LI Zhao-qun ,YUAN Ting-ting ,CUI Shao-wei ,ZHAO Ying-jie ,SHAO Yuan-hai ,SHANG Jian-nong ,LUO Zong-xiu,CAI Xiao-ming,BIAN Lei,CHEN Zong-mao

1 Key Laboratory of Biology,Genetics and Breeding of Special Economic Animals and Plants of Ministry of Agriculture and Rural Affairs,Tea Research Institute,Chinese Academy of Agricultural Science,Hangzhou 310008,P.R.China

2 Tea Variety Research Institute of Wuxi,Wuxi 214122,P.R.China

3 Agricultural Technology Extension Service Center of Xihu District of Hangzhou,Hangzhou 310063,P.R.China

Abstract The tea tussock moth (Euproctis pseudoconspersa) is one of the most destructive chewing pests in tea plantations and causes a serious allergic reaction on the skin of tea plantation workers.The sex pheromone components of its Japanese population were first identified as 10,14-dimethylpentadecyl isobutyrate (10Me14Me-15:iBu) and 14-methylpentadecyl isobutyrate (14Me-15:iBu),with a stereogenic center.Only 10Me14Me-15:iBu has been identified in the Chinese E.pseudoconspersa population.However,field tests have shown that 10Me14Me-15:iBu cannot meet the demand of effective pest control in China.To develop a high-efficiency E.pseudoconspersa sex pheromone formula,electroantennogram (EAG) recordings of (S)-and (R)-enantiomers of 10Me14Me-15:iBu and 14Me-15:iBu were obtained in the present study.The results demonstrated that the EAG responses of male antennae to (R)-enantiomers were significantly higher than responses to the (S)-enantiomers,and 14Me-15:iBu also elicited EAG activity.Field tests showed that the catch numbers of male moths by (R)-enantiomers were significantly higher (P＜0.05) than those of (S)-enantiomers.Addition of 14Me-15:iBu significantly increased the catch numbers of both the (S)-and (R)-enantiomers.The efficient pheromone formula containing 0.75 mg (R)-10Me14Me-15:iBu and 0.1 mg 14Me-15:iBu showed significantly higher attractiveness than commercial pheromone products.Our study demonstrated that (R)-10Me14Me-15:iBu was the major sex pheromone component of E.pseudoconspersa,and 14Me-15:iBu might be the minor sex pheromone component.Furthermore,a high-efficiency sex pheromone formula for E.pseudoconspersa control was defined in this study.

Keywords: Euproctis pseudoconspersa,sex pheromone,chirality,electroantennogram

1.lntroduction

Tea is one of the most popular beverages worldwide,and China is the largest tea-planting and -producing country(Chen and Lin 2015).The tea tussock moth (Euproctis pseudoconspersa) is one of the most destructive chewing pests in the tea plantations of China.Its larvae damage tender and mature leaves of tea plants,causing severe yield losses.Moreover,it causes severe allergic reactions on human skin,making it difficult for workers to pick tea leaves and spray pesticides (Wanget al.2021).In recent years,the occurrence ofE.pseudoconspersahas expanded considerably.

Female moths secrete volatile compounds,the sex pheromones,which are utilized for interspecific communication (Witzgallet al.2010).Synthetic sex pheromones have been widely used for monitoring,mating disruption,and mass trapping of pests (Liebhold and Tobin 2008;Knightet al.2012;Luoet al.2020).Based on their chemical structure,pheromones are classified into four groups: Type I,unsaturated straight-chain hydrocarbons with an ester linkage,alcohols,or aldehydes as terminal functional groups (Andoet al.2004);Type II,unsaturated straight-chain hydrocarbons and their epoxide derivatives;Type III,similar to Type I and Type II with one or more methyl branches (Lofstedtet al.2016);and Type 0,a proposed group containing short-chain methylcarbinols and methylketones (Jurenka 2021).One of the most important among them is methyl-branched sex pheromones because they include stereogenic centers (Mori 2007;Ando and Yamakawa 2015).

