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The sex pheromone of Stathmopoda auriferella (Walker), an important pest of kiwifruit in Korea, was studied. Two candidate pheromone components detected in the gland extracts of females were identified as (E)-5-hexadecenyl acetate (E5-16:OAc) and (E)-5-hexadecenol (E5-16:OH) in a ratio of 75:25 by mass spectral analysis of natural pheromone components and dimethyldisulfide adducts, and retention index comparisons with synthetic standards. In the kiwifruit orchards, E5-16:OAc alone was attractive to S. auriferella males and caught significantly more males than live virgin females. However, addition of E5-16:OH strongly inhibited attraction to E5-16:OAc. These results suggest that the major component of the female-produced sex pheromone of S. auriferella is E5-16:OAc. This hexadecenyl acetate is a novel moth sex pheromone component.[PUBLICATION ABSTRACT]
J Chem Ecol (2013) 39:555558 DOI 10.1007/s10886-013-0269-0
RAPID COMMUNICATION
(E)-5-Hexadecenyl Acetate: A Novel Moth Sex Pheromone Component From Stathmopoda auriferella
Chang Yeol Yang & Kyung San Choi & Myoung Rae Cho
Received: 6 January 2013 /Revised: 15 February 2013 /Accepted: 20 February 2013 /Published online: 13 March 2013 # Springer Science+Business Media New York 2013
Abstract The sex pheromone of Stathmopoda auriferella (Walker), an important pest of kiwifruit in Korea, was studied. Two candidate pheromone components detected in the gland extracts of females were identified as (E)-5-hexadecenyl acetate (E5-16:OAc) and (E)-5-hexadecenol (E5-16:OH) in a ratio of 75:25 by mass spectral analysis of natural pheromone components and dimethyldisulfide adducts, and retention index comparisons with synthetic standards. In the kiwifruit orchards, E5-16:OAc alone was attractive to S. auriferella males and caught significantly more males than live virgin females. However, addition of E5-16:OH strongly inhibited attraction to E5-16:OAc. These results suggest that the major component of the female-produced sex pheromone of S. auriferella is E5-16: OAc. This hexadecenyl acetate is a novel moth sex pheromone component.
Keywords Stathmopoda auriferella . Sex pheromone . (E)-5-hexadecenyl acetate . Lepidoptera . Stathmopodidae . Insect pest
Introduction
The stathmopodid moth, Stathmopoda auriferella (Walker) (Lepidoptera: Stathmopodidae) is distributed in Korea, China, Japan, India, Nigeria, and Greece. The larvae of this species feed on the fruit surface of kiwifruit, Actinidia deliciosa (Actinidiaceae), rendering the fruits unmarketable in Korea (Park et al., 1994). Although the light is an attractant for S. auriferella, light traps are relatively nonspecific (Park et al., 1994). Therefore, there is a need to develop a
species-specific monitoring system for this pest. Identifying the sex pheromone of S. auriferella could lead to a useful monitoring tool and to environmentally friendly control by mass trapping, attract-and-kill, or mating disruption.
A previous field screening test conducted in Japan showed that male S. auriferella (formerly known as S. theoris) were attracted to Z11-14:OAc, Z7-16:OAc, or a mixture of Z7, Z11-16:OAc and Z7,E11-16:OAc (Nagano et al., 1979). However, traps baited with these compounds were not effective in catching S. auriferella males in Korean kiwifruit orchards (C. Y. Yang, personal observation). The principal objective of this study was to identify the sex pheromone of S. auriferella based on chemical analyses of the pheromone gland extracts of females, and the responses of males to synthetic pheromone blends in the field.
Methods and Materials
Insects Larvae of S. auriferella were collected from commercial kiwifruit orchards in July and August of 2011 at Hwaseong, Korea (37.1N, 126.4E). Larvae were reared on kiwifruits in screen cages (30 30 30 cm), and maintained at 23 C under a photoperiod of L14:D10. Emerging adults were collected each day, and the sexes were separated based on the morphological difference at the ventral side of terminal abdominal segments: a partly-extended ovipositor can easily be seen in female moths. Females were kept individually in plastic bottles (7 cm high and 2.5 cm diam) and provided with 10 % sucrose solution as food.
Chemicals Authentic standards of E5-16:OAc, Z5-16:OAc, E5-16:OH, and Z5-16:OH were purchased from Pherobank (Wageningen, The Netherlands; >99 % purity). Dimethyldisulfide (DMDS), iodine, and sodium thiosulfate were obtained from Sigma-Aldrich (St. Louis, MO, USA).
