TY - JOUR
AU - G, Millar, Jocelyn
AB - Abstract In field trials testing attraction of cerambycid beetles to a blend of known pheromone components plus host plant volatiles, several species in the subfamily Spondylidinae were attracted to baited traps, suggesting that one or more components of the blend might constitute their pheromones. Here, we describe laboratory and field experiments aimed at identifying the actual pheromone components produced by these species. Analysis of headspace odors collected from male Tetropium abietis (Fall) (Coleoptera: Cerambycidae) contained (S)-fuscumol as a single component, whereas Asemum nitidum (LeConte) (Coleoptera: Cerambycidae) males produced both (S)-fuscumol and geranylacetone, and Asemum caseyi (Linsley) (Coleoptera: Cerambycidae) produced only geranylacetone. In field trials testing fuscumol, fuscumol acetate, and geranylacetone as individual components or in blends, in combination with host plant volatiles, A. nitidum were attracted to blends of fuscumol and geranylacetone, T. abietis were attracted to fuscumol alone, and A. caseyi were attracted to geranylacetone alone. Fuscumol acetate did not appear to be either attractive or inhibitory. These results, along with previous catches of spondylidine species in traps baited with fuscumol, provide evidence that fuscumol and geranylacetone are likely to be relatively common pheromone structures for species in the subfamily Spondylidinae. longhorned beetle, aggregation-sex pheromone, pheromone bioassay Cerambycid beetles in the subfamily Spondylidinae are distributed globally and are integral components of many coniferous forest systems (e.g., Majka and Ogden 2010, Swift et al. 2010, Kundu 2015, Özbek et al. 2015, Monné 2016). However, there are several examples of ecologically disruptive invasive spondylidines, such as the brown spruce longhorn beetle Tetropium fuscum (Fabricius) (Coleoptera: Cerambycidae), which was accidentally introduced into eastern Canada from Europe (Smith and Hurley 2000, Dearborn et al. 2016), and several invasive Arhopalus (Serville) (Coleoptera: Cerambycidae) species across Australia (Wang and Leschen 2012). Spondylidines are also possible vectors of the nematode Bursephelencus xylophilus (Steiner & Buhrer) (Aphelenchida: Parasitaphelenchidae) (Linit et al. 1983), the causative agent of the devastating pine wilt disease, as well as congeneric nematodes (Robertson et al. 2008) and other plant pathogens (Jacobs et al. 2003). The first reported pheromone from the subfamily, (2S,5E)-6,10-dimethyl-5,9-undecadien-2-ol (henceforth fuscumol) was produced by males and attracted both sexes of the invasive European T. fuscum and Tetropium castaneum (Linnaeus) (Coleoptera: Cerambycidae), as well as the native North American Tetropium cinnamopterum (Kirby) (Coleoptera: Cerambycidae) (Silk et al. 2007, Sweeney et al. 2010). To date, this appears to be the only confirmed pheromone identified from the subfamily, although fuscumol has been reported to attract at least eight other spondylidine species (Hanks and Millar 2013, Sweeney et al. 2014, Collignon et al. 2016). In addition, most spondylidine species infest conifers, and it has been shown that at least some species are attracted to lures based on terpenes and other host plant volatiles (HPVs; Chénier and Philogène 1989, Sweeney et al. 2004, Miller 2006). Host plant volatiles also have been shown to synergize attraction of some species to fuscumol, particularly when the HPVs are released at high rates (Sweeney et al. 2010, Collignon et al. 2016). Fuscumol, its corresponding acetate ((E)-6,10-dimethyl-5,9-undecadien-2-yl acetate), and (E)-6,10-dimethyl-5,9-undecadien-2-one (geranylacetone) form a pheromone motif that is also shared by a number of species in the subfamily Lamiinae (Mitchell et al. 2011, Hanks et al. 2012, Wong et al. 2012, Hanks and Millar 2013, Wickham et al. 2014, Hayes et al. 2016, Hughes et al. 2016, Meier et al. 2016). However, fuscumol acetate has not been identified from or shown to be attractive to any spondylidine species. Fuscumol acetate was first identified from males of the South American lamiine Hedypathes betulinus (Klug) (Coleoptera: Cerambycidae), which produces (R)-fuscumol acetate, (R)- and (S)-fuscumol, and geranylacetone (Fonseca et al. 2010, Vidal et al. 2010). However, field trials to determine which blend or subset of these components might be attractive have not yet been reported. Field screening trials in southern California’s San Bernardino National Forest (SBNF) of a generic cerambycid pheromone blend that included racemic fuscumol, fuscumol acetate, 3-hydroxyhexan-2-one, 2,3-hexanediol, 2-methylbutanol, and monochamol [2-(undecyloxy)ethanol] demonstrated the blend’s efficacy in attracting a variety of cerambycid species. Traps baited with this mixture of pheromones in combination with high release rate terpene lures caught significant numbers of three spondylidine and one lamiine species (Collignon et al. 2016). Of these, the lamiine species Monochamus clamator (Leconte) (Coleoptera: Cerambycidae) was likely attracted to the monochamol in the blend, because monochamol is a known pheromone of a number of Monochamus species (Hanks and Millar 2016). Thus, the goal of the research described here was to follow up on these field screening results, and identify the actual pheromones of the three spondylidine species. The specific objectives of this research were: 1) to determine if fuscumol is necessary and sufficient