Wakamuraet al.(1994) first identified the sex pheromones ofE.pseudoconspersa(Japanese biotype)as 10,14-dimethylpentadecyl isobutyrate (10Me14Me-15:iBu) and 14-methylpentadecyl isobutyrate (14Me-15:iBu).According to their chemical structures,these components belong to the Type III pheromone group.The major component (10Me14Me-15:iBu) attracts wild male moths,and the attractant activity increases when it is blended with the minor component (14Me-15:iBu).10Me14Me-15:iBu is a chiral methyl-branched pheromone.The EAG tests have showed that the (R)-enantiomer induced a larger response than the (S)-enantiomer.However,field studies of the Japanese population have reported that the catch number difference between the (R)-enantiomer,(S)-enantiomer,and the racemic mixture was insignificant (Ichikawaet al.1995;Wakamuraet al.1996).

Only a single electroantennogram (EAG)-active compound,10Me14Me-15:iBu,has been identified from the sex pheromone gland of the Chinese biotype ofE.pseudoconspersa(Zhaoet al.1996).Researchers believe that 10Me14Me-15:iBu is responsible for most of the biological activity associated with the sex-attractant pheromone of this species.The racemate of 10Me14Me-15:iBu has been used for commercial lure production.When used for mass trapping ofE.pseudoconspersa,the synthetic racemate of 10Me14Me-15:iBu reduces larval and egg densities by only 27.87–50.85% and 38.89–51.11%,respectively (Wanget al.2005).Therefore,the attractiveness of syntheticE.pseudoconspersasex pheromones requires further improvement.In this study,we evaluated the EAG-active component and field attractiveness of the two enantiomeric forms of 10Me14Me-15:iBu and 14Me-15:iBu to develop a highefficiencyE.pseudoconspersasex pheromone formula.

2.Materials and methods

2.1.lnsects

Larvae ofE.pseudoconspersawere collected from a tea plantation of the Tea Variety Research Institute of Wuxi,Jiangsu Province,China (120.27°E,31.46°N).The larvae were reared on fresh tea shoots in climatecontrolled rooms with a 14-h light:10-h darkness photoperiod at (25±1)°C and (70±5)% relative humidity.After pupation,male and female pupae were separated into different cages (50 cm×50 cm×50 cm)and kept in darkness until emergence.Adult moths were then provided with a 10% honey solution.Virgin male moths (2 days old) were used for EAG recordings.

2.2.Chemicals

The sex pheromone components (S)-10Me14Me-15:iBu,(R)-10Me14Me-15:iBu,and 14Me-15:iBu were synthesized by Zequan Bio-technology Co.,Ltd.(Hangzhou,China).(S)-10Me14Me-15:iBu and (R)-10Me14Me-15:iBu were synthesized from (S)-and (R)-citronellols,respectively,according to previously reported methods (Ichikawaet al.1995).The NMR data of the components were: (1) (S)-10Me14Me-15:iBu1H NMR(400 MHz,Chloroform-d) δ 4.10 (t,J=6.7 Hz,2H),2.58 (p,J=7.0 Hz,1H),1.70–1.62 (m,2H),1.56 (m,1H),1.43–1.24(m,17H),1.21 (d,J=7.0 Hz,6H),1.19–1.05 (m,4H),0.91 (d,J=6.6 Hz,6H),0.88 (d,J=6.5 Hz,3H);(2) (R)-10Me14Me-15:iBu:1H NMR (400 MHz,Chloroform-d) δ 4.10 (t,J=6.7 Hz,2H),2.58 (p,J=7.0 Hz,1H),1.72–1.62(m,2H),1.56 (m,1H),1.43–1.25 (m,17H),1.21 (d,J=7.0 Hz,6H),1.20–1.05 (m,4H),0.92 (d,J=6.6 Hz,6H),0.89(d,J=6.5 Hz,3H);(3) 14Me-15:iBu:1H NMR (400 MHz,Chloroform-d) δ 4.09 (t,J=6.7 Hz,2H),2.57 (p,J=7.0 Hz,1H),1.71–1.61 (m,2H),1.54 (m,1H),1.40–1.27 (m,20H),1.20 (d,J=7.0 Hz,6H),1.19–1.15 (m,2H),0.90 (d,J=6.6 Hz,6H).Gas chromatography was used to ensure that the purity of each chemical was greater than 99%.

