J Chem Ecol (2009) 35:1448–1460 DOI 10.1007/s10886-009-9736-z Sex Pheromone of the Pine False Webworm Acantholyda erythrocephala Joseph K. Staples & Robert J. Bartelt & Allard A. Cossé & Douglas W. Whitman Received: 24 September 2009 /Revised: 16 December 2009 /Accepted: 16 December 2009 /Published online: 12 January 2010 # Springer Science+Business Media, LLC 2010 Abstract Females of the pine false webworm Acantholyda Keywords Sex pheromone . Pine false webworm . Sawfly. erythrocephala (L) produce the sex pheromone (Z)-6, Acantholyda erythrocephala . Cuticular hydrocarbon . 14-pentadecadienal, which attracts flying males in the field. Abitotic oxidation . Nonanal . Decanal . (Z)-6,14- By using gas chromatography coupled with electroantenno- pentadecadienal . Pentacosane . (Z)-9-pentacosene . graphic detection (GC-EAD) and mass spectrometry (GC-MS), (Z)-1,9-pentacosadiene . (Z,Z)-1,9,15-pentacosatriene . we detected (Z)-6,14-pentadecadienal in volatile collec- Hymenoptera . Pamphiliidae tions and in whole body extracts of female A. erythroce- phala. Females, but not males, also exhibited a 25-carbon cuticular hydrocarbon, (Z,Z)-1,9,15-pentacosatriene, Introduction which can oxidize to (Z)-6,14-pentadecadienal upon exposure to air and sunlight. (Z,Z)-1,9,15-Pentacosatriene Acantholyda erythrocephala (L) (Hymenoptera: Pamphilii- and (Z)-6,14-pentadecadienal identifications were corrob- dae), the pine false webworm (PFW), is common to northern orated by comparison with synthetic standards. (Z)-6, Europe and Asia (MiddleKauff 1958). It was reported in 14-Pentadecadienal is the second pheromone identified for North America during the early 1900’s where it has become pamphilliid sawflies, and the first to elicit strong field a significant pest of white pine (Pinus strobus L.), red pine attraction, and thus offer potential as a pheromone lure to (P. resinosa Ait), and Scots pine (P. sylvestris L) in both aid in control of this forest pest. forests and plantations (Syme 1981; Johnson and Lyon 1991; Allen 2000). Outbreaks in the U.S. and Canada can encompass thousands of hectares and persist for 20 yr or more (Asaro and Allen 1999;Allen2000, personal observa- tion), resulting in a growth reduction and increased mortality J. K. Staples (*) of host trees (MiddleKauff 1958;Syme1981;Moody1990; Department of Environmental Science, Lyons 1994;AsaroandAllen2001;Mayfieldetal.2005). University of Southern Maine, 37 College Avenue, Numerous strategies considered as a means of control for Gorham, ME 04038, USA this pest have met with variable success including: applica- e-mail: [email protected] tion of synthetic pesticides (Lyons et al. 1993), natural toxins such as neem extracts (Helson et al. 1999; Lyons et al. 1996, R. J. Bartelt : A. A. Cossé Crop Bioprotection Research Unit, USDA, 1998, 2003), biological control (Wilson 1984; Lyons 1999; ARS National Center for Agricultural Utilization Research, Kenis and Kloosterman 2001; Thompson et al. 2003). Peoria, IL 61604, USA However, many of these strategies require precise knowledge of the distribution and phenology of PFW over large tracts of D. W. Whitman 4120 School of Biological Sciences, Illinois State University, forest. As such, a pheromone lure could be valuable for Normal, IL 61790, USA monitoring, trapping, or disrupting A. erythrocephala. J Chem Ecol (2009) 35:1448–1460 1449 Prior to this research, laboratory studies that used a two moist, silica sand, 3 cm to 4 cm deep. Trays containing insects choice olfactometer found that male A. erythrocephala were then placed in growth chambers under total darkness at exhibit a series of characteristic behaviors in response to 4°C and ∼70% RH for 2 mo. Pupation was initiated by raising female derived volatiles, as well as to decanal. The latter the temperature to 12°C for 10–12 d. can be obtained from aerations and whole body extracts of Pupae and pre-emergent adults also were collected from both sexes (Staples 1999). Male behaviors included the Sand Ridge site in mid March through early April and antennation, upwind flight, and courtship behavior that used for chemical analyses, behavioral studies, and field involved abdominal flexing accompanied by substrate tests. Some adults were kept in 26×39×23 cm plastic boxes vibration (Staples 1999). These responses were similar to fitted with aluminum screen covers and maintained in the laboratory and field data from another Pamphilliid sawfly, dark at 4°C and ∼70% RH. Under these conditions, groups the web-spinning larch sawfly, Cephalcia lariciphila,in of healthy male or female sawflies (200 individuals per response to female derived odors and to a likely pheromone box) could be kept alive for up to 30 d. component, ortho-aminoacetophenone (Borden et al. 1978; Baker et al. 1983). Volatile Collection Volatiles were collected from aerations In this paper, we report that female A. erythrocephala of individual and multiple male or