Sex Pheromone Components in the New Zealand Greenheaded Leafroller Planotortrix excessana (Lepidoptera: Tortricidae) R. A. Galbreath Entomology Division, D.S.I.R., Private Bag, Auckland, New Zealand M. H. Benn Chemistry Department, University of Calgary, Alberta, Canada, on leave at Entomology Divi­ sion, D.S.I.R. H. Young Horticulture and Processing Division, D.S.I.R., Auckland, New Zealand V. A. Holt Entomology Division, D.S.I.R., Auckland, New Zealand Z. Naturforsch. 40 c, 266-271 (1985); received October 30, 1984 Sex Pheromone, Tetradecenyl Acetates, Planotortrix excessana, Tortricidae, Sibling Species Planotortrix excessana was found to include moths of two distinct pheromone-types which were not mutually attractive. Tetradecyl acetate and (Z)-8-tetradecenyl acetate were identified as pheromone components in one, and two other tetradecenyl acetates, probably (Z)-5- and (Z)-7- tetradecenyl acetate, in the other. By contrast with other pheromones reported from the tribe Archipini, A 11-tetradecenyl compounds were not found in either pheromone-type. Introduction locally available host plant, Acmena smithii (Poiret), The greenheaded leafroller, Planotortrix exces­ in place of alfalfa leaf meal. Pupae were removed, sana (Walker), along with the brownheaded leaf­ sexed, and separated accordingly. Pheromone ex­ roller Ctenopseustis obliquana (Walker) and the tract was collected from virgin female moths as light-brown apple moth Epiphyas postvittana (Walker) previously described [4], Male moths were main­ are predominant among the complex of leafroller tained separately under a natural light cycle until pests of horticulture in New Zealand. All three required for bioassay. moths are classified in the Tortricidae, subfamily Electroantennogram (EAG) responses of antennae Tortricinae, tribe Archipini [1]. Sex pheromone of male moths were determined by the method of components have been identified in the Australian Roelofs [5] with standards presented as previously E. postvittana [2]. We have examined the other two described [4]. A field cage bioassay, following that species, which are endemic to New Zealand, and of Tamaki et al. [6] was also used. In this bioassay here describe the identification of pheromone com­ the pheromonal activity of fractions was determined ponents in P. excessana and give evidence for a by captures of released male moths in traps con­ second pheromone-type within the species as cur­ taining the fractions. Test fractions in pentane solu­ rently defined. tion were spotted on filter paper strips which were placed in sticky traps (Pherocon IC; Zoecon Corp., USA) hung 1 m above the ground in a 7 x 10x2m Materials and Methods field cage. The amount of extract or fraction per A colony of P. excessana was established from trap was 20 female-equivalents. Male moths were moths collected at Auckland. They were reared in­ released in the cage each evening shortly before dividually on a diet similar to that of Roelofs and dusk, just prior to their normal period of activity. Feng [3], substituting dried and powdered leaf of a Counts of moths trapped were made the next morn­ ing. Synthetic materials were tested either on filter Reprint requests to R. A. Galbreath. paper in the same way as extracts, or applied in 0341-0382/85/0300-0266 $01.30/0 pentane solution to 5 mm sleeve-type rubber caps Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. R. A G albreath et al. ■ Sex Pheromone Components in the New Zealand Greenheaded Leafroller 267 (1780-B10, Arthur H. Thomas Co., USA), which had been kept at 40 °C overnight it was washed were used for the duration of the test. with aqueous Na2S20 3 (50 (il), centrifuged, and the Materials on filter paper were replaced nightly. aqueous layer carefully removed. The pentane solu­ Field tests of synthetic materials used rubber caps tion so obtained was evaporated to dryness under a in sticky traps hung 1.5 m above the ground and gentle stream of nitrogen. Hexane (2 nl) was added 15 m or more apart in areas infested with P. exces- to give a solution that was analyzed by GC-MS sana. The trap positions were rotated weekly when using a DB-1 bonded phase WCOT column (30 m x the catches were recorded. For statistical compar­ 0.3 mm fused silica, J & W Scientific Inc., USA). ison trap catches were transformed to ]/(/ + 1/2) and The MS was scanned continuously and the spectra differences between means tested by analysis of stored on disc and later searched for the molecular variance (with fractions, replicates and successive and major fragment ions expected for derivatized night’s catches treated as separate factors) and alkenyl acetates. Duncan’s new multiple range test. In the tables, the means listed are of untransformed catches. Results