Joumal ofChemical Ecology, Vol. 27, No.9, September 2001 (@ 2001)

IDENTIFICATION OF A FEMALE-SPECIFIC, ANTENNALLY ACTIVE VOLATILE COMPOUND OF THE CURRANT STEM GIRDLER*

ALLARD A. COSSE,I.t ROBERT J. BARTELT,! DAVID G. JAMES,2 and RICHARD J. PETROSKI'

I USDA Agricultural Research Service National Centerfor Agricultural Utilization Research Bioactive Agents Research Unit 1815 N. University Street, Peoria, Illinois 61604 21rrigated Agriculture Research and Extension Center Washington State University 24106 North BUlllI Road Prosser, Washington 99350

(Received November 30,2000; accepted May 12,2001)

Abstract-We identified (Z)-9-octadecen-4-olide as a female-specific, anten­ nally active compound from the currant stem girdler Janus integer Norton. Fe­ male specificity was demonstrated by gas chromatographic comparison of liq­ uid chromatography fractions of male and female volatile emissions and whole body extracts. The y-lactone was identified by coupled gas chromatographic­ electroantennographic detection (GC-EAD), coupled gas chromatographic-mass spectrometric (GC-MS) analysis, microchemical reactions, and GC and MS comparison with authentic standards. GC-EAD analysis offemale volatile emis­ sions and cuticular extracts showed a single peak of activity on male antennae, which was not present in male-derived materials. Female antennae did not re­ spond to any of the tested materials. The hydrogenation product of the natural EAD-active material was a known saturated y-lactone. The mass spectrum of the dimethyl disulfide derivative of the natural y-lactone was consistent with a double bond present in the 9 position. Comparison ofthe natural y-Iactone and a synthesized racemic mixture of (Z)-9-octadecen-4-0Iide on a chiral GC column showed the presence of a single enantiomer in the natural material.

*Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. tTo whom correspondence should be addressed. e-mail: [email protected]

1841

0098·0331/0 1I0900·184IS19.50/0 '£' 2001 Plenum Publishing Corporation 1842 COSSE, BARTELT, JAMES, AND PETROSKI

Key Words-Jan liS illteger, sawfly, Hymenoptera, Cephidae, sex pheromone, (Zl-9-octadecen-4-o1ide, coupled gas chromatographic-electroantennographic detection (GC-EAD).

INTRODUCTION

The currant stem girdler, Janus integer Norton (Hymenoptera: Cephidae), is an occasional pest of red currant (Ribes spp,) in North America (Crassweller et aI., 1997), In spring, egg laying female 1. integer can make numerous punctures in canes, resulting in drooping and wilting of new shoots, Further damage occurs as emerging larvae tunnel within the canes, This insect also attacks poplar and willow trees, and damage to currants usually is more severe near stands of these trees (Crassweller et aI., 1997), Preliminary field observations indicated that caged virgin females attracted male 1. integer (D, G, James, unpublished data), thus 1. integer could utilize a female-produced sex pheromone for mate location, Currently, the only studied cephid pheromone is that of the wheat stem sawfly, Cephus cinctus Norton (Cosse et aI., unpublished data), The pheromone may be beneficial to current pest control practices, especially since insecticides are ineffective against larvae inside the currant stems, This paper describes the isolation, structure determination, and electroan­ tennographic activity of a female-specific volatile compound and compares this with other sawfly pheromones,

MATERIALS AND METHODS

Insects, Overwintering currant stem girdler larvae present in dormant red cur­ rant stems were collected near Prosser, Washington, during January 2000, Larva­ containing stems were placed individually into glass vials, capped with a moist piece ofcloth-covered cotton, and sent to NCAUR. Upon arrival in Peoria, Illinois, the vials were kept at 25°C under a 16:8 (LD) hour photoperiod regime, Adults emerged about three weeks later. Adults used in this study were 1- to 4-days old, Volatile Collections and Cuticular Extracts, One- to three-day volatile collec­ tions were made from individuals or groups of 4 male or female insects. Volatiles were trapped on Super Qporous polymer (80-100 mesh, Alltech, Deerfield, IL) or on solid-phase microextraction (SPME) fibers (l00 !Lm poly(dimethyl)siloxane, Supelco, Bellefonte, PA). Volatile collections were made by methods and equip­ ment generally described by Cosse and Bartelt (2000), Cuticular extracts were obtained by a single rub of the abdomen of individual male or female insects with a SPME fiber, In addition, individuals or groups of 8 male or female insects were killed by freezing when 1-4 days old, and soaked overnight in 1 ml hexane, FEMALE-SPECIFIC, COMPOUND OF THE CURRANT STEM GIRDLER 1843