C. Y. Yang (*) : K. S. Choi : M. R. ChoNational Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 440-706, Republic of Korea e-mail: [email protected]
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Pheromone Extraction Preliminary observations revealed that S. auriferella usually mates during the second half of the scotophase. Therefore, the terminal abdominal segments including the pheromone gland were excised from 1- to 2-d-old females during the eighth to ninth hour of the scotophase under a binocular microscope. Batches of three pheromone glands were immersed in 10 l of hexane in a0.3-ml conical glass vial (Wheaton, Millville, NJ, USA) for 10 min. The hexane extract was transferred into another vial and stored at 20 C until chemical analysis.
Chemical Analysis Pheromone gland extracts were analyzed on an Agilent 6890 N GC interfaced to an Agilent 5975C mass-selective detector. Samples were run on DB-5MS and DB-Wax columns (30 m0.25 mm ID, 0.25 m film thickness, J&W Scientific, Folsom, CA, USA). For the DB-5MS column, the oven temperature was programmed from 80 C (1-min hold) to 250 C at 10 C/min, and held at the final temperature for 10 min. For the DB-Wax column, the oven temperature was maintained at 80 C for 1 min, increased to 180 C at 10 C/min, then to 220 C at 5 C/min, and held for 10 min. Injector and transfer line temperatures were 250 C. Electron ionization mass spectra were recorded from m/z 50 to 500 at 70 eV with the ion source temperature of 230 C. GC retention times are quoted as retention indices relative to those of n-alkanes. Compounds from pheromone gland extracts were identified by comparisons of their retention indices and mass spectra with those of authentic standards on two different columns. Quantities of compounds were calculated by using hexane solution of synthetic E5-16:OAc as an external standard.
Dimethyldisulfide Derivatization Determination of double-bond positions in unsaturated compounds of the gland extract was achieved by reaction with DMDS (Buser et al., 1983). DMDS derivatization of a 30-female gland extract was carried out as previously described by Yang et al. (2011). The DMDS adduct was analyzed with a DB-5MS column, using a temperature program of 80 C to 300 C at 10 C/min, with other GC conditions as those described above.
Field Test A field trial was carried out to test the attraction of S. auriferella males to various combinations of E5-16: OAc and E5-16:OH from August 310, 2012 in commercial kiwifruit orchards, where the larvae were collected. Delta traps (Green Agro Tech, Gyeongsan, Korea) were baited with white rubber septa (8 mm OD; Aldrich Chemical, Milwaukee, WI, USA) loaded with synthetic pheromone blends. In addition, traps baited with two 0- to 1-d-old virgin females in small mesh cages were used as positive controls, and traps without baits were used as negative controls. Traps were hung in kiwifruit orchards at a height of approximately1.5 m. A randomized complete block design was used with
four replicates of each treatment. The distance between traps within a block was at least 10 m. Males captured in traps were counted at intervals of 23 d, and sticky inserts and virgin females were replaced at each count.
Numbers of males caught per treatment were transformed to log (x+1) to normalize the variance and submitted to oneway analysis of variance (ANOVA). Treatments that failed to capture males were not included in the analyses to avoid violating assumptions of ANOVA. Means were separated with Tukeys HSD test at =0.05 (SAS Institute Inc., 2010).
Results and Discussion
GC-MS analyses on pheromone gland extracts of female S. auriferella indicated the presence of two candidate pheromone components (compounds 1 and 2) along with straight-chain alkanes frequently found in lepidopteran glands (Fig. 1a). One minor compound (compound x) also was detected in the extracts, but mass spectra did not have fragment ions of the typical moth pheromone components (Ando and Yamakawa, 2011).
The mass spectrum of compound 1 had diagnostic fragment ions m/z 222 (M+-60), 194, 166, and 61 (CH3COOH2+), which suggested a hexadecenyl acetate. Analysis of the DMDS-derivative revealed an adduct of hexadecenyl acetate with a molecular ion at m/z 376 (282+MeSSMe) and diagnostic ions at m/z 201 (C11H22SMe), and 175 (CH3COOC5H9SMe)
indicative of the acetate with the double bond at the 5 position (Fig. 1b) (Buser et al., 1983). The retention indices of compound 1 on both columns coincided with those of synthetic E5-16:OAc (DB-5MS: 1990, DB-Wax: 2328), but not with Z5-16:OAc (DB-5MS: 1984, DB-Wax: 2325).
