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. 2). The chemical ionization spectra of we assumed a ring, and, because it is an ester, a lactone structure compound 1 with either ammonia or isobutane as the reagent gas seemed likely. 1 showed m/z 195 [M+H]+ as the base peak (Fig. S1), indicating The H-NMR spectrum conspicuously lacked a methyl group a molecular weight of 194, in good agreement with the EI and nonolefinic methine groups (Fig. 3). Also, the COSY spec- spectrum. The fragmentation pattern did not offer many clues to trum indicated that each carbon–carbon double bond was di- the structure but retrospectively provided support for the final substituted and that each olefinic carbon was bonded to one structure. The vapor-phase IR spectrum revealed a strong car- hydrogen atom (Fig. S3). Taken together, this information led to − bonyl peak at 1,752 cm 1, consistent with a nonconjugated ester the conclusion that the compound is a macrocyclic lactone with function. The IR spectrum also showed good evidence of both two isolated double bonds and no branching; in other words, the fi aliphatic and olefinic C—H stretching with multiple peaks compound is a 13-member ring ester. The positions and con g- − around 3,000 cm 1 (Fig. S2). The final structure of compound 1 urations of the double bonds were the only remaining questions. was elucidated largely from NMR experiments. The position of one double bond (carbons numbered 11 and 12 in Fig. 3) could be determined directly from the COSY spectrum (Fig. S3) and the chemical shift of the methylene doublet at 4.41 ppm. The COSY spectrum showed correlation EAD 1 between the proton on carbon 12 at 5.64 ppm and the methylene ECOLOGY group at 4.41 ppm. The chemical shift of this methylene group “ ” EAD 2 was consistent with its being sandwiched between the ester oxygen and a carbon–carbon double bond. Olefinic proton 11 showed correlation to methylene group 10. The position of the other double bond was determined anal- 4 ogously. The COSY spectrum showed a correlation between 3 olefinic proton 4 at 5.62 ppm and methylene group 3 at 2.84 ppm 1 (Fig. S3). The chemical shift of methylene group 3 must be be- CHEMISTRY 2 FID tween the ester carbonyl and the other double bond. The COSY and total correlation spectroscopy (TOCSY) spectra (Figs. S3– S6) allowed assignment of all of the protons listed in Table S1. 15 16 17 18 19 20 The configuration of double bonds at positions 4 and 11 was Retention time (min) determined by simultaneous decoupling of methylene group 6 Fig. 1. Characterization of an extract of the sex pheromone glands of and methylene group 10. When methylene group 6 was decou- Parcoblatta lata virgin females by coupled GC-EAD. FID is flame ionization pled from proton 5, the only remaining coupling was to proton 4. detector response; EAD electroantennographic response; numbers 1–4 de- The coupling constant was 10.5 Hz, which allowed only the note electrophysiologically active peaks consistently found in P. lata extracts. Z-configuration. Similar reasoning was applied to the decoupling

Eliyahu et al. PNAS | Published online December 19, 2011 | E491 Downloaded by guest on September 27, 2021 10.5 Hz 10.9 Hz 12 4 11 5

5.65 5.60 5.55 5.50 5.45 ppm 5.65 5.60 5.55 5.50 5.45 ppm Homo decoupled

O 7,8,9 13 O 3 Natural 1 2 13 3 12 4 (Z) (Z) 5 6 10 11 10 7 6

89 2.00 2.00 1.00 1.00 4.01 2.01 6.00

Impurity

Synthetic 2.02 6.87 2.00 1.01 1.00 2.00 4.02 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

Fig. 3. NMR of compound 1 purified from an extract of the sex pheromone glands of P. lata virgin females. (Top)Olefinic region (Left) and homo decoupling experiment with both sets of protons 6 and 10 decoupled simultaneously (Right). (Middle) Natural compound, (Bottom) Synthetic parcoblattalactone.