Hexane extracts were stored at -20°C Extracts were concentrated under a gentle stream of nitrogen and fractionated on open columns of silica gel (0.5 x 3 cm). Two-milliliter fractions were collected for each of the following elution solvents: hexane; 5%, 10%, and 25% ether in hexane (by volume); and ether. Fractions were concentrated to a volume similar to the non-fractionated samples prior to any further analysis. Electrophysiology. Coupled gas chromatographic-electroantennographic (GC-EAD) analyses were made by methods and equipment generally described by Cosse and Bartelt (2000). GC-EAD connections were made by inserting a glass­ pipette Ag-AgCl-grounding electrode into the back of an excised sawfly head. A second glass-pipette Ag-AgCl-recording probe was placed in contact with the dis­ tal end of one antenna. Both pipettes were filled with Beadle-Ephrussi (Ephrussi and Beadle, 1936) saline. Instrumentation. Volatile collections, extracts, and liquid chromatography (LC) fractions were analyzed by gas chromatography with flame-ionization de­ tection (GC-FID) and coupled GC-mass spectrometry (GC-MS, electron impact and chemical ionization). Samples were injected in splitless mode using Hewlett Packard 6890 instruments fitted with 30 meter EC-l capillary columns (0.25 mm LD., 0.25 {Lm film thickness, Alltech, Deerfield, IL). Temperature programs were from 50°C to 275°C at lQoC per min (GC-MS) or at lSOCpermin (GC-EAD). Injec­ tor temperatures were maintained at 250°C, and GC-EAD effluent interface from post-column splitter was kept at 275°C Injectors were fitted with SPME injector liners (Supelco, Bellefonte, PA) for SPME analysis. Mass spectrometry was per­ formed with a Hewlett Packard 5973 instrument (electron impact, 70 eV). Chemical ionization spectra (isobutane) were obtained on a Thermo Quest Trace GC 2000 quadrupole mass spectrometer. The Wiley mass spectral library, with 275,821 spectra, was available on the MS data system (Wiley, 1995). Chiral resolution was achieved on a capillary column having a diacetyl-derivatized ,B-cyclodextrin phase, ,B-DEX 225 (30 m length, 0.25 mm ID, 0.25 {Lm film thickness; Supelco, Bellefonte, PA) operated at 200°C Microchemical Reactions. Hydrogenation of GC-EAD-active LC fractions was used to confirm the number of carbon-carbon double bonds. Samples (100 {Ll) were stripped to dryness under a stream of nitrogen and resuspended in 100 {Ll ethanol to which was added "-'0.5 mg of 10% Pd on charcoal. Reduction was accomplished by bubbling a gentle stearn of hydrogen through the sample for 5 min at room temperature. The reduced sample was filtered and analyzed by GC-MS and GC-EAD. Dimethyl disulfide (DMDS) derivatives were prepared for determination of double bond locations of EAD-active material (Carlson et aI., 1989). Samples (lOa {Ll) were stripped to dryness under a stream of nitrogen, and DMDS and 5% iodine in ether were added (equal volumes, about 25 {Ll each). The samples were heated at 45°C for 1-2 hr, then diluted with hexane and worked up with 1844 COSSE, BARTELT, JAMES, AND PETROSKI aqueous sodium thiosulfate to destroy the iodine. The organic layer was dried over sodium sulfate, evaporated under nitrogen, resuspended in about 10 III hexane, and analyzed by GC-MS. Oven temperature was programmed up to 300°C, and MS scanning range was 40-600 amu. The antenally active 10% etherlhexane LC fractions contained two major free fatty acids, linoleic- and oleic acid, which were methylated to the corresponding methyl esters with diazomethane. Approximately 5 mg of N-methyl-N-nitroso­ p-toluenesulfonamide (Diazald, Aldrich, Milwaukee, WI) were added to a 1: 1 mixture of ether and a mixture of 5% aqueous KOH and methanol 0:1). Ten micro liters of the yellow ether layer were added to 100 ILl of the EAD-active LC fraction. After 5 min, the mixture was evaporated and the residue dissolved in hexane for GC-MS and GC-EAD analysis.