female sawflies at the produce an attractant pheromone, (Z)-6,14-pentadecadienal, National Center for Agricultural Utility Research, Peoria, to which only males respond. We demonstrate that the IL, as per Cossé and Bartelt (2000) and Cossé et al. (2001). synthetic compound attracts male sawflies in the field, and Sawflies were aerated in 150 ml glass tubes (3×30 cm), and provide preliminary evidence indicating that (Z)-6,14- the resulting volatiles were collected on Super Q traps (80– pentadecadienal can be produced via abiotic oxidation of 100 mesh, Alltech Deerfield, IL, USA) (1×0.5 cm diam) a long-chain unsaturated hydrocarbon found exclusively in plugged with silanized glass wool. These were placed at the females. inlet and outlet of each chamber. Airflow was held at 50 ml min−1 via valve-controlled vacuum. Sawflies were given access to water in 2 ml glass vials plugged with Methods and Materials cotton and placed inside each chamber. Volatiles were collected from 1 d to 4 d under florescent light at 27°C and Life History In North America, adult Acantholyda eryth- a 16 L:8D photoperiod. Outlet traps from each aeration rocephala emerge from underground in spring (March to May were rinsed with 500 μl of HPLC grade hexane (Fisher depending on latitude) to mate and oviposit (MiddleKauff Scientific) and concentrated to 50 μl under a steady stream 1958; Lyons 1994;AsaroandAllen1999). Flight season of nitrogen and stored at −70°C. lasts for approximately 3 wk, depending on local climate and weather conditions. Females oviposit on the previous year’s Whole Body Extracts Whole body extracts were obtained needles. Larvae hatch approximately 10 d later and feed from adult A. erythrocephala by soaking groups of six male gregariously from within protective webs for 18–21 d or six female sawflies in 2 ml hexane for 2 min. The (Middlekauff 1958). In May to July (depending on latitude), extracts then were concentrated to 200 μl under a stream of last-instars drop to the forest floor and burrow 1–15 cm into nitrogen. A series of fractions (1.5 ml of each) of increasing the soil (Jahn 1967; MiddleKauff 1938;AsaroandAllen polarity were eluted on a silica gel column (0.6 cm ID× 1999). Larvae overwinter in earthen cells as bright green 2 cm height, gravity flow), beginning with, hexane, pronymphs. Pupation occurs in the following spring (Jahn followed by 5%, 10%, and 25% diethyl ether (redistilled) 1967), followed by adult emergence. Adult A. erythroce- in hexane, and finally 100% ether. Fractions were concen- phala are strongly phototropic and readily bask in direct trated to 30 μl under a stream of nitrogen, and analyzed as sunlight on cool days. As such, population densities usually described below for specific chemical constituents. are greater along sunny forest edges or in well-lit patches on the forest floor. Chemical Analysis Volatile collections and chromatograph- ic fractions of whole body washes were analyzed by using Collection and Rearing of Live Insects Most A. erythroce- coupled gas chromatography-mass spectrometry (GC-MS). phala used in this research were collected as pronymphs in Equipment consisted of a Hewlett-Packard model 6890 gas Sand Ridge State Forest (Mason County, IL, USA) during chromatograph interfaced with a Hewlett-Packard model fall 2001, 2002, and 2003, by digging with a shovel under 5973 mass selective detector. Injections were made through defoliated trees to a depth of approximately 15 cm. The a split/splitless inlet, operated in splitless mode. Columns bright green pronymphs were identified readily and collected used included DB-5 (30×0.25 mm ID with 0.25 µm film, by hand. Pronymphs were transported to the laboratory and J&W Scientific, Folsom, CA, USA), DB-1 (15×0.25 mm placed in 31×8×6 cm plastic trays, 100–200 per tray, on ID with 0.10 µm film, J&W Scientific), and a 30 m EC-5 1450 J Chem Ecol (2009) 35:1448–1460 (0.25 mm ID, 0.25 µm film thickness, Alltech Associates, using Wittig condensations as the key step (Fig. 1). Reactions Deerfield, IL, USA). A typical temperature program was 50°C were monitored by GC-MS. Attempts were not made to for 1 min, increasing at 10°C min−1 to 300°C, and with a final optimize yields. The reagents were obtained from Aldrich hold time of 20 min. GC-MS settings were modified as Chemical Co., Milwaukee, WI, USA, and solvents were of necessary for specific analyses; however, the mass spec- HPLC grade. trometer scanning range was typically 40 amu to 550 amu Five Wittig salts were prepared by refluxing the corre- (EI, 70 eV), except in analysis of dimethyl disulfide (DMDS) sponding alkyl halides with triphenylphosphine in acetonitrile derivatives where the maximum mass was increased to (Sonnet 1974). Three of these (4, 5,and6) had simple alkyl 650 amu. The 1995 version of the Wiley mass spectral groups of 1, 10, and 16 carbons, respectively, and two had library was available on the data system (Wiley 1995).