2.3.EAG recordings

Solutions of (R)-10Me14Me-15:iBu,(S)-10Me14Me-15:iBu,and 14Me-15:iBu were prepared in hexane at different concentrations (0.001,0.01,0.1,1.0,and 10 μg μL–1).Both the basal and distal segments of the antenna of 2-day-old virgin males were removed.The antenna was then connected to Ag-AgCl electrodes filled with saline solution.For each tested chemical compound,a filter paper strip (0.5 cm×5.0 cm) containing 10 μL of test solution was placed into a 13.5-cm long Pasteur pipette.The tip end of the Pasteur pipette was connected to a hole in a glass tube,which directed a charcoal-filtered and humidified airstream (800 mL min–1) by an air flow controller (CS-55;Syntech Inc.,Netherlands).The base of the Pasteur pipette was linked to a pulse airflow pipe.Each test compound was expelled into the airstream when the baseline of the EAG signal remained stable.Nonanal was selected as the reference compound.The recording trial was performed in the following sequence: n-hexane,reference compound,test compound (compounds were tested from low to high dosage),reference compound,and n-hexane.Each dosage was tested for at least five antennae,with a 30-s interval to allow the antenna to recover.The electrical signal was amplified and converted to a digital signal by IDAC (Syntech Inc.,Netherlands).The digital signals were recorded with EAGPro Software(version 2.0;Syntech Inc.,Netherlands).

2.4.Field trapping tests

The field trapping tests were performed in the tea plantations of Wuxi City (Jiangsu,China;120.27°E,31.46°N) and Guilin (Guangxi,China;110.17°E,25.32°N)between June and September in 2019 and 2020.The synthetic sex pheromone compounds,(R)-10Me14Me-15:iBu,(S)-10Me14Me-15:iBu,and 14Me-15:iBu,were dissolved in distilled hexane (10 μg μL–1).The sex pheromone solutions were then added to white rubber septa (8 mm outside diameter;Sigma Aldrich Inc.,St.Louis,MO) that were used as delivery materials.Control rubber septa were loaded with 100 μL of n-hexane.After the volatilization of n-hexane,each group of septa was packed and stored at–20°C until their next use.The commercialE.pseudoconspersasex pheromone lures used in this study were purchased from Ningbo Newcon Biotechnology Inc.(Ningbo,China),Pherobio Technology Co.,Ltd.(Beijing,China),Hangzhou Zhongcha Technical Service Co.,Ltd.(Hangzhou,China),and Zhangzhou Enjoy Agricultural Technology Co.,Ltd.(Zhangzhou,China).A combination of septa and commercial sex pheromone lures were placed in wing traps and hung separately 20 cm above tea plants at intervals of 12 m.Four replicates of each treatment were tested,and the captured males were counted every week.

2.5.Statistical analyses

SPSS (v.26.0 2019,IBM Corp.,Armonk,NY) was used for the statistical analyses.The data was analyzed using a one-way analysis of variance and compared using Tukey’s honestly significant difference (HSD) test withP-values adjusted for multiple comparisons.To homogenize the variance,means were transformed using the log(x+1)transformation.