HO(CH2)60H PPTS 1hexane, acetonitrile HO(CH2)60THP ~ PDC, CH 2CI 2

OHC(CH2)sOTHP nonyl(triphenyl)phosphonium bromide, 1THF,BuLi CH3(CH2hCH=CH(CH2hOTHP (mostly Z) ~ Dowex SOW x 8, MeOH

CH3(CH2hCH=CH(CH2)SOH ~ PDC, CH 2C1 2

CH3(CH2hCH=CH(CH2)4CHO 1. Br(CH2hC02Et, SmI2, HMPA, THF 12. HCI CH3(CH2hC=C(CH2)4~O + (E)-isomer II HH

(Z)-9-octadecen-4-olide FIG. 1. Synthesis of (Z)-9-octadecen-4-o1ide (see text for abbreviations). FEMALE-SPECIFIC, COMPOUND OF THE CURRANT STEM GIRDLER 1845

Synthesis of (Z)-9-octadecen-4-0Iide. A preliminary synthesis to confirm structure was conducted by one of us (RJP), based on well-known reactions (Figure I). One hydroxy group of 1,6-hexanediol (Aldrich, Milwaukee, WI) was protected as a tetrahydropyranyl (THP) ether by using pyridinium p-toluenesulfo­ nate (PPTS) as catalyst (Miyashita et aI., 1977). Monoprotection of the diol was accomplished by modification of the published procedure (Miyashita et aI., 1977). In a biphasic mixture ofacetonitrile and hexane, the reactants were primarily in the more polar (acetonitrile) phase, but the mono ether migrated to the hexane phase, reducing the chance offurther derivatization to the diether. The free hydroxyl was oxidized with pyridinium dichromate (PDq in CH2Ch to give the aldehyde (Corey and Schmidt 1979). The aldehyde was reacted with nonyl(triphenyl)phosphonium bromide to give, predominantly, the Z-olefin (Sonnet, 1974). The THP protective group was easily removed by acid catalysis (Dowex 50 W x 8) in MeOH (Beier and Mundy, 1979). The alcohol was oxidized to the aldehyde as described above. Samarium iodide mediated the coupling of Br(CH2hC02Et with the aldehyde, to form the 4-hydroxy ester. This cyclized to the desired y-lactone during the acidic workup (Otsubo et aI., 1987). The Sm1]-induced coupling of an unactivated organic halide with an aldehyde is promoted by hexamethylphosphoric triamide (HMPA). Both enantiomers are formed.

RESULTS AND DISCUSSION

Cuticular Extracts and Volatile Collections. Comparisons of the GC pro­ files of extracted materials obtained by SPME rub and hexane soak showed a high degree of similarity (Figure 2). GC-EAD analyses (N = IS) of cuticular extracts (SPME rub and hexane soak) from individuals or groups of male or fe­ male insects were obtained by using male and female antennae. EAD activity occurred consistently at one GC retention time (21.8 min) when cuticular extracts from female insects (N = 6) (Figure 2) were tested on male antennae, but not on female antennae. Male and female antennae showed no EAD activity with cuticu­ lar extracts derived from males (N = 9). Similarly, GC-EAD analyses (N = 10) of volatiles collected (Super Q and SPME) from individuals or groups of female insects (N = 5) consistently indicated the same GC retention time, with EAD ac­ tivity only on male antennae. Male and female antennae showed no EAD activity with volatiles derived from males (N = 5). Liquid Chromatography. TheEAD-activity offemale cuticular extracts eluted from silica gel with 10% ether in hexane, suggested an oxygenated compound for the EAD-active material. No other antennal activity was noted for any of the other silica gel fractions (Figure 3). Comparison ofGC-MS analyses of 10% etherlhexane fractions ofmale versus female cuticular extracts demonstrated a female specific compound, as shown in 1846 COSSE, BARTELT, JAMES, AND PETROSKI

