The moth Euzophera pyriella (Lepidoptera: Pyralididae) is one of the important fruit pests in the pear orchards of Xinjiang, China. Extracts from the sex pheromone gland of virgin female moths were analyzed with gas chromatography-mass spectrometry (GC-MS). Two components, (Z)-8-Dodecenyl acetate (Z8-12:Ac) and (Z,E)-9,12-Tetradecadien-1-ol (Z9E12-14:OH), were identified in extracts. Z9E12-14:OH was the most active in electroantennogram (EAG) and field attraction studies. Z8-12:Ac and other common components identified in Euzophera (including Z9-14:Ac, Z9-14:OH and Z9E12-14:Ac) neither elicited significant EAG responses nor attracted many males in field tests. Binary mixtures of Z9E12-14:OH and Z8-12:Ac in different ratios were active in field tests, but the number of male moths trapped did not differ between these binary mixtures and Z9E12-14:OH alone. We conclude that Z9E12-14:OH is a major component of the female sex pheromone of E. pyriella and could be used as an attractant for monitoring the populations of this moth species.
Pyrus × sinkiangensis T. T. Yu (Rosales: Rosaceae), Xinjiang fragrant pear, is a major economic crop in Xinjiang, China (Li et al. 2007; Chen et al. 2007; Jia et al. 2009), and Euzophera pyriella Yang (Lepidoptera: Pyralididae) is one of the most important insect pests in Xinjiang fragrant pear orchards (Hou et al. 2011). Euzophera pyriella is widely distributed in Xinjiang (Yang 1994) and has caused serious damage to pear trees, especially in Korla and Aksu areas, in recent years. The larvae not only attack pear trees, but also damage other crops, including Malus pumila Mill, Ziziphus jujuba Mill, Prunus armeniaca Linn, Amygdalus persica Linn, Ficus carica Linn (Song et al. 1994; Yang et al. 2011). Thus, determining methods to monitor population dynamics of E. pyriella is a high priority for orchard managers.
Euzophera pyriella has 3 generations per yr. Adults of the first generation emerge in late April, those of the second generation in early June and the third generation adults emerge in late July. Adults eclose mainly during the day, but mate at night. Mated females lay eggs singly or in small groups in wounds and crevices of trees. Larvae feed on phloem and xylem by boring tunnels filled with yellow or black feces, resulting in hollow tree limbs and trunks. The larva surrounded by a thin, gray cocoon overwinters in feeding tunnels, fractures of the trunk, and tree holes (Ma et al. 2013). Sometimes fragrant pear fruits also are heavily damaged with crop losses exceeding 90%.
Larvae live beneath the tree bark, which makes them both difficult to detect and difficult to control by insecticide sprays. An alternate way to control E. pyriella may be to reduce the population by trapping adults or disrupting mating behavior. The motivation for this study was to identify key components of the E. pyriella sex pheromone with the ultimate goal of using this pheromone to manage populations.
Materials and Methods
Insects
Euzophera pyriella larvae were collected in fragrant pear orchards in Korla (N 86.06° E 41.68°) and Aksu (N 80.29° E 41.15°) (Xinjiang Uygur Autonomous Region, northwestern China) in spring (Mar–Apr, 2013). In the laboratory, larvae were transferred to plastic containers (4.5-cm diam × 4.0-cm high), and reared until pupation in an artificial climate box at 22 °C, 75–80% RH and 14:10 h L:D. Pupae were separated by sex: female pupae have a short (65∼70µm long), smoothsided suture in the middle of the 8th abdominal segment and 2 sides of the suture were smooth, whereas male pupae have a longer (100∼110µm long) suture with a lump on its sides formed by the genital pore on the 9th abdominal segment. Emerging adults were collected daily and kept individually in plastic dishes (9.0-cm diam), and fed 10% sugar-water solution.
Pheromone Gland Extraction
Female moths have a large sex pheromone gland located at the intersegmental membrane between the 8th and 9th abdominal segment. Pheromone glands of 2–3 day-old virgin females were excised during the calling period (about 50 min into the scotophase, Fig. 1). Calling females were cooled to -20 °C for at least 3 min prior to gland excision. Pheromone glands were excised and placed in hexane (10 µl hexane per abdominal tip) and extracted at room temperature for 50–60 min, and then transferred to a clean glass vial (Agilent, USA). Hexane extracts were stored at -20°C until they were analyzed. Sex pheromones were extracted from virgin females, while males were used for electroantennogram (EAG) studies.