for attraction of the three spondylidine species; 2) to determine if fuscumol acetate is utilized by any of the attracted spondylidines, either alone or in combination with fuscumol; 3) to determine whether additional spondylidine and lamiine species would be attracted to lures containing fuscumol, fuscumol acetate, or a blend of the two. After the first season of field testing and collection of headspace volatiles from live beetles, we added a fourth objective, to determine whether geranylacetone is also a pheromone component for spondylidines, either alone or in combination with the other two pheromone candidates. Methods and Materials Field Bioassays Testing Fuscumol and Fuscumol Acetate The first field bioassays were conducted at two sites approximately 0.5 km apart in the SBNF in San Bernardino Co., California, USA. The sites were near Jenks Lake (34°09′45.8′′N 116°54′08.6′′W) and are dominated by Ponderosa pine (Pinus ponderosa Douglas) and white fir (Abies concolor [Gordon]) (Pinales: Pinaceae), with some western black oak (Quercus kelloggii Newbury), canyon live oak (Quercus chrysolepis Liebm.) (Fagales: Fagaceae), big-cone Douglas-fir (Pseudotsuga macrocarpa [Vasey]) (Pinales: Pinaceae), and incense cedar (Calocedrus decurrens Torr.) (Pinales: Cupressaceae). Black cross-vane intercept traps (Alpha Scents, Portland, OR) coated with Fluon (Graham et al. 2010) were hung on 1.5 m tall, L-shaped stands made from PVC pipe. Traps were placed 10–15 m apart in transects, with treatments initially assigned randomly to traps. Traps were checked twice weekly and their order was rerandomized at every check. Beetles were live trapped so that they could be used for pheromone collection. Voucher specimens of all species have been deposited in the Entomology Museum at UC Riverside. Two sequential assays were run. For the first, the treatments consisted of a HPV blend + ethanol as a positive control, and HPV + ethanol with racemic fuscumol, racemic fuscumol acetate, or racemic fuscumol + fuscumol acetate. The pheromones (Bedoukian Research, Danville, CT; 1 ml of a 50 mg/ml solution of each compound in isopropanol) were dispensed from 5 × 7.5 cm, 2 mil (= 0.051 mm) wall thickness low-density polyethylene resealable bags (Fisher Scientific, Pittsburgh, PA). The HPV lure was a previously developed synthetic conifer volatiles blend (Table 1; Collignon et al. 2016) which was released from open-topped glass jars (4.3 cm tall × 4.3 cm outer diameter × 3.1 cm opening, 25 ml capacity), reloaded with 10 ml of conifer blend twice per week. Ethanol was released from 10 × 15 cm, 2 mil (= 0.051 mm) wall thickness low-density polyethylene resealable bags (Fisher Scientific) loaded with 50 ml ethanol. Traps were deployed from 12 May through 24 June 2016 to target Neospondylis upiformis (Mannerheim) (Coleoptera: Cerambycidae). For the second assay, the conifer blend and ethanol lures were changed to a 2:1 (S):(R)-α-pinene lure (same release device as for the conifer blend) without ethanol from 24 June through 3 August 2016 to target Asemum nitidum Linnaeus (Coleoptera: Cerambycidae). These two HPV blends were most attractive to the respective species in a previous study (Collignon et al. 2016). Table 1. Synthetic blend composition for the host volatiles blend, with each compound’s contribution expressed as milliliters per 100 ml Compound Enantiomeric ratio ml/100 ml Source α-pinene 2:1 (S:R) 17.2 Sigma-Aldrich camphene racemic 1 Sigma-Aldrich β-pinene pure (S) 17.2 Alfa-Aesar myrcene - 17.2 Acros Organics 3-carene racemic 17.2 Sigma-Aldrich limonene 2:1 (R:S) 17.2 Alfa-Aesar 1,8-cineole - 4 Alfa-Aesar borneol pure (S) 2 Alfa-Aesar camphor 2:1 (S:R) 2 Alfa-Aesar 4-allylanisole - 1 Alfa-Aesar (-)-trans-β-caryophyllene - 4 Acros Organics Compound Enantiomeric ratio ml/100 ml Source α-pinene 2:1 (S:R) 17.2 Sigma-Aldrich camphene racemic 1 Sigma-Aldrich β-pinene pure (S) 17.2 Alfa-Aesar myrcene - 17.2 Acros Organics 3-carene racemic 17.2 Sigma-Aldrich limonene 2:1 (R:S) 17.2 Alfa-Aesar 1,8-cineole - 4 Alfa-Aesar borneol pure (S) 2 Alfa-Aesar camphor 2:1 (S:R) 2 Alfa-Aesar 4-allylanisole - 1 Alfa-Aesar (-)-trans-β-caryophyllene - 4 Acros Organics For chiral compounds, ratios of enantiomers are listed. View Large Table 1. Synthetic blend composition for the host volatiles blend, with each compound’s contribution expressed as milliliters per 100 ml Compound Enantiomeric ratio ml/100 ml Source α-pinene 2:1 (S:R) 17.2 Sigma-Aldrich camphene racemic 1 Sigma-Aldrich β-pinene pure (S) 17.2 Alfa-Aesar myrcene - 17.2 Acros Organics 3-carene racemic 17.2 Sigma-Aldrich limonene 2:1 (R:S) 17.2 Alfa-Aesar 1,8-cineole - 4 Alfa-Aesar borneol pure (S) 2 Alfa-Aesar camphor 2:1 (S:R) 2 Alfa-Aesar 4-allylanisole - 1 Alfa-Aesar (-)-trans-β-caryophyllene - 4 Acros Organics Compound Enantiomeric ratio ml/100 ml Source α-pinene 2:1 (S:R) 17.2 Sigma-Aldrich camphene racemic 1 Sigma-Aldrich β-pinene pure (S) 17.2 Alfa-Aesar myrcene - 17.2 Acros Organics 3-carene racemic 17.2 Sigma-Aldrich limonene 2:1 (R:S) 17.2 Alfa-Aesar 1,8-cineole - 4 Alfa-Aesar borneol pure (S) 2 Alfa-Aesar camphor 2:1 (S:R) 2 Alfa-Aesar 4-allylanisole - 1 Alfa-Aesar (-)-trans-β-caryophyllene - 4 Acros Organics For chiral compounds, ratios of enantiomers are listed. View Large Field Assays Testing Fuscumol, Fuscumol Acetate, and Geranylacetone Because geranylacetone was identified from headspace volatiles collected from A. nitidum in 2016, a second field bioassay was conducted 25 May through 7 