Unusual macrocyclic lactone sex pheromone of Parcoblatta lata, a primary food source of the endangered red-cockaded woodpecker Dorit Eliyahua,b,1,2, Satoshi Nojimaa,b,1,3, Richard G. Santangeloa,b, Shannon Carpenterc,4, Francis X. Websterc, David J. Kiemlec, Cesar Gemenoa,b,5, Walter S. Leald, and Coby Schala,b,6 aDepartment of Entomology and bW. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC 27695; cDepartment of Chemistry, College of Environmental Science and Forestry, State University of New York, Syracuse, NY 13210; and dDepartment of Entomology, University of California, Davis, CA 95616 Edited by May R. Berenbaum, University of Illinois at Urbana–Champaign, Urbana, IL, and approved November 28, 2011 (received for review July 20, 2011) Wood cockroaches in the genus Parcoblatta, comprising 12 species Identification of the sex pheromone of P. lata has important endemic to North America, are highly abundant in southeastern implications in biological conservation and forest management pine forests and represent an important prey of the endangered practices. This species and seven related species in the genus red-cockaded woodpecker, Picoides borealis. The broad wood cock- Parcoblatta inhabit standing pines, woody debris, logs, and snags roach, Parcoblatta lata, is among the largest and most abundant of in pine forests of the southeastern United States, and they rep- the wood cockroaches, constituting >50% of the biomass of the resent the most abundant arthropod biomass in this habitat (4). woodpecker’s diet. Because reproduction in red-cockaded wood- Most importantly, P. lata constitutes a significant portion peckers is affected dramatically by seasonal and spatial changes (>50%) of the diet of the endangered red-cockaded wood- P. lata in arthropod availability, monitoring populations could serve pecker, Picoides borealis (5–7). The red-cockaded woodpecker is as a useful index of habitat suitability for woodpecker conservation especially sensitive to habitat disturbance, and suitable habitats and forest management efforts. Female P. lata emit a volatile, long- fi include old-growth pine with nest cavities, dying and dead pines, distance sex pheromone, which, once identi ed and synthesized, and no hardwood understory and overgrowth (8). Moreover, could be deployed for monitoring cockroach populations. We de- reproduction in the red-cockaded woodpecker is highly de- scribe here the identification, synthesis, and confirmation of the pendent on seasonal and spatial changes in the arthropod com- chemical structure of this pheromone as (4Z,11Z)-oxacyclotrideca- 4,11-dien-2-one [= (3Z,10Z)-dodecadienolide; herein referred to as munity on bark, with successful reproduction occurring when “parcoblattalactone”]. This macrocyclic lactone is a previously un- arthropod production is high and failure to produce viable identified natural product and a previously unknown pheromonal clutches occurring in poorer foraging habitats (9). Because P. structure for cockroaches, highlighting the great chemical diversity lata nymphs develop beneath the bark of dead standing or re- fi that characterizes olfactory communication in cockroaches: Each cently fallen trees, this species also has very speci c habitat long-range sex pheromone identified to date from different genera requirements. These requirements may result in spikes in the belongs to a different chemical class. Parcoblattalactone was bio- abundance of wood cockroaches several years after environ- logically active in electrophysiological assays and attracted not only mental disturbances such as hurricanes or ice storms, which can P. lata but also several other Parcoblatta species in pine forests, knock down large numbers of pines. Availability of a synthetic underscoring its utility in monitoring several endemic wood cock- sex pheromone for P. lata and other Parcoblatta species therefore roach species in red-cockaded woodpecker habitats. could serve as a useful, low-cost, and ecologically sound tool (i) to monitor cockroach populations, (ii) to assess the suitability of pheromone monitoring | surveillance | wildlife conservation foraging habitats, and (iii) to guide management decisions in Southern forests to maximize habitat for arthropod communities. any animals—especially nocturnal insects—have evolved The sex pheromone could be used to monitor spatial and espe- Msexually dimorphic sex pheromones as an efficient and cially temporal changes in the major food source of the red- relatively private communication channel for mate attraction and cockaded woodpecker, ensuring a robust and healthy overall mate choice (1). Sex pheromones also function in species dis- food web. crimination, and therefore pheromone blends also play prom- inent roles in premating reproductive isolation of closely related species and in speciation (1, 2). Author contributions: D.E., S.N., F.X.W., D.J.K., C.G., W.S.L., and C.S. designed research; D.E., Most cockroach species are nocturnal, and they profoundly S.N., R.G.S., S.C., F.X.W., D.J.K., W.S.L., and C.S. performed