3.Results

3.1.EAG responses of males

The sex pheromone components,(R)-10Me14Me-15:iBu and (S)-10Me14Me-15:iBu,were individually tested to verify whether maleE.pseudoconspersashowed different EAG responses to both the (S)-and (R)-enantiomers.The results demonstrated that the EAG responses of male antennae to (S)-10Me14Me-15:iBu,(R)-10Me14Me-15:iBu,and 14Me-15:iBu were sigmoidal dose-response curves (Fig.1-A and B).However,EAG responses of (R)-enantiomers elicited significantly higher EAG activity in maleE.pseudoconspersathan those of (S)-enantiomers at 1 and 10 μg doses (Fig.1-A).

Fig.1 Electroantennographic (EAG) responses of male Euproctis pseudoconspersa antennae to synthetic pheromone components.A,EAG responses to the (S)-and (R)-enantiomers of 10Me14Me-15:iBu.B,EAG responses to 14Me-15:iBu.Data are mean±SE(n=6).Asterisks above the lines indicate significant differences assessed via ANOVA followed by by Student’s t-test (P＜0.05).

3.2.Field trapping tests

To study the attractiveness of (S)-and (R)-enantiomers ofE.pseudoconspersamajor sex pheromone components,(R)-10Me14Me-15:iBu and (S)-10Me14Me-15:iBu were tested individually and in combination with 14Me-15:iBu in the tea garden of Wuxi (Fig.2).The result showed thatE.pseudoconspersawere captured with both the (S)-and (R)-enantiomers.However,the number of males captured with the (R)-enantiomer was significantly higher than those captured with the (S)-enantiomer.Moreover,almost no males were captured with only 14Me-15:iBu during the test period.Blending with 14Me-15:iBu significantly increased the number of males captured with both (S)-and (R)-enantiomers.Even after blending with 14Me-15:iBu,the catch numbers of (R)-enantiomers were significantly higher than those of (S)-enantiomers.

Fig.2 Catch numbers of Euproctis pseudoconspersa males with different synthetic pheromone components.Data are mean±SE(n=4).Letters above the bars indicate significant differences assessed via ANOVA followed by Tukey’s HSD test (P＜0.05).

The dose effect of (R)-10Me14Me-15:iBu (blended with 14Me-15:iBu) on the sex pheromones ofE.pseudoconspersawas then examined in the tea gardens of both Guilin and Wuxi (Fig.3).The results showed that male catch numbers significantly increased when the dose of (R)-10Me14Me-15:iBu was raised from 0 to 0.75 mg per septum.However,the catch numbers declined at the 1 mg dose level.Furthermore,the male catch number with (R)-10Me14Me-15:iBu was compared with the racemic mixture of 10Me14Me-15:iBu blended with 0.1 mg per septum of 14Me-15:iBu (Fig.4).The comparative analysis showed that catch numbers with(R)-10Me14Me-15:iBu were significantly greater than those with the racemic mixture.

Fig.3 Catch numbers of Euproctis pseudoconspersa males with different dosages of (R)-10Me14Me-15:iBu in the tea gardens in Wuxi and Guilin,China.Data are mean±SE (n=4).Letters above the bars indicate significant differences assessed via ANOVA followed by Tukey’s HSD test (P＜0.05).

Fig.4 Catch numbers of Euproctis pseudoconspersa males with(R)-10Me14Me-15:iBu and a racemic mixture of 10Me14Me-15:iBu in the tea gardens in Wuxi and Guilin,China.Data are mean±SE (n=4).Letters above the bars indicate significant differences assessed via ANOVA followed by Tukey’s HSD test (P＜0.05).

The sex pheromone formula combining different doses of 14Me-15:iBu (0,0.1,0.5,and 1.0 mg per septum)and 1.0 mg per septum of (R)-10Me14Me-15:iBu were evaluated for their attractiveness toE.pseudoconspersamale moths in Wuxi (Fig.5).The trap test catch numbers demonstrated that 0.1 mg per septum of 14Me-15:iBu blended with (R)-10Me14Me-15:iBu showed the highest attractiveness,and that the male catch number declined at higher doses of 14Me-15:iBu.