of methylene group 10 from proton 11, which gave a coupling P = 0.035) (Fig. S9). Adhesive-covered (sticky) traps also were constant of 10.9 Hz and therefore also a Z-configuration. significantly more effective than pitfall traps (t test, t = 3.438, The structure of compound 1 was proposed as (4Z,11Z)- n =8,P = 0.011) (Fig. S9). In June 2011 we conducted dose– oxacyclotrideca-4,11-dien-2-one (= (3Z,10Z)-dodecadienolide), response studies at Lake Johnson Park, Raleigh, NC. Sticky traps which we named “parcoblattalactone” to account for its origin baited with 10, 100, and 1,000 ng parcoblattalactone in rubber from Parcoblatta and lactone structure. This compound was lures attracted significantly more P. lata males than did control synthesized, as detailed in Fig. S7. The synthesis took advantage traps that were loaded with hexane solvent only (Fig. 5). Parco- of the symmetric placement of the double bonds and the oxygen blattalactone also attracted three other Parcoblatta species, most atoms. The key step was the deconjugation of the triple bond from commonly P. virginica and Parcoblatta caudelli. Although P. the carboxylic acid. Thus, the diyne (compound 7)wascyclizedto pennsylvanica nymphs and adults were found in this forest, none the corresponding diyne lactone and semihydrogenated to the were trapped by parcoblattalactone-baited traps. Interestingly, desired compound 1 (Fig. S7). The identity of the sex pheromone rubber septa baited with headspace volatiles from aerations of of P. lata was proved unambiguously: The NMR spectra of com- virgin P. lata females attracted P. lata males and the other Par- pound 1 and the natural compound were identical (Fig. 3). coblatta species in approximately the same ratio as synthetic Synthetic parcoblattalactone was tested in EAG dose–response parcoblattalactone. No immature Parcoblatta were found on any experiments with antennae of adult male P. lata, Parcoblatta of the traps; one or two adult females were trapped occasionally, virginica, and Parcoblatta pennsylvanica that were freshly col- but equally in baited and unbaited traps. lected in a pine forest in Raleigh, NC. Both P. lata and P. virginica responded to the pheromone at 0.1 ng to ∼1 μgonfilter paper in Discussion a log dose–response manner, but P. pennsylvanica antennae The recovery, conservation, and management of habitats of an responded only at very high loadings of parcoblattalactone (Fig. endangered species necessitate detailed knowledge of its eco- 4). At high doses the EAG amplitude declined, but EAG re- logical requirements. The red-cockaded woodpecker, for exam- covery to baseline was considerably slower (i.e., the EAG peak ple, requires open pine woodlands and savannahs with large old was broader at high doses). pines as cavity trees for nesting and roosting (8) as well as In a mixed pine-hardwood forest (Fig. S8), parcoblattalactone abundant, nonfragmented, contiguous foraging habitat, including was significantly more attractive in overnight trapping using mature pines with an open canopy, few or no midstory trees, rubber septa than filter-paper dispensers (t test, t = 2.398, n =7, abundant native bunchgrass and forb groundcovers, and coarse

E492 | www.pnas.org/cgi/doi/10.1073/pnas.1111748109 Eliyahu et al. Downloaded by guest on September 27, 2021 PNAS PLUS

Fig. 4. EAG responses of isolated antennae of three Parcoblatta species. The photograph shows P. lata (Upper)andP. pennsylvanica (Lower) nymphs (Left), adult females (Center), and adult males (Right). The small squares on the grid are 1/8 inch (3.17 mm) on a side, and the large squares are 1 inch (25.4 mm) on a side.