Gas chromatography and mass spectrometry in­ strumentation and procedures were as previously In initial tests, traps containing either virgin described [4], Female tip extract was fractionated by female moths or abdominal tip extracts both caught preparative GC using OV-1 (3% on Chromosorb male P. excessana. The abdominal tip extract was W-HMDS 100/120 mesh in a 2 m x 5.3 mm ID stain­ then subjected to preparative GC on OV-1, collect­ less steel column), with 10% of the effluent split to ing successive 1 min fractions over a time-span en­ the detector and 90% to the collector port [4], Under compassing Rt s of simple alkyl compounds up to the operating conditions used the retention times about C20 chain length. These fractions were then (R T) of dodecyl, tetradecyl, and hexadecyl acetates screened using the EAG response of a male an­ were 4.7 min, 7 min, and 9.5 min respectively. Col­ tenna. Maximal response was obtained for the lections were made from 3 to 6 min (A), 6 to 8.5 min fraction corresponding to the Rr s of tetradecyl/ (B) and 8.5 to 11 min (C). tetradecenyl acetates (14: Ac, U 14: Ac). Female abdominal tip extract was also fraction­ When female abdominal tip extract was frac­ ated by column chromatography on Florisil [7], tionated by column chromatography on Florisil only Some fractions were saponified by 10% KOH in the “esters” fraction, eluted with 5% diethyl ether/ 80% ethanol/water overnight, or acetylated by acetic pentane, was active in the field-cage bioassay anhydride and pyridine. Preparative thin layer (Table I (a)). Furthermore, saponification of the chromatography (TLC) was carried out on silica gel “esters” fraction destroyed its activity, which was G impregnated with silver nitrate (30% w/w) and then restored by acetylation (Table 1(b)), in accord the plates developed with benzene. Zones cor­ with the principal pheromone components being responding to the alkyl, £-alkenyl, Z-alkenyl and acetates. When the “esters” fraction was further alkadienyl acetates were located by running refer­ fractionated by silica gel/silver nitrate TLC, only ence substances in the margins of the plate and the Z-alkenyl acetate fraction was active in the field locating these with a fluorescein spray. cage bioassay (Table 1(c)), and this activity was not Double bond location was determined by a proce­ significantly increased by recombining it with the dure based on that of Francis and Veland [8] as saturated, £-alkenyl and alkadienyl acetate fractions modified by Buser et al. [9], involving bis-thiome- (Table 1(d)). thylation of the alkenyl acetates with dimethyl di- When tip extract was fractionated by preparative sulphidc in the presence of iodine. Female tip- GC on OV-1 a major signal was observed with Rr extract w'as first fractionated by preparative GC on matching tetradecyl/tetradecenyl acetates, and upon OV-1 as described above. Derivatization was then field-cage bioassay only fraction B, incorporating carried out in a 100 |il conical reaction vial. A pen- this region, was active (Table 1(e)). This activity tane solution of the relevant fraction was evaporated was not significantly increased by any recombina­ to about 20 (il and then treated with dimethyl di­ tion of fractions (Tables 1(e) and (f)). On the other sulphide (1 |il) and a solution of iodine in diethyl hand, fraction B or combinations with it were ether (60 mg ml“ 1, 3 |il). After this reaction mixture significantly more active than crude tip extract, 268 R. A. G albreath et al. • Sex Pheromone Components in the New Zealand Greenheaded Leafroller Table I. Field cage bioassays: catches of male P. excessana (corresponding to CH 3 CO 2 H 2 species formed from in traps containing fractions of female abdominal tip acetates), and m /z 196 and 194 (corresponding to extract (20 female equivalents per trap): (a) fractionation by Florisil chromatography (2 replicates; 130 moths re­ M-60 fragment ions from 14: Ac and U14:Ac re­ leased, 100 trapped); (b) saponification and acetylation of spectively). Tip extract was fractionated by prep­ active fraction from (a) (2 replicates; 130 moths released, 93 trapped); (c) fractionation by silica gel/silver nitrate arative GC on OV- 1 as above and the combined TLC (2 replicates; 100 moths released, 73 trapped); fractions A, B and C subjected to GC-MS analysis (d) recombinations of silver nitrate TLC fractions (4 re­ on a Carbowax 20M SCOT column. Strong signals plicates; 160 moths released, 56 trapped); (e) fractionation by GLC on OV-1 (2 replicates; 150 moths released, were detected with RT's corresponding to 14: Ac 137 trapped); (f) recombinations of GLC fractions (2 re­ (m /z 61 and 196 channels) and a U14:Ac (m /z 61 plicates, 350 moths released, 316 trapped).