July 2017, testing the attractiveness of geranylacetone alone and in combination with the other pheromone candidates. The bioassays were conducted at the same two sites located near Jenks Lake, and four additional sites. These additional sites consisted of a third site located near Jenks Lake, roughly two miles from the other two, a site near the base of Onyx Peak (34°11′41.9′′N 116°43′06.9′′W), and two sites located near Crestline in the SBNF (34°15′56.3′′N 117°17′28.3′′W). At all six sites, black cross-vane intercept traps coated with Fluon were hung from the lower branches of Ponderosa pine trees. Traps were placed roughly 10–15 m apart in transects, and one of eight treatments was assigned randomly to each trap. Traps were checked weekly, and the order of treatments along a given transect was rerandomized after each check. As in the previous year, beetles were trapped live so that they could be used for pheromone collection in the laboratory. For this bioassay, all treatments consisted of the conifer HPV blend (Table 1) + one of eight pheromone lures. The pheromone lures consisted of 1) racemic fuscumol, 2) racemic fuscumol acetate, 3) geranylacetone (all from Bedoukian Research, Danville, CT), 4) fuscumol + fuscumol acetate, 5) fuscumol + geranylacetone, 6) fuscumol acetate + geranylacetone, 7) a mixture of all three compounds, and 8) a solvent control. The pheromones were dispensed from polyethylene bags as in 2016, except that ethanol rather than 2-propanol was used as the solvent. Geranylacetone was used at a concentration of 25 mg/ml rather than 50 mg/ml because it is a single compound, in comparison to the two enantiomers present in the racemic fuscumol and fuscumol acetate. The HPV blend was released from uncapped 20 ml wide-mouth glass vials hung next to the pheromone lures. The HPV lures were recharged weekly, and the pheromone lures were replaced roughly every 10 d. Identification of Beetle-emitted Volatiles Live beetles were sampled for pheromone emission individually or in groups of up to five beetles (Table 2). A total of seven aerations of N. upiformis with ponderosa pine sprigs were made, including some with 10% sugar water provided as nutrition. Headspace volatiles were collected from a single unsexed Tetropium abietis with a white fir sprig, and with two males and a female, with wire mesh perches and 10% sugar water provided. Six headspace collections were made from male A. nitidum beetles held with white fir twigs and sugar water, or sugar water alone. Four headspace collections were made from Asemum caseyi held with sugar water, one each with a single female or male, and two with two males. Volatiles were collected for a week or until beetles died. No beetles were resampled if they lived longer than 1 wk. The host substrates used were the preferred hosts of these species’ populations in the SBNF (Linsley and Chemsak 1997). Control host plant twigs were aerated under the same conditions. Volatile collections were conducted using wide-mouth 500 ml Teflon containers (Thermo Scientific, Fisher Scientific; #24030250), with the screw-cap lids fitted with Swagelok bulkhead unions (Swagelok, Solon, OH) to connect inlet and outlet tubes. Air was pulled through the system by vacuum at 500 ml/min. Incoming air was cleaned by passage through granulated activated charcoal (14–16 mesh; Fisher Scientific). Volatiles were collected onto ~50 mg of thermally-desorbed activated charcoal (50–200 mesh; Fisher Scientific) held between glass wool plugs in a short piece of glass tubing. Aerations were conducted under ReptiSun 10.0 UVB lights (Zoo Med Laboratories Inc., San Luis Obispo, CA) which provide a full spectrum of light mimicking natural daylight, with a 16:8 h L:D cycle. Volatiles were eluted from the charcoal with 1 ml dichloromethane. Table 2. Collections of headspace odors from spondylidine species Species No. individuals Host substrate Sugar water Pheromone detected Neospondylis upiformis 1 Ponderosa pine No No N. upiformis 1 Ponderosa pine No No N. upiformis 1 Ponderosa pine No No N. upiformis 4 Ponderosa pine No No N. upiformis 4 Ponderosa pine No No N. upiformis 5 Ponderosa pine No No N. upiformis 4 Ponderosa pine Yes No Asemum nitidum 1♂ White fir No No A. nitidum 1♂ White fir No (S)-fuscumol, geranylacetone A. nitidum 1♂ White fir No No A. nitidum 2 White fir No (S)-fuscumol, geranylacetone A. nitidum 2 White fir Yes No A. nitidum 2♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone A. nitidum 2♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone A. nitidum 1♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone Asemum caseyi 1♀ white fir No No A. caseyi 3♂ n/a (wire mesh perch) Yes Geranylacetone A. caseyi 4♂ n/a (wire mesh perch) Yes Geranylacetone A. caseyi 1♂ n/a (wire mesh perch) Yes Geranylacetone Tetropium abietis 1 White fir No No T. abietis 2♂,1♀ n/a (wire mesh perch) Yes (S)-fuscumol Species No. individuals Host substrate Sugar water Pheromone detected Neospondylis upiformis 1 Ponderosa pine No No N. upiformis 1 Ponderosa pine No No N. upiformis 1 Ponderosa pine No No N. upiformis 4 Ponderosa pine No No N. upiformis 4 Ponderosa pine No No N. upiformis 5 Ponderosa pine No No N. upiformis 4 Ponderosa pine Yes No Asemum nitidum 1♂ White fir No No A. nitidum 1♂ White fir No (S)-fuscumol, geranylacetone A. nitidum 1♂ White fir No No A. nitidum 2 White fir No (S)-fuscumol, geranylacetone A. nitidum 2 White fir Yes No A. nitidum 2♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone A. nitidum 2♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone A. nitidum 1♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone Asemum caseyi 1♀ white fir No No A. caseyi 3♂ n/a (wire mesh perch) Yes Geranylacetone A. caseyi 4♂ n/a (wire mesh perch) Yes Geranylacetone A. caseyi 1♂ n/a (wire mesh perch) Yes Geranylacetone Tetropium abietis 1 White fir No No T. abietis 2♂,1♀ n/a (wire mesh perch) Yes (S)-fuscumol Individuals with unspecified sex were not sexed. View Large Table 2. Collections of headspace odors from spondylidine species Species No. individuals Host substrate Sugar water Pheromone detected Neospondylis upiformis 1 Ponderosa pine No No N. upiformis 1 Ponderosa pine No No N. upiformis 1 Ponderosa pine No No N. upiformis 4 Ponderosa pine No No N. upiformis 4 Ponderosa pine No No N. upiformis 5 Ponderosa pine No No N. upiformis 4 Ponderosa pine Yes No Asemum nitidum 1♂ White fir No No A. nitidum 1♂ White fir No (S)-fuscumol, geranylacetone A. nitidum 1♂ White fir No No A. nitidum 2 White fir No (S)-fuscumol, geranylacetone A. nitidum 2 White fir Yes No A. nitidum 2♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone A. nitidum 2♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone A. nitidum 1♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone Asemum caseyi 1♀ white fir No No A. caseyi 3♂ n/a (wire mesh perch) Yes Geranylacetone A. caseyi 4♂ n/a (wire mesh perch) Yes Geranylacetone A. caseyi 1♂ n/a (wire mesh perch) Yes Geranylacetone Tetropium abietis 1 White fir No No T. abietis 2♂,1♀ n/a (wire mesh perch) Yes (S)-fuscumol Species No. individuals Host substrate Sugar water Pheromone detected Neospondylis upiformis 1 Ponderosa pine No No N. upiformis 1 Ponderosa pine No No N. upiformis 1 Ponderosa pine No No N. upiformis 4 Ponderosa pine No No N. upiformis 4 Ponderosa pine No No N. upiformis 5 Ponderosa pine No No N. upiformis 4 Ponderosa pine Yes No Asemum nitidum 1♂ White fir No No A. nitidum 1♂ White fir No (S)-fuscumol, geranylacetone A. nitidum 1♂ White fir No No A. nitidum 2 White fir No (S)-fuscumol, geranylacetone A. nitidum 2 White fir Yes No A. nitidum 2♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone A. nitidum 2♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone A. nitidum 1♂ n/a (wire mesh perch) Yes (S)-fuscumol, geranylacetone Asemum caseyi 1♀ white fir No No A. caseyi 3♂ n/a (wire mesh perch) Yes Geranylacetone A. caseyi 4♂ n/a (wire mesh perch) Yes Geranylacetone A. caseyi 1♂ n/a (wire mesh perch) Yes Geranylacetone Tetropium abietis 1 White fir No No T. abietis 2♂,1♀ n/a (wire mesh perch) Yes (S)-fuscumol Individuals with unspecified sex were not sexed. View Large Extracts were analyzed by coupled gas chromatography-mass spectrometry (GC-MS) with either an Agilent 7820A gas chromatograph fitted with an autosampler and coupled to an Agilent 5977E mass selective detector (Agilent Technologies, Santa Clara CA), or a Hewlett-Packard 6890 gas chromatograph coupled to a Hewlett-Packard 5973 GC. The Agilent 7820A GC was equipped with a DB-5MS column (30 m × 0.25 mm ID × 0.25 micron film; Agilent), and the oven temperature was programed from 40°C/1 min, then increased 5°C/min to 280°C, final time 5 min; the injector and transfer line temperatures were set to 280°C. The HP6890 GC was equipped with a DB-17MS column (30 m × 0.25 mm ID × 0.25 micron film; Agilent), and the oven temperature was programed from 40°C/1 min, then increased 10°C/min to 280°C, final time 10 min; the injector and transfer line temperatures were set to 280°C. For both instruments, one microliter aliquots of extracts were injected in splitless mode. Chromatograms from insect aerations were checked against those from the Ponderosa pine and white fir controls to identify insect-specific peaks. The insect-produced compounds were identified by matching retention times and mass spectra with those of authentic standards of fuscumol, fuscumol acetate, and geranylacetone that were available from previous studies. The absolute configuration of fuscumol acetate was determined by analysis of extracts on an HP5890 GC fitted with a Cyclodex-B chiral stationary phase column (30 m × 0.25 mm ID × 0.25 micron film; J&W Scientific, Folsom, CA). Analyses were conducted using a temperature program of 50°C/1 min, then 5°C/min to 220°C, hold 20 min, with injector and detector temperatures of 150°C and 250°C, respectively. Racemic and enantiomerically-enriched standards of fuscumol acetate, available from previous work (Hughes et al. 2016) were run under the same conditions. The (S)-enantiomer eluted first, and the two enantiomers were separated to baseline. The enantiomers of fuscumol do not resolve on this column, and so extracts containing fuscumol were first derivatized to the corresponding acetates by adding 10 μl of 10% pyridine + 1% 4-dimethylaminopyridine solution in dichloromethane to 100 μl of an aeration extract, followed by addition of 20 μl of 4.5% acetyl chloride in dichloromethane, followed by vortexing, then stirring overnight. A standard of racemic fuscumol was derivatized as a control. The reaction was quenched by addition of 5 μl ethanol and stirring for 3 h, after which 0.5 ml of pentane was added, followed by addition of ~50 μl saturated aqueous sodium bicarbonate solution and vortexing. The top, organic layer was transferred to a clean vial containing 5–10 mg anhydrous sodium sulfate, vortexed and stirred for 1 h, and then transferred to a new vial. The esterified