woody debris that supports arthropod availability to bark-for- date represent three divergent classes of chemicals. Several aging birds (4). The assessment of habitat quality and prey highly attractive epoxygermacranoid compounds have been availability entails exceptional investment in broad-scale moni- identified from various Periplaneta and Blatta species (family toring with nonselective traps, such as burlap bands on trees or Blattidae), and these compounds, in various blends, also appear cardboard, pitfall and light traps. Because the availability of large to function in reproductive isolation (13–17). An exceptionally prey to feed nestlings significantly increases the reproductive attractive α-pyrone (supellapyrone) was identified from the success of red-cockaded woodpecker breeding groups, and brown-banded cockroach Supella longipalpa (Blattellidae, Pseu- P. lata and related wood cockroaches comprise the most com- dophyllodromiinae), and this pheromone appears to function as mon large item fed to nestlings—26–62% of the diet (5)—the a single compound and not as part of a pheromone blend (18– newly identified P. lata sex pheromone holds promise as an im- 20). A substituted quinone (blattellaquinone), from the German portant, economically and ecologically sound tool to monitor the cockroach Blattella germanica (Blattellidae, Blattellinae) (21), quality and suitability of foraging habitats for red-cockaded represents another chemical class. The only volatile sex attrac- woodpeckers. tant identified in the family Blaberidae is a mixture of rather Sex pheromones may be separated functionally into two broad simple hydroxyketones, phenols, and thiazolidines (collectively classes: long-distance volatile pheromones that attract mates named “seducins”), produced by male lobster cockroaches, from a distance, and short-range or contact sex pheromones used Nauphoeta cinerea (22). Thus, cockroaches have undergone in courtship, mate assessment, and mate choice (1). Despite their adaptive radiations in their sexual communication signals, pro- great biological and ecological diversity, and the recognition that ducing highly diverse and often novel sexual attractants and several species are major pests with public health and veterinary consequently fashioning relatively “private” channels of olfactory impacts, the sex pheromones of only a small number of cock- communication. Our understanding of their contact-based che- roach species have been identified chemically so far (12). In most mosensory sexual communication is limited to only a few species, cockroach species, sexual attractants are emitted by the female— but in this context, too, cockroaches evolved a diverse chemical the heavier and shorter-winged sex—attracting the slimmer, arsenal of “aphrodisiacs” to elicit courtship and to bias mate long-winged male. The volatile sex pheromones identified to choice and acceptance (23, 24). ECOLOGY CHEMISTRY

Fig. 5. Field trapping of Parcoblatta species using synthetic parcoblattalactone loaded in rubber septa dispensers.

