cing bars driven partway into the ground. Basins of traps used in graph (Shimadzu Corp., Kyoto, Japan) fitted with a capillary column field bioassays contained saturated aqueous NaCl solution with a few (Rtx-1; 30 m × 0.25 mm i.d. × 0.25 μm film; Restek,