Synthesis and Field Evaluation of the Sex Pheromone Analogues to Soybean Pod Borer Leguminivora glycinivorella

In order to develop efficient lures for soybean pod borer Leguminivora glycinivorella (Matsumura) in China, (E,E)-8,10-dodecadienyl acetate (EE-8,10-12:Ac), the main component of the pheromone of L. glycinivorella, and 12 structurally-related compounds were synthesised in good overall yields, regiospecificities, and stereo-selectivities via coupling reactions catalysed by Li2CuCl4. The effect of different synthetic compounds, alone or in combination with EE-8,10-12:Ac, on numbers of captured L. glycinivorella males was evaluated. EE-8,10-12:Ac, (E)-10-dodecenyl acetate(E-10-12:Ac), (E)-8-dodecenol (E-8-12:OH), tetradecyl acetate (14:Ac), and (Z)-9-tetradecenyl acetate (Z-9-14:Ac) alone displayed different attractiveness to L. glycinivorella males. 14:Ac, E-8-12:OH, E-10-12:Ac, (E,E)-8,10-dodecadienal (EE-8,10-12:Ald), (E)-8-dodecenal (E-8-12:Ald), (E)-10-dodecenal (E-10-12:Ald) and Z-9-14:Ac all showed a synergistic effect to EE-8,10-12:Ac at certain dosages. The binary mixtures of EE-8,10-12:Ac and E-10-12:Ald, Z-9-14:Ac,14:Ac, E-8-12:Ald, EE-8,10-12:Ald, E-8-12:OH, or E-10-12:Ac in suitable ratios give 17.00-, 10.98-, 10.67-, 6.73-, 5.54-, 4.30- and 4.50-fold increases in trap catch, respectively, over the standard pheromone lure, and as novel pheromone blends, demonstrated potential use in pheromone traps to monitor or control L. glycinivorella populations in China.


Introduction
Leguminivora glycinivorella is one of the most destructive soybean pests in North China. Larvae feed mainly on the pod hulls, especially of young beans, severely reducing crop yield and quality. (E,E)-8,10-Dodecadienyl acetate (EE-8,10-12:Ac) is the main sex pheromone of L. glycinivorella [1]. In 2008 Wu reported the synthesis of EE-8, 10-12:Ac by the use of Suzuki-Miyaura cross-coupling reaction between potassium (E)-1-propenyl trifluoroborate and (E)-9-iodo-1-(2-tetrahydropyranyloxy)-8-nonene in the presence of PdCl 2 (dppf), CH 2 Cl 2 and Cs 2 CO 3 [2]. However, this method suffers from high cost and difficulties associated with industrial scale-up. Alternatively, Wang et al. have implemented a field experiment on the attraction of L. glycinivorella using a mixture of EE-8,10-12:Ac and (E)-10-dodecenyl acetate (E10-12:Ac) at Gongzhuling and Dehui, Jilin Province, China [3]. A strong attraction to male moths by a mixture of EE-8,10-12:Ac and E10-12:Ac in a ratio of 2:5 was observed. Nevertheless, this reported pheromone lure does not work well in Heilongjiang Province, China. This discrepancy may attribute to the different habitats of L. glycinivorella in Heilongjiang and Jilin, the different climates (including temperature and humidity) and the planting structures.
Monounsaturated C12 and C14 alcohols, acetates and aldehydes are commonly found in lepidopteran pheromone glands [4], often as minor components, and some have been shown to synergize the attraction to the main pheromone component(s). For example, EE-8, 10-12:Ald added at 0.002 mg to 2 mg EE-8,10-12:OH (the main component of the pheromone of the codling moth Cydia pomonella) increases the number of codling moth males caught in traps [5].