Eliyahu et al. PNAS | Published online December 19, 2011 | E493 Downloaded by guest on September 27, 2021 The P. lata pheromone (4Z,11Z)-oxacyclotrideca-4,11-dien-2- EAG assays. Unfortunately, there are no phylogenetic studies of one, a 13-member macrocyclic lactone, represents a newly this genus, so we cannot speculate on whether the cross-attraction identified natural product and a new class of cockroach pher- of P. lata, P. virginica, and P. caudelli has a basis in a common omones that is radically different from blattellaquinone, the only evolutionary history. Interestingly, however, P. virginica and P. other sex pheromone identified in the subfamily Blattellinae. caudelli also were attracted to volatile emissions of P. lata females Although the P. lata pheromone shares a lactone structure with that contained the full blend of pheromone components. This supellapyrone, also isolated from the family Blattellidae, the unusual observation suggests that other species-isolating mecha- latter includes only a six-member ring structure. Lactones are nisms may operate in this genus, such as temporal and spatial ubiquitous in microbes, plants, and animals, where they serve partitioning of sexual activity of different species within the for- diverse biological functions including communication and quo- est. It also is possible that, although we loaded the volatile rum-sensing (25), as defensive compounds, toxins, and insecti- emissions P. lata females into the rubber septa dispensers, they cides (26), and even as structural elements of insect nests (27). might have released an “off-blend” of the pheromone blend, thus However, macrocyclic lactones similar to parcoblattalactone compromising species specificity. have been found in only a handful of other animals, where they Parcoblattalactone thus contributes to our fundamental un- have multiple functions. A closely related macrolide, but with derstanding of the evolution of chemical communication in a double bond between carbons 7 and 8 rather than 11 and 12 cockroaches. It also provides a practical tool for monitoring [(4Z,7Z)-oxacyclotrideca-4,7-dien-2-one = (3Z,6Z)-3,6-dodeca- populations of insects that comprise an important food source dienolide] was isolated as an aggregation pheromone of the for an endangered bird species, whose protection hinges on sawtoothed grain beetle, Oryzaephilus surinamensis, an important habitat conservation through appropriate management. Future stored-product pest (28). Aerations of the flat grain beetle, research should elucidate the pheromone blends of all Parco- Cryptolestes pusillus, also contained this 13-member macrolide, blatta species on which the red-cockaded woodpecker preys. but apparently it was not part of the aggregation pheromone These blends would serve as valuable tools in determining the blend; instead, a similar lactone with only one double bond, richness and abundance of prey and, together with other habitat (4Z)-oxacyclotrideca-4-en-2-one [=(3Z)-3-dodecenolide)], is features (e.g., dead trees, boles, snags, downed trees, understory the major aggregation pheromone component in this species complexity), could be essential in preserving appropriate habitat (29). Related 12- and 14-member unsaturated macrolides have for this endangered species. By attracting three of the most been isolated as aggregation pheromones of stored-products common Parcoblatta species within the red-cockaded wood- beetle pests (28, 29). Interestingly, (3Z)-3-dodecenolide also is pecker habitat, parcoblattalactone represents a significant ad- thought to serve as a sex pheromone in the emerald ash borer, vance toward this goal. Agrilus planipennis (30), a devastating invasive beetle that has killed tens of millions of ash trees in the United States. During Materials and Methods fl copulation, male Heliconius butter ies transfer several 12- and Insects and Pheromone Extraction. Parcoblatta lata nymphs were collected 14-member macrolides to the female, including both the 13R- during the spring in the years 2003–2007 from felled pines and snags in and 13S-configured (9Z,11E)-octadeca-9,11-dien-13-olide, and forests in and around Raleigh, Wake County, NC. The nymphs were reared to these lactones apparently serve as antiaphrodisiacs that allow the adulthood and separated by sex. We were able to raise 1,400 female nymphs male to monopolize the female (31, 32). Stritzke et al. (33) found to the adult stage, and abdominal tergites 1–7 of 6- to 7-d-old virgin females an unusual 13-member sesquiterpene macrolide in the abdomi- were dissected and immediately extracted in n-hexane for 30 min, followed nal androconial organs of a danaid butterfly. Saturated 14- by a second hexane extraction. The two extracts were combined for further fi member lactones also have been characterized as sex pher- pheromone puri cation. omones of the stink bug Piezodorus hybneri (34) and aggregation Flash Chromatography. Hexane extracts (∼200 female equivalents) were re- pheromones in ants (35). Much larger macrocyclic lactones, up duced under a gentle stream of N2 to ∼200 μL and were loaded onto Pasteur to 24-member rings, have been isolated as defensive secretions of fi – ’ pipette minicolumns lled with 200 mg of chromatographic silica gel (100 Amitermes termites (36) and from the Dufour s glands of hal- 200 mesh; Fisher Scientific). The column was eluted successively with two ictine (sweat) bees, where they appear to function in recognition rinses of hexane (2 mL each) and 2%, 5%, and 10% (vol/vol) diethyl-ether in of females by males (37). Very large macrolides produced by hexane. Each fraction was tested for biological activity with EAG, and the many bacteria and fungi also have antibiotic and broad anti- active fraction from different batches (second hexane fraction) was reduced parasitic spectra (e.g., avermectins) (38). The possibility that again under N2 and refractionated on a silica-gel column impregnated with parcoblattalactone might have biological characteristics other 20% (wt/wt) silver nitrate. This column was eluted with three rinses each of than its role as a sex pheromone deserves further investigation. 2 mL hexane, 1%, 3%, 4%, 5%, 10%, and 100% diethyl ether. Each fraction was bioassayed with EAG, and the active fractions (3% and 4% ether) were Although we isolated parcoblattalactone from P. lata,italso fi elicited antennal EAG responses from P. virginica and attracted combined for further puri cation by HPLC and preparative GC. males of P. virginica, P. caudelli, and a third unidentified Parco- HPLC. Active fractions from silver nitrate silica-gel columns were purified blatta species. Interestingly, the antennae of P. pennsylvanica did further on a HP1050 HPLC (Hewlett-Packard) equipped with a 1-mL sample not respond to parcoblattalactone, and this species was not loop and two silica-gel columns connected in series (Econosphere Silica, 5 μm, attracted to pheromone-baited traps, although it co-occurs in 4.6 mm i.d. × 250 mm; Alltech Associates, Inc.). The solvent system was pine-hardwood forest with the other species. Parcoblattalactone hexane and diethyl ether programmed as follows: 100% hexane for 3 min, is a major component of the P. lata pheromone, but male an- then a linear gradient of 0.5% diethyl ether/min to 10% diethyl ether, then tennae also responded to three additional components in extracts a linear gradient of 20% diethyl ether/min to 50% diethyl ether, and a 5-min of P. lata female pheromone glands. These observations suggest hold at 50% diethyl ether. The flow rate was set at 1 mL/min; the effluent that the 12 species in the genus Parcoblatta likely use species- was monitored at 210 and 254 nm, and 1-min fractions were collected in specific multicomponent pheromone blends, as is common for glass vials. many other insects. Moreover, P. pennsylvanica, and possibly GC-EAD. An HP 5890 Series II gas chromatograph, equipped with either an EC- other Parcoblatta species that were not attracted to parco- × μ fi fi 5 or EC-WAX capillary column (30 m 0.25 mm, 0.25- m lm thickness; blattalactone, either have greater delity to their multicomponent Alltech Associates, Inc.), was modified and used for the GC-EAD analyses blend that might include parcoblattalactone or may use a differ- (39). Helium was used as the carrier gas at a head pressure of 115 kPa and ent major component in their pheromone blend. The latter a flow rate of 1.5 mL/min. Samples were injected in splitless mode, and the possibility is suggested by the observation that P. pennsylvanica purge valve was opened after 1 min. Oven temperature was set at 50 °C for antennae were relatively unresponsive to parcoblattalactone in 2 min, then increased at 15 °C/min to 250 °C and held for 10 min. The tem-