samples were analyzed on the Cyclodex-B column as described above. Statistical Analyses Bioassay data were tested separately for each species, with replicates based on both spatial and temporal replication. Temporal replicates equalled the number of times the traps were checked. Replicates where no beetles of a given species were trapped in any trap—usually due to inclement weather or being outside the species’ flight period—were not included in analyses. Differences between treatment means were tested using the nonparametric Friedman’s test (PROC FREQ, option CMH; SAS Institute 2011) because data violated homoscedasticity assumptions of analysis of variance (ANOVA) (Sokal and Rohlf 1995). In cases of a significant overall Friedman’s test, pairs of treatments were compared with a REGWQ means separation test, controlled for maximum experiment-wise error rates (PROC GLM, SAS Institute 2011). Results Identification of Possible Pheromone Components In the analyses of headspace volatiles collected from live beetles, five A. nitidum samples were found to contain (S)-fuscumol and geranylacetone—one from an aeration of a single male with white fir, one from an individual of undetermined sex with white fir, and three from aerations of males with only sugar water. One T. abietis sample from a mixed-sex group of beetles contained (S)-fuscumol. Fuscumol (of undetermined chirality) had previously been detected in an aeration of a male T. abietis (J.G.M. and A.M. Ray, unpublished data). No fuscumol was detected in Ponderosa pine or white fir aerations. Insect-specific compounds were not found in any of the N. upiformis headspace collections. In 2017, headspace volatiles from three samples from A. caseyi males—one group of three males, one group of four males, and one male alone—were found to contain geranylacetone. Control aerations of sugar water vials, and a headspace extract from a female A. caseyi did not contain geranylacetone, indicating that this compound was likely male-specific. Field Bioassays Testing Racemic Fuscumol and Fuscumol Acetate Five spondylidine species were trapped during the course of the 2016 bioassays, T. abietis, N. upiformis, A. nitidum, A. striatum (Linnaeus) (Coleoptera: Cerambycidae), and A. caseyi, but only the first three were caught in numbers sufficient to warrant statistical analyses. T. abietis were only caught during the first part of the experiment in which conifer volatiles were used as a coattractant. A total of 22 beetles were trapped in 13 treatment replicates, and only in traps baited with lures containing fuscumol as a component (Fig. 1A). N. upiformis were also only trapped during the first part of the experiment, with 33 beetles trapped in eight treatment replicates; there were no significant differences among any of the four treatments (Fig. 1B). A. nitidum were trapped in the second part of the experiment using α-pinene as a coattractant, with 47 beetles trapped in 14 treatment replicates (Fig. 1C). Fuscumol acetate was least attractive, whereas treatments containing fuscumol or fuscumol acetate with host plant volatiles were not significantly different than the host plant volatiles alone. No lamiine species were caught in any of the traps, despite fuscumol and fuscumol acetate being known lamiine pheromone components. Fig. 1. View largeDownload slide Mean (± 1SE) numbers of Tetropium abietis, Neospondylis upiformis, and Asemum nitidum caught in 2016 in traps baited with HPV (see Table 1) and ethanol alone or in combination with fuscumol (F), or fuscumol acetate (FA). For each species, means with different letters are significantly different (REGWQ means separation test, P < 0.05). Fig. 1. View largeDownload slide Mean (± 1SE) numbers of Tetropium abietis, Neospondylis upiformis, and Asemum nitidum caught in 2016 in traps baited with HPV (see Table 1) and ethanol alone or in combination with fuscumol (F), or fuscumol acetate (FA). For each species, means with different letters are significantly different (REGWQ means separation test, P < 0.05). Field Bioassays Testing Fuscumol, Fuscumol Acetate, and Geranylacetone In the 2017 bioassays, four spondylidine species were trapped, including A. caseyi, A. nitidum, T. abietis, and N. upiformis, but only the first two species were caught in numbers sufficient to warrant statistical analyses. A total of 69 A. caseyi were trapped in 17 treatment replicates. The largest number of beetles were attracted to the treatment with geranylacetone and HPV, whereas all other treatments were not significantly different than the HPV control (Fig. 2A). A total of 34 A. nitidum were trapped in 14 treatment replicates. Only the treatment containing fuscumol, geranylacetone, and the HPVs was significantly more attractive than the HPV control (Fig. 2B). Although too few T. abietis individuals were trapped to allow for statistical analysis (15 beetles total), beetles of this species were only caught in traps baited with treatments containing fuscumol, as in the previous year. Only four N. upiformis individuals were trapped, precluding any meaningful analysis. Fig. 2. View largeDownload slide Mean (± 1SE) numbers of Asemum caseyi and Asemum nitidum caught in 2017 in traps baited with HPV (see Table 1) and ethanol alone or in combination with fuscumol (F), fuscumol acetate (FA), or geranylacetone (GA). For each species, means with different letters are significantly different (REGWQ means separation test, P < 0.05). Fig. 2. View largeDownload slide Mean (± 