Chemical Analysis
Pheromone gland extracts were analyzed on an Agilent 7890A GC interfaced to an Agilent 5975C mass-selective detector. Samples were run on DB-5 capillary columns (30 m × 0.25 mm ID, 0.25 µm film thickness, Agilent Technologies, USA). The column oven was maintained at 50 °C for 2 min, and then the temperature was increased to 270 °C at 10 °C /min, at which point it was held for 10 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min and GC inlet temperature was 260 °C. Electron ionization mass spectra were recorded from m/z 40 to 330 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 comparison of their retention indices and mass spectra with those of known standards, and a series of n-alkanes (C10— C20) were used to determine Kováts indices (IK) of analytes (Kováts 1965).
Electrophysiological Analyses
EAG recordings were made with a Syntech EAG, consisting of a probe/micromanipulator (MP-15), a data acquisition interface box (IDAC-2) and a stimulus air controller (CS-55). An excised male antenna was mounted onto an antenna holder for the EAG probe (PRG-2) with an electrode gel (Sigma). EAG responses (mV) to different stimuli were measured consecutively from the antenna. The process was repeated 3 times with the same antenna, for each of 5 antenna. Stimuli were applied to a strip of filter paper (0.5 × 3.0 cm), inserted into a 15-cm long Pasteur pipette. The tip of the pipette was introduced into the main airflow tube (8-mm diam), in which a moistened air stream (100 mL/min) was flowing continuously toward the antenna. Stimuli were applied to the antenna by puffing the humidified air for 0.3 s from the Pasteur pipette.
The antenna was stimulated by different sex pheromone standards, applied in random order, with at least 40 s between stimulations. Several sex pheromone standards diluted to 0.1% per µL with hexane were tested. The EAG values obtained from each standard were averaged.
Field Tests
Field trapping tests were conducted in April, and from 3 Jul to 2 Sep, 2013 in Korla (N 41.68° E 86.06°) and Aksu (N 41.15° E 80.29°) with delta traps (25-cm long × 20-cm wide) and blank dispensers (small green rubber septa, 8-mm diam) (Pherobio Technology Co. Ltd., China). Pheromone compounds were dissolved in hexane, and the solutions were pipetted into the groove of the blank dispenser. The solutions of the lure were completely infiltrated and stored at 4 °C until used. Each lure was placed at the center of a Delta trap 1 cm above the sticky board, and the traps were hung 1.5–2.0 m above the ground at intervals of 20–30 m in the fragrant pear orchards. Five replicates were arranged randomly at intervals of 100–200 m. The lure was replaced after 10 days. The number of captured males was counted every 2 days.
Chemicals
Chemicals used in identification, electrophysiological analyses and field tests are provided by Sigma-Aldrich (America), Chemos GmbH (Germany), and Pherobio Technology Co. Ltd. (China).
Statistical Analyses
Kováts indices (IK) were calculated by the standard formula (Marques et al. 2000). Data Analyses for significant differences were conducted by one-way AN OVA (analysis of variance [ANOVA]), and multiple comparisons were conducted by Duncan's multiple range test. The level of significance in all tests was set at α = 0.05.
Results
GC-MS Analysis of Sex Pheromone Extracts
GC-MS analyses on crude sex pheromonegland extracts of E. pyriella females revealed the presence of 2 suspected pheromone components (peaks 1 and 2) found in other Lepidoptera (Fig. 2) . Peak 1 and 2, respectively, represent Z8-12:Ac and Z9E12–14:OH, with a compatibility >97%. Retention times of these 2 peaks (Table 1), and the mass spectra of their diagnostic fragment ions were consistent with known standards (Fig. 3) .
Mass chromatograms of M+ at m/z 226 characteristic fragment ions of acetate (ions at m/z 67) indicated an occurrence of Z8-12:Ac, RT 15.324 min and M+ at m/z 210 characteristic fragment ions of double-bond tetradecanol (ions at m/z 81, 95) indicated the occurrence of Z9E12-14:OH, RT 16.198 min in the extract.
EAG Responses of Male E. pyriella to Synthetic Sex Pheromone Components
Figure 4 shows the EAG responses of E. pyriella males to the 2 sex pheromone components identified by GC-MS (Z8–12:Ac and Z9E12–14:OH) and 3 other common compounds previously identified in Euzophera (Z9–14:Ac, Z914:OH, Z9E12–14:Ac). Z9E12–14:OH elicited the strongest response, followed by Z9–14:OH and Z8–12:Ac. Two other acetates, Z9–14:Ac and Z9E12-14:Ac, did not elicit significant EAG responses in E. pyriella males.