research; S.N., S.C., and F.X.W. rely on pheromones for intraspecific communication. For ex- contributed new reagents/analytic tools; D.E., S.N., F.X.W., D.J.K., W.S.L., and C.S. analyzed ample, females of the broad wood cockroach, Parcoblatta lata, data; and D.E., S.N., R.G.S., S.C., F.X.W., D.J.K., C.G., W.S.L., and C.S. wrote the paper. fl a species endemic to pine forests of the southeastern United The authors declare no con ict of interest. States, have short wings, are incapable of flight, and therefore This article is a PNAS Direct Submission. have limited dispersal capability. However, by emitting a volatile Freely available online through the PNAS open access option. sex pheromone sexually receptive females are able to recruit 1D.E. and S.N. contributed equally to this work. males, which are excellent flyers, efficiently (3). Females and 2Present address: Center for Insect Science, University of Arizona, Tucson, AZ 85721. nymphs thus are able to remain in the relatively protected hab- 3Present address: Shin-Etsu Chemical Co., Ltd., Tokyo, Japan. itat beneath the sloughing bark of decaying logs, whereas males 4Present address: Department of Chemistry, Yale University, New Haven, CT 06520. fi 5 incur the greater energetic cost of mate- nding and predation Present address: Departament de Producció Vegetal i Ciència Forestal, Universitat de that is associated with greater movement. Our earlier behavioral Lleida, Lleida, Spain. observations suggested the presence of a female sex pheromone, 6To whom correspondence should be addressed. E-mail: [email protected]. emitted during a “calling” display, and anatomical and electro- See Author Summary on page 2705 (volume 109, number 8). fi fi physiological studies con rmed that female-speci c tergal pher- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. omone glands produced the pheromone (3). 1073/pnas.1111748109/-/DCSupplemental. E490–E496 | PNAS | Published online December 19, 2011 www.pnas.org/cgi/doi/10.1073/pnas.1111748109 Downloaded by guest on September 27, 2021 Results 67 PNAS PLUS Our previous analysis showed that the sex pheromone is pro- duced in the anterior seven tergites of sexually mature virgin 54 MW = 194.13 females, and biological activity is contained in hexane extracts or C H O collections of headspace volatiles (3). Gas chromatography (GC)- 12 18 2 electroantennographic detection (GC-EAD) analyses of hexane extracts of tergites from virgin female P. lata consistently revealed 81 four EAD-active peaks, denoted 1, 2, 3,and4 in Fig. 1. The electrophysiologically active compounds eluted in a nonpolar fraction (second hexane fraction) when this extract was frac- Abundance tionated on a silica-gel column. This fraction then was chroma- 93 134 tographed on a silver nitrate-impregnated silica-gel column, and the resulting fractions were monitored by electroantennography (EAG). Compound 1 was found in the 3% and 4% diethyl ether 107 121 194 in hexane-fractions, which were combined and then separated on 152 165 normal-phase HPLC, yielding compound 1 in a semipure fraction that eluted at 22–23 min. This combined fraction (∼100 μL 50 100 150 200 250 hexane representing the extract of 1,400 females) was separated m/z in several sequential preparative GC procedures (10), accumu- 1 Fig. 2. EI mass spectrum of compound 1, also showing its molecular weight lating all collections of compound in the same megabore cap- and deduced molecular formula. illary trap. Finally, the ∼2 μg of compound 1 collected in the capillary trap was transferred directly into an NMR micro- capillary tube using benzene D6, as described by Nojima et al. The proton spectrum was remarkably clean (Fig. 3), high- (11). The NMR sample was prepared at North Carolina State lighting the importance of minimizing solvent and the advantage University, Raleigh, NC, and then was hand-delivered immedi- of using benzene-D6 to elute compound 1 in preparative GC. If ately to the College of Environmental Science and Forestry, State we assume that the olefinic protons are in a 2:1:1 ratio, then University of New York, Syracuse, NY for NMR analyses. In this there are a total of 18 hydrogen atoms. The 18 hydrogen atoms study, we did not pursue the purification and chemical identifi- taken with the probable ester function (i.e., two oxygen atoms) cation of GC peaks 2, 3,and4, which gave relatively weak EAD gave a molecular formula of C12H18O2, with four sites or degrees responses and would require many more females because they of unsaturation. The carbonyl accounted for one unsaturated were present in minute amounts in the extracts. site and two carbon–carbon double bonds (obtained from the The GC-electron impact (EI)-mass spectrum of compound 1 correlation spectroscopy (COSY) NMR spectrum; Fig. S3) showed a base peak at m/z 67 and characteristic fragments at m/z accounted for two other unsaturated functions. Because neither (% intensity relative to the base peak) 54 (87), 134 (27), 152 the IR nor the NMR spectrum indicated other multiple bonds, (2.4), and 194 (7.3) (Fig.