1SE) numbers of Asemum caseyi and Asemum nitidum caught in 2017 in traps baited with HPV (see Table 1) and ethanol alone or in combination with fuscumol (F), fuscumol acetate (FA), or geranylacetone (GA). For each species, means with different letters are significantly different (REGWQ means separation test, P < 0.05). Discussion Individuals of four of the species in this study—N. upiformis, T. abietis, A. nitidum, and A. caseyi—had been previously caught in field bioassays testing host plant volatiles in combination with a generic blend of cerambycid pheromones which included fuscumol, fuscumol acetate, monochamol, 3-hydroxyhexan-2-one, 2,3-hexanediol, and 2-methyl-butan-1-ol (Collignon et al. 2016). The work described here was intended to clarify which components of the generic blend were actually pheromone components for these species. For N. upiformis, no clarification was possible because too few beetles of this species were caught, and there were no significant differences among any of the lures tested. It remains unclear why so few of this species were caught in both 2016 and 2017, given that they had been caught in significant numbers with a generic pheromone lure in previous studies (Collignon et al. 2016). It is possible that one of the other components of the blend (i.e., monochamol, 2-methylbutanol, syn-2,3-hexanediol, and 3-hydroxyhexan-2-one) might have contributed to attraction of N. upiformis in that earlier study. This may only be clarified if volatiles can be collected successfully from N. upiformis, to determine exactly what they produce. Multiple attempts to collect volatiles failed because, for reasons unknown, the survival of N. upiformis was poor, with many beetles dying in the few hours between collecting them from traps and setting them up in aeration chambers, and the majority dying within 24 h of beginning collection of headspace volatiles. Consequently, none of the seven headspace extracts prepared from these beetles had detectable quantities of any insect-produced compounds. For T. abietis, the field bioassays and the analytical results provided support for (S)-fuscumol being the major and possibly only pheromone component for this species, with fuscumol being identified from a mixed-sex aeration, and in a sample from a male that was collected and analyzed several years earlier. Based on these data and precedents from other members of the genus (T. fuscum, T. cinnamopterum, and T. castaneum; Silk et al. 2007, Sweeney et al. 2010), it seems likely that (S)-fuscumol is a male-produced aggregation-sex pheromone for this species. The 2016 field bioassay data supported this conclusion, with fuscumol in combination with HPVs being significantly attractive, whereas fuscumol acetate with HPVs, or HPVs alone, were not attractive. While catches of T. abietis were low in 2017, only treatments that contained fuscumol in the lure blend attracted any beetles of this species. Furthermore, fuscumol acetate was neither synergistic nor inhibitory in the 2016 assay, possibly because fuscumol acetate may not be a pheromone component for related spondylidine species. To date, there is no evidence that spondylidine species produce or are attracted to fuscumol acetate (reviewed in Hanks and Millar 2016). For A. nitidum, aeration samples containing detectable amounts of insect-produced compounds were obtained from four samples from males and one sample containing individuals of undetermined sex. Both geranylacetone and (S)-fuscumol were detected in all of these samples. The 2016 field bioassays for A. nitidum provided no information to help clarify the pheromone because there were no significant differences between the fuscumol-containing treatments and the host plant volatiles control. However, the 2017 field bioassays, which included geranylacetone in the treatments, showed that a combination of fuscumol + geranylacetone with HPVs was more attractive to A. nitidum adults than the host plant volatiles control, whereas each component individually (with HPVs) was no more attractive than the HPV control. These data suggest that both compounds are required components of the pheromone of this species. One possible reason for the relatively low trap catches overall for both T. abietis and A. nitidum may be that trials were carried out with racemic fuscumol, whereas analyses showed that both species produced only the (S)-enantiomer of fuscumol. However, in previously reported work, three other Tetropium species, (T. fuscum, T. cinnamopterum, and T. castaneum) were found to be equally attracted to either (S)-fuscumol or racemic fuscumol (Sweeney et al. 2010). Furthermore, among species in the subfamily Lamiinae which use fuscumol or fuscumol acetate as pheromone components, synergism between enantiomers of fuscumol or fuscumol acetate has been reported (Meier et al. 2016), but to our knowledge, there have not yet been any reports of one enantiomer of fuscumol or fuscumol acetate antagonizing response to the other enantiomer. The analytical data and the field bioassays suggested that geranylacetone is the sole component of the pheromone of A. caseyi, and that attraction of this species is antagonized by fuscumol. This suggests a mechanism by which A. caseyi can remain reproductively isolated from related species which use either fuscumol, or a blend of fuscumol and geranylacetone as their pheromone. In summary, geranylacetone appears to have a dual role in the pheromones of cerambycids. For example, it has been