Compound 2 showed a molecular ion m/z 240, and diagnostic fragment ions m/z 222 (M+-18) and 194, suggesting a hexadecenol. The mass spectrum of a DMDS adduct with M+ at m/z 334, derived from the hexadecenol in gland extract, showed 201 (C11H22SMe), and 133 (HOC5H9SMe), placing the double bond at position 5. The retention indices of compound 2 on the DB-5MS and DB-Wax columns were identical to those of the synthetic E5-16:OH (1868 and 2415, respectively) rather than Z5-16:OH (1867 and 2418, respectively). Accordingly, the structures of compounds 1 and 2 in the pheromone glands were determined to be E5-16:OAc and E5-16:OH. Based on the batch extracts (N=10), the amount (SE) of the major component E5-16:OAc present in the pheromone gland extract was estimated as 2.10.4 ng per female, and the relative ratio of E5-16:OAc and E5-16:OH was 75:25.
A total of 270S. auriferella males were captured in a field experiment with no catch in unbaited traps. E5-16:OAc as a single component was attractive to males, whereas E5-16: OH was unattractive alone (Fig. 2). Traps baited with 1 mg
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a
b
Fig. 1 GC-MS analysis of sex pheromone of Stathmopoda auriferella. a Total ion chromatogram of female pheromone gland extracts on a DB-Wax column. Compound identities as follows: C23)
tricosane; x) unknown;1) E5-16:OAc; C24)
tetracosane; 2) E5-16:OH; C25)
pentacosane. b Mass spectrum of compound 1 after DMDS derizativation
C
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E5-16:OAc captured approximately three times more males than traps with caged virgin females. Addition of 5 to 100 % of E5-16:OH to the major component, E5-16:OAc, significantly inhibited attraction of male S. auriferella. Male captures gradually decreased in response to increasing amounts of the alcohol relative to E5-16:OAc. Regression analysis indicated a negative linear relationship between trap catch and proportion of E5-16:OH in binary blends (F=12.57, P=0.038, r2=0.81). However, it remains to be determined whether attractiveness of E5-16:OAc could be affected by addition of smaller amounts (<5 % of the major component) of E5-16:OH.
Chemical analyses and field test showed that the sex pheromone of S. auriferella consists of a single major component, E5-16:OAc. The only species for which a female sex pheromone has been reported previously in the genus Stathmopoda is the persimmon fruit moth, S. masinissa (Naka et al., 2003). This species uses (4E,6Z)-4,6-hexadecadienyl acetate (E4,Z6-16:OAc) as its sex pheromone. However, E4,Z6-16:OAc and other structurally related dienes were not detected in
pheromone gland extracts of female S. auriferella. These studies suggest that the sex pheromones of moths in the genus Stathmopoda generally are 16-carbon compounds with one or more double bonds.
Hexadecenyl acetates with double bonds at positions 7, 9, 10, or 11 had been reported previously as a component of female sex pheromones for 45 other moths, including Euxoa messoria, Helicoverpa assulta, Pseudexentera spoliana, Leucinodes orbonalis, and Plutella xylostella (El-Sayed., 2012). E5-16:OAc has been discovered to be an attractant for one oecophorid, Bisigna procerella in a field screening test in the Netherlands (Willemse et al., 1987), but had not been identified from conspecific females. Therefore, this acetate is a novel structure of sex pheromone identified in Lepidoptera.
As noted above, E5-16:OH were detected in pheromone gland extracts of females, but addition of this to the main pheromone component, E5-16:OAc, strongly inhibited attraction for males. This result suggests that the alcohol is not a pheromone component. The inhibitory effect of other
60
Fig. 2 Catches of male Stathmopoda auriferella in traps baited with E5-16:OAc and E5-16:OH singly and in various blends in kiwifruit orchards in Hwaseong, Korea. Bars with the same letter are not significantly different at P<0.05 by Tukeys HSD test. VF = two virgin females
50
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compounds found in gland extracts of conspecific females has been reported for other moths (McElfresh and Millar, 1999; Gibb et al., 2007; Yang et al., 2009). However, it remains unknown whether E5-16:OH is actually released by calling females. This alcohol may be simply a precursor to E5-16:OAc in the pheromone glands. Additional electro-physiological and chemical studies with volatile emissions from S. auriferella females are required to determine whether E5-16:OH and/or other possible pheromone components are emitted by calling females.
During attractant screening experiments for moth species in Japan, Nagano et al. (1979) found that S. auriferella males were attracted to Z11-14:OAc, Z7-16:OAc, or a mixture of Z7,Z11-16:OAc and Z7,E11-16:OAc. However, we found no evidence that females from the Korean population of S. auriferella produced these acetates. Further examination of pheromone gland extents from different populations of this species will help to clarify the situation.
Acknowledgments We thank Prof. Seunghwan Lee for identification of Stathmopoda auriferella. We also acknowledge the assistance of Seok Bum Keon and Sang Hwan Oh with field work.
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