Our goal is to develop efficient lures for monitoring and controlling soybean pod borer population in China. Thus, EE-8,10-12:Ac and its chain-elongated, monounsaturated and aldehyde analogues were efficiently synthesised from the acetate and Grignard reagent via coupling reactions catalyzed by dilithium tetrachlorocuprate (Li 2 CuCl 4 ), followed by evaluation of the effect of twelve different analogues, alone or in combination with EE-8,10-12:Ac, on numbers of L. glycinivorella males captured in traps.
The effect on catch when other synthetic compounds were combined with EE-8,10-12:Ac varied with the compound and dose. In 2006, it has been reported that E-8-12:Ac strongly inhibited the attractiveness of EE-8,10-12:Ac to L. glycinivorella in Japan [1]. When tested as a single compound, E-8-12:Ac was not attractive to L. glycinivorella males (Table 1). It significantly decreased the mean catch when added to traps baited with EE-8,10-12:Ac at dosage 0.05, 0.1, 0.01 mg/septum and as the mixture of natural type I (EE-8,10-12:Ac (30 ng) and E-8-12:Ac (34 ng)) [8] (Figure 1). Our results show that E-8-12:Ac is one of the inhibitors of the sex pheromone EE-8,10-12:Ac to L. glycinivorella in Heilongjiang Province, China. Thus, as pheromone antagonist, antipheromone and inhibitor, E-8-12:Ac showed broad prospects, especially when coupled with mating disruption of L. glycinivorella.  The combination of EE-8,10-12:Ac and E-10-12:Ac in a suitable ratio enhanced the attractiveness to L. glycinivorella males in field applications compared with EE-8,10-12:Ac administered alone. Wang showed that lures baited with a mixture of EE-8,10-12:Ac and E-10-12:Ac in a ratio of 2:5 (natural type II) were extremely attractive to L. glycinivorella at Gongzhuling and Dehui, Jilin Province, China [3]. Contrarily, the present work, which was carried out in Harbin, Heilongjiang Province, China, observed a decreased attractiveness with a number of captured male moths of only 4.0 trap −1 day −1 when using the mixture of natural type II compared with singly administered EE-8,10-12:Ac (10.21 trap −1 day −1 ). However, a significantly enhanced attractiveness (55 trap −1 day −1 ) of male moth was observed for a molecular ratio of 10:1 for a binary blend of EE-8,10-12:Ac and E-10-12:Ac ( Figure 2). This result suggests the possibility that the sex pheromone of this species varies geographically. Nevertheless, a 10:1 synthetic mixture of EE-8,10-12:Ac and E-10-12:Ac could be used to efficiently monitor and/or control the pod borer field population in China.
Compound 12:Ac has been detected in a gland extract of L. glycinivorella pheromone, and the GC-EAD showed that it was antennally active [1]. When tested as a single compound, 12:Ac alone was not attractive to L. glycinivorella males (Table 1), and significantly decreased mean catch when added to traps baited with EE-8,10-12:Ac at doses of 0.01 and 0.5 mg per septum ( Figure 3). Interestingly, however, doses of 0.5 and 0.01 mg of 12:Ac were inhibitory, but an intermediate dose of 0.1 mg was not. This can be explained as follows: generally, only the concentration of pheromone in the field which is similar to the concentration of pheromone in natural female moth can be received as a signal and effectively attract the male moth. Additionally, field catches can be affected by various factors, such as humidity, temperature, and the wind force, etc.  Up to now, there were no reports on E-8-12:OH being attractive to L. glycinivorella. In the present study, E-8-12:OH alone displayed a significant attractiveness to the borer males (Table 1), and also showed a synergistic effect with EE-8,10-12:Ac (Figure 4). Catches followed by different letters within one column are significantly different at p = 5% by Tukey's HSD [capture data were transformed to log(x + 1) before performing analysis].