shown to be a biosynthetic precursor to fuscumol in both lamiines (H. betulinus; Zarbin et al. 2013) and spondylidines (T. abietis; Mayo et al. 2013), and it has also been found in headspace volatiles from males of two lamiine species, Astylidius parvus (LeConte) (Coleoptera: Cerambycidae) and Lepturges angulatus (LeConte) (Coleoptera: Cerambycidae). In the latter species, geranylacetone has been confirmed to be a pheromone component (Meier et al. 2016), and in the data presented here, geranylacetone appears to be part of the pheromone blend of the spondylidine A. nitidum and the sole pheromone component of the congener A. caseyi. It has also been detected in headspace volatiles from males of the spondylidine Arhopalus productus (LeConte) (Coleoptera: Cerambycidae) (J.G.M., unpublished data) and the lamiine Moneilema semipunctata (LeConte) (Coleoptera: Cerambycidae) (R.M.C., unpublished data). Thus, it appears likely that geranylacetone will prove to be a pheromone component for additional species in both the lamiine and spondylidine subfamilies. Acknowledgments We thank Tom Coleman and Stacy Hishinuma of the U.S. Forest Service for access to field sites. We also thank Jacqueline Serrano for help with field work. This research was supported by grants from the United States Department of Agriculture Animal and Plant Health Inspection Service (14-8130-1422-CA, 15-8130-1422-CA, 16-8130-1422-CA) to J.G.M. and USDA-AFRI-NIFA Predoctoral Fellowship Program to R.M.C. (2016-67011-25097). References Cited Chénier , J. V. R. , and B. J. R. Philogène . 1989 . Field responses of certain forest Coleoptera to conifer monoterpenes and ethanol . J. Chem. Ecol . 15 : 1729 – 1745 . Google Scholar Crossref Search ADS PubMed Collignon , R. M. , I. P. Swift , Y. Zou , J. S. McElfresh , L. M. Hanks , and J. G. Millar . 2016 . The influence of host plant volatiles on the attraction of longhorn beetles to pheromones . J. Chem. Ecol . 42 : 215 – 229 . Google Scholar Crossref Search ADS PubMed Dearborn , K. W. , S. B. Heard , J. Sweeney , and D. S. Pureswaran . 2016 . Displacement of Tetropium cinnamopterum (Coleoptera: Cerambycidae) by its invasive congener Tetropium fuscum . Environ. Entomol . 45 : 848 – 854 . Google Scholar Crossref Search ADS PubMed Fonseca , M. G. , D. M. Vidal , and P. H. Zarbin . 2010 . Male-produced sex pheromone of the cerambycid beetle Hedypathes betulinus: chemical identification and biological activity . J. Chem. Ecol . 36 : 1132 – 1139 . Google Scholar Crossref Search ADS PubMed Graham , E. E. , R. F. Mitchell , P. F. Reagel , J. D. Barbour , J. G. Millar , and L. M. Hanks . 2010 . Treating panel traps with a fluoropolymer enhances their efficiency in capturing cerambycid beetles . J. Econ. Entomol . 103 : 641 – 647 . Google Scholar Crossref Search ADS PubMed Hanks , L. M. , and J. G. Millar . 2013 . Field bioassays of cerambycid pheromones reveal widespread parsimony of pheromone structures, enhancement by host plant volatiles, and antagonism by components from heterospecifics . Chemoecology 23 : 21 – 44 . Google Scholar Crossref Search ADS Hanks , L. M. , and J. G. Millar . 2016 . Sex and aggregation-sex pheromones of cerambycid beetles: basic science and practical applications . J. Chem. Ecol . 42 : 631 – 654 . Google Scholar Crossref Search ADS PubMed Hanks , L. M. , J. G. Millar , J. A. Mongold-Diers , J. C. H. Wong , L. R. Meier , P. F. Reagel , and R. F. Mitchell . 2012 . Using blends of cerambycid beetle pheromones and host plant volatiles to simultaneously attract a diversity of cerambycid species . Can. J. Forest Res . 42 : 1050 – 1059 . Google Scholar Crossref Search ADS Hayes , R. A. , M. W. Griffiths , H. F. Nahrung , P. A. Arnold , L. M. Hanks , and J. G. Millar . 2016 . Optimizing generic cerambycid pheromone lures for Australian biosecurity and biodiversity monitoring . J. Econ. Entomol . 109 : 1741 – 1749 . Google Scholar Crossref Search ADS PubMed Hughes , G. P. , L. R. Meier , Y. Zou , J. G. Millar , L. M. Hanks , and M. D. Ginzel . 2016 . Stereochemistry of fuscumol and fuscumol acetate influences attraction of longhorned beetles (Coleoptera: Cerambycidae) of the subfamily Lamiinae . Environ. Entomol . 45 : 1271 – 1275 . Google Scholar Crossref Search ADS PubMed Jacobs , K. , K. Seifert , K. Harrison , and T. Kirisits . 2003 . Identity and phylogenetic relationships of ophiostomatoid fungi associated with invasive and native Tetropium species (Coleoptera: Cerambycidae) in Atlantic Canada . Can. J. Bot . 81 : 316 – 329 . Google Scholar Crossref Search ADS Kundu , S . 2015 . An analysis of the influence of land use and selected land cover parameters on the distribution of certain longhorn beetles (Coleoptera: Cerambycidae) in Sweden . M.Sc. thesis, Southern Sweden Forest Research Centre , Alnarp, Sweden . pp. 1 – 68 . Linit , M. J. , E. Kondo , and M. T. Smith . 1983 . Insects associated with the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae), in Missouri . Environ. Entomol . 12 : 467 – 470 . Google Scholar Crossref Search ADS Linsley , E. G. , and J. A. Chemsak . 1997 . The Cerambycidae of North America, part VIII: bibliography, index, and host plant index . University of California Press , Berkeley . Majka , C. G. , and J. Ogden . 2010 . New records of Cerambycidae in Nova Scotia . J. Acad. Entomol. Soc . 6 : 12 – 15 . Mayo , P. D. , P. J. Silk , M. Cusson , and C. Béliveau . 2013 . Steps in the biosynthesis of fuscumol in the longhorn beetles Tetropium fuscum (F.) and Tetropium cinnamopterum Kirby . J. Chem. Ecol . 