Field Attraction of E. pyriella Males by Synthetic Dispensers
Figure 5 shows that of the 8 sex attractants tested, only the dispensers having Z9E12–14:OH attracted the most E. pyriella males. The dispensers with a mixture of Z9E12–14:OH and Z8–12:Ac also attracted males, but the number was much smaller than that of males attracted to using Z9E12–14:OH separately.
Lures baited with 200µg Z9E12–14:OH attracted more E. pyriella males than did either lower or higher concentrations (Fig. 6). When Z8–12:Ac was added to Z9E12–14:OH, decreasing the ratio of Z9E12–14:OH, the number of E. pyriella males caught decreased in the test (Fig. 7).
Table 1.
Retention times and Kováts indices of suspected sex pheromone components.
Discussion
Sex pheromones of Lepidoptera have been identified from nearly 630 species (Ando 2012); of these, close to 200 are from moths in the superfamily Pyraloidea (El-sayed 2012); Lepidopteran sex pheromones usually are straight-chain compounds. Their structures are determined by the degree of unsaturation, carbon chain length, functional group, and geometry and position of the double bond (Shibasaki et al. 2013). Lepidopteran pheromones are divided into 2 groups according to their chemical structure (Yamakawa hydes, and their derivatives. Type II pheromones, which have been detected from species in the families Lymantriidae, Arctiidae, Noctuidae, and Geometridae, are comprised of longer chain C17 to C23 compounds.
Fig. 5.
Trap catches of Euzophera pyriella males baited with various sex attractants. Means followed by the same lower case letters are not significantly different (Duncan s test, P < 0.05).
Fig. 6.
Attraction of Euzophera pyriella males by traps baited with Z9E12-14:OH in different doses. Means followed by the same lower case letters are not significantly different (Duncans test, P < 0.05).
In our study, we detected 2 pheromones: Z8–12:Ac and Z9E12–14:OH, both Type I pheromones. Z9E12–14:OH, the main sex pheromone, is a prevalent pheromone component within other Euzophera species (Ando 2012), including E. hatangensis Caradja (Wen et al. 2009), E. pinguis Haworth (Ortiz 2002), E. punicaella Moore (Bestmann et al. 1993b) and E. semifuneralis Walker (Biddinger et al. 1993). It also has been detected as a part of the sex pheromones of noctuid moths (Rynchaglea scitula [Butler] and Spodoptera exigua [Hübner]) and the crambid moth (Anania verbascalis [Denis & Schiffermüller]).
Fig. 7.
Attraction of Euzophera pyriella males to traps baited with synthetic mixtures of the 2 pheromone components, Z9E12–14:OH and Z8–12:Ac, in different ratios. Means followed by the same lower case letters are not significantly different (Duncans test, P < 0.05).
In contrast, Z8–12:Ac was found in the sex pheromone-gland extracts by GC-MS, but alone it neither elicited antennal responses in EAG experiments nor attracted males in the field. Inhibitory effects of other compounds found in pheromone gland extracts of conspecific calling females have been reported for other moths (Gibb et al. 2007; Yang et al. 2009; Yang et al. 2013), but when Z8–12:Ac was added to Z9E12–14:OH to make a binary mixture, the trap catch was not significantly higher from the trap catch with Z9E12–14:OH alone. However, it remains unknown whether Z8-12:Ac is actually emitted by E. pyriella calling females or whether is simply a precursor compound to other acetates, and it may be active as a sex pheromone component over short distance. Behavioral function over short distance in chemical communication needs to be further studied (Hu & Du 2005).
In summary, we conclude that Z9E12–14:OH is a major sex pheromone component of E. pyriella calling females. When applied to rubber septum at a dose of 200 µg, Z9E12-14:OH is a potent lure that may be used as a convenient and reliable tool for monitoring, mass trapping, or possibly mating disruption in integrated pest management. Further chemical and electrophysiological analyses, including GC-EAD (Zhang et al. 2012), GC × GC/TOFMS (Kalinová et al. 2006), and GC/FT-IR (Leal et al. 2001; Visser 2002) with gland extracts and entrained volatile collections from E. pyriella females are needed to determine if other pheromone components are released by calling females. These could be used in subsequent behavioral and field tests to establish the optimum composition and dose for lures.
Acknowledgments
We heartily thank Fei Xiao and Shirong Zhong for help with collecting insects and with the field experiments in Xinjiang; Cuiwei Liu at Pherobio Technology Co. Ltd. (China) for help with providing the chemicals; Dr. Aaron Ellison (Harvard University) and Dr. Changlu Wang (Rutgers University) for English editing. This research was supported by the Special Fund for Agro-Scientific Research in the Public Interest, China (No. 201203036).
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