A

GC

EAD

B

GC 21.00 21.33 21.67 22.0022.33 22.67

EAD

o 3 6 9 12 15 18 21 24 27 30 Time (min) FIG. 2. Simultaneously recorded gas chromatographic (GC) and electroantennographic detection (EAD) of typical male antennae to a hexane extract of female J. integer (A) and a SPME cuticular rub of female J. integer (B). Insert shows the GC retention time of EAD-activity. FEMALE-SPECIFIC, COMPOUND OF THE CURRANT STEM GIRDLER 1847

A

GC

EAD ~{i~~.. I~~~ri/'lli-oII{Nr"MI"'I"t

8 21.8 I

1 I 1 I , 1 1 1 16 17 18 19 20 21 22 23 24 25 26 Time (min)

FIG. 3. Simultaneously recorded gas chromatographic (GC) and electroantennographic detection (EAD) of typical male antennae to liquid chromatography fraction (10% ether in hexane) of female J. integer hexane extract (A), and synthetic (Z)-9-octadecen-4-olide (B). Arrows denotes the GC retention times of EAD-activity.

Figure 4. This compound corresponded to the EAD-active material, based on comparisons ofGC-MS and GC-EAD elution profiles (same column, but different temperature ramps). The mass spectrum of the EAD-active compound indicated a molecular weight of 280 (Figure 5), which was confirmed by the chemical ionization mass spectrum (base peak at mlz 281), but its EI spectrum did not match any of the spectra in the MS library. 00 .j:o. 00 a~ ~\¥b

22.6 1

() o en en S'1' to ;J> ~ ~ ~ 20.0 20.5 21.0 21.5 220 22.5 23.0 23.5 24.0 24.5 25.0 m !" Time (min) ~ o FIG. 4. The gas chromatographic comparison of liquid chromatography fractions (10% ether in hexane) of female (upper) and male (lower) 1. integer hexane extracts. Arrows denotes the presence of linoleic (a) and oleic acids (b). ~ o en f5 FEMALE-SPECIFIC, COMPOUND OF THE CURRANT STEM GIRDLER 1849

67 100 81

55 80 H 95 CH H3C-(CR 2)4J(J0* u 60 '"c "0 185 HH '"C :J ..0« 40 109 136

20 280 I 220 ii 182 I 262 oIt 1,1 •• 1, 40 60 80 100 120 140 160 180 200 220 240 260 280 Mass/Charge

153 100

173 80

u '"c 60 "0 '"C :J 135 «..0 40 201 374

': u, j,,1u, ,," .1, 1 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 Mass/Charge

FIG. 5. EI-Mass spectrum of (Z)-9-octadecen-4-olide from female J. integer (upper) and EI-mass spectrum of the DMDS derivative of this compound, showing origins of major fragments (lower).

A molecular weight of 280 suggested C19H360 (2 double bonds or ring equivalents) or ClsH3202 (3 double bonds or ring equivalents) as likely molec­ ular formulas. In fact, the silica gel fraction with the female-specific compound contained two relatively abundant fatty acids, which were identified by MS as 1850 COSSE, BARTELT, JAMES, AND PETROSKI linoleic- and oleic acid (Figure 5). The female 10% ether/hexane fraction was treated with several chemical reagents to further elucidate the chemical composi­ tion of this EAD-active material. Methylation. The addition of diazomethane to the female 10% ether/hexane fraction converted the carboxylic acids to the corresponding methyl esters, but did not affect the EAD-active compound, indicating it did not have a free carboxyl group. Hydrogenation. The mass spectrum of the EAD-active compound after hy­ drogenation showed a molecular ion at m/z 282, indicating the presence of one carbon-carbon double bond. Furthermore, the MS library picked y-stearolactone as a likely match for the hydrogenation product. This MS library match was confirmed by comparison of the mass spectra and GC retention times of the hydrogenation product and an authentic sample of y-stearolactone (Cermak and Isbell, 2000). Similar to the natural product, this synthetic standard also had a frag­ ment ion at m/z 85, which would be expected for a y-Iactone (cleavage where side chain joins ring), suggesting that the natural compound was probably a y-Iactone with one carbon-carbon double bond in the side chain; the fragment ion at m/z 85 would not be present if the carbon-carbon double bond was residing in a ring. The presence of a double bond might also be important for antennal activity, because GC-EAD analysis ofthe hydrogenation product showed a complete loss ofantennal activity. In addition, GC-EAD analysis prior to the diazomethane treatment showed no antennal activity for the oleic acid: surprisingly however, the methyl ester of oleic acid was antennally active. DMDS Derivatization. The mass spectrum of the DMDS (MW = 94) deriva­ tive of the EAD-active compound is presented in Figure 5. The readily formed derivative showed a molecular ion at m/z 374 (= 280 + 94), and the fragment ions (m/z 173 and 201) were consistent with a double bond being present in the 9 position. Synthetic (Z)-9-octadecen-4-olide. The synthetic y-Iactone matched the natu­ ral female-derived 1. integer compound exactly by mass spectrum, mass spectrum of DMDS derivative, and by GC-EAD analyses (Figure 3). The Wittig synthe­ sis (Figure I) created both the (Z) and (E) configurations at the double bond. The Z-isomer was recognized because it eluted slightly earlier by GC on the non-polar column (Z-isomer at 21.82 min, E-isomer at 21.87 min) and because it was more abundant (ca. 70:30), as expected for the Wittig reaction conditions (Sonnet. 1974). GC analysis established that the natural material contained only the earlier-eluting Z-isomer, and by GC-EAD the synthetic Z-isomer was antennally active (Figure 6). The synthetic y-Iactone was synthesized as a racemic mixture and its two enantiomers showed base-line separation on the chiral GC column (Figure 7). The earlier eluting enantiomer had the same GC retention time as the natural compound. Future chiral synthesis of the y-Iactone will determine the absolute configuration of the natural y-Iactone. FHvIALE-SPECIFIC. COMPOUND OF THE CURRA.NT STEM GIRDLER 1851