39 : 377 – 389 . Google Scholar Crossref Search ADS PubMed Meier , L. R. , Y. Zou , J. G. Millar , J. A. Mongold-Diers , and L. M. Hanks . 2016 . Synergism between enantiomers creates species-specific pheromone blends and minimizes interspecific attraction for two cerambycid beetle species . J. Chem. Ecol . 42 : 1181 – 1192 . Google Scholar Crossref Search ADS PubMed Miller , D. R . 2006 . Ethanol and (-)-alpha-pinene: attractant kairomones for some large wood-boring beetles in southeastern USA . J. Chem. Ecol . 32 : 779 – 794 . Google Scholar Crossref Search ADS PubMed Mitchell , R. F. , E. E. Graham , J. C. H. Wong , P. F. Reagel , B. L. Striman , G. P. Hughes , M. A. Paschen , M. D. Ginzel , J. G. Millar , and L. M. Hanks . 2011 . Fuscumol and fuscumol acetate are general attractants for many species of cerambycid beetles in the subfamily Lamiinae . Entomol. Exp. Appl . 141 : 71 – 77 . Google Scholar Crossref Search ADS Monné , M . 2016 . Catalogue of the Cerambycidae (Coleoptera) of the neotropical region. Part III. Subfamilies Lepturinae, Necydalinae, Parandrinae, Prioninae, Spondylidinae and families Oxypeltidae, Vesperidae and Disteniidae . Available from http://cerambyxcat.com. Accessed 4 October 2016. Özbek , H. , H. Özdikmen , and F. Aytar . 2015 . Contributions of the longhorned beetles knowledge of Turkey by the subfamilies Aseminae, Saphaninae, Spondylidinae, Cerambycinae and Stenopterinae (Coleoptera: Cerambycidae) . Munis Entomol. Zool . 10 : 291 – 299 . Robertson , L. , A. García-Álvarez , S. C. Arcos , M. A. Dez-Rojo , J. P. Mansilla , R. Sauz , C. Martinez , M. Escuer , L. Castresana , A. Notario , et al. 2008 . Potential insect vectors of Bursaphelenchus spp. (Nematoda: Parasitaphelenchidae) in Spanish pine forests , pp. 221 – 233 . In M. M. Mota and P. Vieira (eds.), Pine wilt disease: a worldwide threat to forest ecosystems . Springer Science , Dordrecht, Netherlands . SAS Institute . 2011 . SAS/STAT 9.3 user’s guide . SAS Institute Inc ., Cary, NC . Silk , P. J. , J. Sweeney , J. Wu , J. Price , J. M. Gutowski , and E. G. Kettela . 2007 . Evidence for a male-produced pheromone in Tetropium fuscum (F.) and Tetropium cinnamopterum (Kirby) (Coleoptera: Cerambycidae) . Naturwissenschaften . 94 : 697 – 701 . Google Scholar Crossref Search ADS PubMed Smith , G. , and J. E. Hurley . 2000 . First North American record of the palearctic species Tetropium fuscum (Fabriculus) (Coleoptera: Cerambycidae) . Coleopt. Bull . 54 : 540 . Google Scholar Crossref Search ADS Sokal , R. R. , and F.J. Rohlf . 1995 . Biometry: 3rd edition . W. H. Freeman , New York, NY . Sweeney , J. , P. De Groot , L. MacDonald , S. Smith , C. Cocquempot , M. Kenis , and J. M. Gutowski . 2004 . Host volatile attractants and traps for detection of Tetropium fuscum (F.), Tetropium castaneum L., and other longhorned beetles (Coleoptera: Cerambycidae) . Environ. Entomol . 33 : 844 – 854 . Google Scholar Crossref Search ADS Sweeney , J. D. , P. J. Silk , J. M. Gutowski , J. Wu , M. A. Lemay , P. D. Mayo , and D. I. Magee . 2010 . Effect of chirality, release rate, and host volatiles on response of Tetropium fuscum (F.), Tetropium cinnamopterum Kirby, and Tetropium castaneum (L.) to the aggregation pheromone, fuscumol . J. Chem. Ecol . 36 : 1309 – 1321 . Google Scholar Crossref Search ADS PubMed Sweeney , J. D. , P. J. Silk , and V. Grebennikov . 2014 . Efficacy of semiochemical-baited traps for detection of longhorn beetles (Coleoptera: Cerambycidae) in the Russian far east . Eur. J. Entomol . 111 : 397 – 406 . Google Scholar Crossref Search ADS Swift , I. P. , L. G. Bezark , E. H. Nearns , A. Solis , and F. T. Hovore . 2010 . Checklist of the Cerambycidae (Coleoptera) of Costa Rica . Insecta Mundi . 131 : 1 – 68 . Vidal , D. M. , M. G. Fonseca , and P. Zarbin . 2010 . Enantioselective synthesis and absolute configuration of the sex pheromone of Hedypathes betulinus (Coleoptera: Cerambycidae) . Tetrahedron Lett . 51 : 6704 – 6706 . Google Scholar Crossref Search ADS Wang , Q. , and R. A. B. Leschen . 2012 . Identification and distribution of Arhopalus species (Coleoptera: Cerambycidae: Aseminae) in Australia and New Zealand . N.Z. Entomol . 26 : 53 – 59 . Google Scholar Crossref Search ADS Wickham , J. D. , R. D. Harrison , W. Lu , Z. Guo , J. G. Millar , L. M. Hanks , and Y. Chen . 2014 . Generic lures attract cerambycid beetles in a tropical montane rain forest in southern China . J. Econ. Entomol . 107 : 259 – 267 . Google Scholar Crossref Search ADS PubMed Wong , J. C. , R. F. Mitchell , B. L. Striman , J. G. Millar , and L. M. Hanks . 2012 . Blending synthetic pheromones of cerambycid beetles to develop trap lures that simultaneously attract multiple species . J. Econ. Entomol . 105 : 906 – 915 . Google Scholar Crossref Search ADS PubMed Zarbin , P. H. , M. G. Fonseca , D. Szczerbowski , and A. R. Oliveira . 2013 . Biosynthesis and site of production of sex pheromone components of the cerambycid beetle, Hedypathes betulinus . J. Chem. Ecol . 39 : 358 – 363 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Fuscumol and Geranylacetone as Pheromone Components of Californian Longhorn Beetles (Coleoptera: Cerambycidae) in the Subfamily Spondylidinae
JF - Environmental Entomology
DO - 10.1093/ee/nvy101
DA - 2018-10-03
UR - https://www.deepdyve.com/lp/oxford-university-press/fuscumol-and-geranylacetone-as-pheromone-components-of-californian-01hCkUpqT3
SP - 1300
VL - 47
IS - 5
DP - DeepDyve
ER -