Up to now, there has been no report on EE-8,10-12:Ald, E-8-12:Ald and E-10-12:Ald being attractants of L. glycinivorella. In the present study, weak attractiveness of these three compounds has been observed when administrated individually (Table 1). Surprisingly, the addition of a small amount of these three compounds significantly enhanced the activity of EE-8,10-12:Ac (Figure 4), especially the compound E-10-12:Ald at 0.1 mg dosage. These significant synergistic effects of EE-8,10-12:Ald, E-8-12:Ald and E-10-12:Ald to EE-8,10-12:Ac indicated their potential usefulness in the biological control of L. glycinivorella males in China. This work also shed light on the application of this kind of compounds as sex attractants of other Lepidopteran insects. No other moth species were captured in significant numbers during the field tests.

General
NMR spectra were recorded at 500 MHz and 125 MHz for 1 H and 13 C, respectively, on a Brüker, ADVANCE III instrument (Brüker BioSpin AG, Fällanden, Switzerland) in CDCl 3 solution with Me 4 Si as internal standard. The GC/MS analysis of the synthetic compounds was performed on a Thermo Trace gas chromatograph (Thermo Finnigan, Miami, FL, USA) coupled to Polaris Q mass spectrometer (Thermo Finnigan, Miami, FL, USA) under the following analytical conditions: HP-5 capillary column (15 m × 0. 53 mm × 0. 25 μm film thickness); helium (0.5 mL/min); programmed temperature 50-220 °C (7 °C/min); injector temperature (250 °C) and interface (230 °C); ionization energy, 70 eV; scan range, 30-500 amu; scan time, 1 s. All solvents were purified by standard procedures. GF 254 TLC plates are used for the monitoring of reaction, and silica gel of 200-300 mesh is used for the column chromatography (Qingdao Haiyang Chemical Co., Qingdao, China). The acetate and aldehyde derivatives were obtained by the acetylation and pyridinium chlorochromate (PCC) oxidation of the corresponding alcohols, respectively [13,14].

Field Trapping Experiments
Trapping experiments were carried out during July and August 2010 and 2011 in two soybean fields separated 30 km in Harbin, Heilongjiang Province, China during the flight period of L. glycinivorella males. Mean temperature, relative humidity, and wind speed during the trapping period were 25 °C, 50%, and 6.0 ms −1 in 2010, and 23 °C, 60%, and 6.5 ms −1 in 2011. Synthetic chemicals were dissolved in ca. 100 μL of dichloromethane and impregnated into rubber septa (0.3 g, 10 mm outside diameter × 16 mm height; Xi'an Dizhai Co., Shaanxi, China) at various doses (see below). The solvent was evaporated at room temperature overnight. A water pot (green, 24 cm diameter × 10 cm depth, Weilu plastic Co., Qingdao, Shandong, China) 3/4 filled with water and 0.5% detergent was employed as the trap. Traps in one replicate were hung from a wire, suspended 1-2 cm above the surface of the water in the trap which was hung on a tripod 1 m above the soil surface at ca. 13 m from each other in a line. Six replicates were used for each treatment. The treatments were completely randomized design and the positions of the traps were rotated to eliminate the position effect. Insects captured on the traps were removed every morning and identified, sexed, and catalogued. The water in the pot was replenished every week. Two types of controls were included: rubber septa impregnated with 100 µL dichloromethane as a solvent control, and untreated rubber septa. In experiment 1, the effect of dose (0.01, 0.1, and 0.5 mg per lure) on capture of L. glycinivorella males in traps baited with thirteen different synthetic compounds was tested from 30 July to 6 August 2010. In experiment 2, we tested nine different synthetic compounds (12:Ac, 14:Ac, E-8-12:OH, E-8-12:Ac, E-10-12:Ac, EE-8,10-12:Ald, E-8-12:Ald, E-10-12:Ald and Z-9-14:Ac) at three doses (0.01, 0.1, and 0.5 mg) added to traps baited with EE-8,10-12:Ac (0.1 mg) for their effect on numbers of L. glycinivorella males captured in traps. This experiment was conducted in the period from 7 August to 17 August 2011.