E494 | www.pnas.org/cgi/doi/10.1073/pnas.1111748109 Eliyahu et al. Downloaded by guest on September 27, 2021 perature of the injector, FID, and EAD transfer line was set at 270 °C. The GC-IR Spectroscopy. Vapor-phase IR spectroscopy was acquired with a Win GC/ PNAS PLUS

column effluent was combined with N2 makeup gas (20 mL/min) using IR Pro (Varian) with a GC/IR interface and a Scimitar FTS 2000 linked to a 6890 a thermal conductivity detector capillary column adapter and then was split Network GC system (Agilent). The transfer line and light pipe were operated 1:1 to the FID and EAD. Charcoal-filtered and humidified air at 250 mL/min at 250 °C. Pheromone extract was separated on a HP-5 column (30 m × 0.32 flushed the EAD outlet over the antennal preparation within a cold-water– mm × 0.25 μm; Agilent) operated at 100 °C for 1 min, increased to 250 °C at jacketed condenser (∼10 °C). a rate of 20 °C/min, and held at this temperature for 5 min. A custom acrylic antennae holder modified from Nojima et al. (21, 39) was used for EAD recordings. A male cockroach head was mounted in a plastic NMR Spectroscopy. A Bruker AVANCE 600-MHz spectrometer equipped with pipette tip, and the distal ends of both antennae were inserted into a glass a 1-mm Bruker triple resonance inverse (TXI) microprobe was used for NMR fi ’ capillary. The pipette tip and capillary tube were lled with Eagle scell analysis. The signal of a trace amount of undeuterated benzene (7.16 ppm), culture medium that made contact with gold electrodes. The output signal which is an unavoidable contaminant in NMR samples, was used as a refer- fi × from the antennae was ampli ed 10 by a custom high-input impedance DC ence. Capillary NMR tubes were obtained from Bruker BioSpin. amplifier and filtered by a high-pass filter with a cutoff frequency of 0.5 Hz fi (21, 39, 40). The ampli er output was routed through a signal acquisition EAG. Adult males were freshly collected in the field, and four antennae of board within the GC and displayed along with the FID signal in Agilent each species were exposed sequentially to the full range of doses. An IDAC-4 ChemStation. Adult males, ∼30 d post eclosion, were used for GC-EAD. amplifier (Syntech) was used to amplify the responses of an isolated antenna to odorants. Parcoblattalactone was loaded in 20 μL hexane onto a strip of Preparative GC. An HP5890 gas chromatograph equipped with a cool on- filter paper. Then hexane was allowed to evaporate for 2 min, and the filter- fi column injector was modi ed as a preparative GC according to Nojima et al. paper dispenser was placed into a glass Pasteur pipette. Purified air was fl (10, 11). Brie y, a split-splitless injection port assembly was installed adjacent delivered over the antenna at 250 mL/min and was diverted through the fi to the FID port and modi ed as a sample collection port. A 30-m deactivated stimulus cartridge for 0.5 s. column (0.53 mm i.d., no stationary phase) (Alltech Associates, Inc.), fol- lowed by a separation column, was installed to permit large-volume cool on- Field Trapping. Parcoblattalactone was loaded in 100 μL hexane into red column injections (41). An EC-5 megabore capillary column (5.0-μm film rubber septa (Wheaton). The hexane was allowed to evaporate; then 100 μL thickness, 0.53 mm i.d. × 30 m) (Alltech Associates, Inc.) was used as the of clean hexane was loaded into the same septum to carry the pheromone separation column and programmed at 30 °C for 2 min, at 10 °C/min to 260 °C, into the septum matrix and was allowed to evaporate. The septa were po- then at 20 °C/min to 280 °C, and held for 30 min. The cool on-column injector sitioned in the centers of adhesive-coated traps vertically attached by thumb temperature was set to track 3 °C higher than the oven temperature. Sec- tacks to pine trees at a height of 1.5–2 m (Fig. 5 and Fig. S8). Trapping was tions of megabore capillary columns (DB-1, 20 cm long, 0.53 mm i.d., 5.0-μm conducted overnight in a mixed pine-hardwood forest at Lake Johnson Park, film thickness) (Agilent Technologies) were used as fraction collection traps Wake County, NC. Six transects of the five treatments were set with traps (10). Before use, these collection traps were rinsed twice with 100 μLof 12–15 m apart. methylene chloride (HPLC and pesticide residue analysis grades; Fisher Sci- fi entific) and dried overnight at room temperature. Traps were not cooled To evaluate the species speci city of their sex-pheromonal signal, lures during collections, and the same trap was used repeatedly to collect the baited with volatile collections of virgin P. lata females were included also. fi same GC fraction. Virgin females were placed in a large glass jar and aerated with puri ed air. The 20-cm capillary traps were eluted directly into microcapillary NMR Volatiles were trapped on SuperQ, which was eluted with hexane, concen- tubes with ∼7 μL of benzene-D6 (99.96% deuteration; Cambridge Isotope trated, and loaded on rubber septa at an approximate dose representing 10 Laboratories), as described by Nojima et al. (11). female-day equivalents.

GC-MS Analyses. An Agilent 5975 mass selective detector coupled to an Agilent ACKNOWLEDGMENTS. We thank members of the C.S. laboratory who 6890 GC was used for GC-MS analyses of EAD-active compounds. The GC was assisted with collecting and rearing Parcoblatta, the City of Raleigh Depart- ment of Parks and Recreation for permission to conduct the field work in operated in splitless injection mode and fitted with a DB-5MS column (30 m × × μ – local parks, and Katalin Böröczky for comments on an earlier draft of the 0.25 mm 0.25 m; Agilent). The oven was programmed from 50 250 °C at manuscript. This work was supported in part by the Blanton J. Whitmire 15 °C/min after an initial delay of 2 min and held at 250 °C for 10 min. Injector Endowment at North Carolina State University, and a portion of the manu- temperature was 270 °C; MS quadrupole temperature was 150 °C; MS source script was written while D.E. was supported by the Center for Insect Science temperature was 230 °C; and transfer line temperature was 250 °C. through National Institutes of Health Training Grant 1K12 GM000708.

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