A

H H H,C, ~ )CH2),J:°~O PI'hi U H

GC

EAD

B

GC

EAD

21.48 21.65 21.82 21.98 22.15 Time (min)

FIG. 6. Simultaneously recorded gas chromatographic (GC) and electroantennographic detection (EAD) of typical male antennae to an isomer mixture ofsynthetic (Z)-, and (£)-9­ octadecen-4-0Iide (A) and liquid chromatography fraction (10% ether in hexane) of female J. integer hexane extract (B). Based on GC-FID peak areas and fraction volumes, the estimated maximum release rate of natural y-lactone was 10 ng/female/day. Sawfly Sex Pheromones. Forthcoming behavioral tests will determine whether this female-specific y-lactone of 1. integer is capable of attracting male sawflies and, thus, be confirmed as a sex pheromone. Little is published on pheromone systems ofsawflies belonging to the family Cephidae. The only studied cephid pheromone is that of the wheat stem sawfly Cephus cinctus Norton (Cosse et aI., unpublished data). Insect Specific y-Lactones. Sex pheromones ofsawflies belonging to the fam­ ily Diprionidae and to one species belonging to the family of Tenthredinidae are well-studied (Bartelt and Jones, 1983; Bartelt et aI., 1983; Anderbrant, 1999). However, no y-lactone containing compounds have been reported as sawfly 1852 COSSE, BARTELT, JAMES, AND PETROSKI

A 65.5 66.4

62 63 64 65 66 67 68 69 70 Time (min)

B 65.5

62 63 64 65 66 67 68 69 70 Time (min) FIG. 7. Chiral separation by gas chromatography of racemic (Z)-9-octadecen-4-olide (A) and (Z)-9-octadecen-4-o1ide (B) from female J. integer.

pheromones, although related y-lactones are known as scarab beetle pheromones (Tumlinson et aI., 1977; Leal, 1999). For example, the sex pheromone of the yellowish elongate chafer, Heptophylla picea Motschulsky was identified as (R,Z)­ 7,lS-hexadecadien-4-o1ide and is probably biosynthesized from stearic acid (Leal et aI., 1996). Similarly, fatty acid derivatives might be involved in the proposed sex pheromone of currant stem girdler.

Acknowledgments-We thank the following colleagues at NCAUR, Peoria. Illinois: Ronald D. Plattner for assistance in obtaining CI mass spectra and Terry Isbell for the sample of y-stearolactone. FEMALE-SPECIFIC, COMPOUND OF THE CURRANT STEM GIRDLER 1853

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U.S. Dent. of Agricultufl~ National Center tor Agncultural Utilization Research, Peona, lUinois