Fig.5 Catch numbers of Euproctis pseudoconspersa males with different dosages of 14Me-15:iBu.Data are mean±SE(n=4).Letters above the bars indicate significant differences assessed via ANOVA followed by Tukey’s HSD test (P＜0.05).

According to the above results,an efficient sex pheromone formula should contain 0.75 mg per septum of (R)-10Me14Me-15:iBu and 0.1 mg per septum of 14Me-15:iBu.To evaluate its attractiveness,the proposed formula was compared with the commercialE.pseudoconspersasex pheromone lures from four companies in the tea gardens of Wuxi and Guilin(Fig.6).The field trapping test showed that theE.pseudoconspersamale catch numbers of the proposed sex pheromone formula were significantly(more than 2 times) higher than those of the 4 commercial sex pheromone lures.The catch numbers among the 4 commercial sex pheromone lures were similar.

Fig.6 Comparison of Euproctis pseudoconspersa male catch numbers with the efficiency pheromone formula and commercial pheromone products in the tea gardens in Wuxi and Guilin,China.Newcon,Newcon commercial pheromone products from Ningbo Newcon Biotechnology Inc.;Yingge’er,Yingge’er commercial pheromone products from Zhangzhou Yingge’er Agricultural Technology Co.,Ltd.;Zhongcha,Zhongcha commercial pheromone products from Hangzhou Zhongcha Technical Service Co.,Ltd.;Pherobio,Pherobio commercial pheromone products from Pherobio Technology Co.,Ltd.;New,efficiency pheromone formula developed by the present study.Data are mean±SE (n=4).Letters above the bars indicate significant differences assessed via ANOVA followed by Tukey’s HSD test (P＜0.05).

4.Discussion

Synthetic sex pheromones have been successfully used in pest control as an alternative to insecticides(Witzgallet al.2010;Cui and Zhu 2016).However,mass trapping experiments have shown that the synthetic sex pheromones ofE.pseudoconspersacannot meet the demands of effective pest control.To improve the attractiveness of syntheticE.pseudoconspersasex pheromones,EAG and field trapping tests were performed to verify the following: (1) whether different chiral isomers have different activities;(2) how the chiral isomers work;and (3) the function of the minor sex pheromone component in field trapping.

The major sex pheromonecomponent ofE.pseudocon-spersa,10Me14Me-15:iBu,is a chiral compound (Ichikawaet al.1995).Enantiomers can influence the activity of the chiral pheromone if: (1) both enantiomers are active;(2) one enantiomer is active and the other is inactive;(3) one enantiomer is active but the other is inhibitory;and (4) enantiomers are active in combination but individually inactive.Our study demonstrated that both the (S)-and (R)-enantiomers of 10Me14Me-15:iBu showed EAG responses and field attractiveness,and the male catch number of the (R)-enantiomers was significantly higher than those of the(S)-enantiomers and racemic mixture.Therefore,the results indicated that (R)-10Me14Me-15:iBu is the major sex pheromone component ofE.pseudoconspersa.In contrast,previous studies on the Japanese biotype ofE.pseudoconspersareported that the catch numbers of the (R)-enantiomers and the racemic mixture were similar(Wakamuraet al.1996).This difference might be related to the pheromone polymorphism between geographical populations.

As a minor sex pheromone component,14Me-15:iBu could increase the male catch numbers of the Japanese biotype ofE.pseudoconspersawhen blended with 10Me14Me-15:iBu (Wakamuraet al.1994).However,it has not been identified from the Chinese biotype of this species (Zhaoet al.1996).Our study showed that 14Me-15:iBu elicited EAG activity of male antennae with a typical sigmoidal dose-response curve.Furthermore,field tests demonstrated that this compound significantly increased the catch numbers with (R)-10Me14Me-15:iBu and (S)-10Me14Me-15:iBu.We speculated that 14Me-15:iBu might also be the minor sex pheromone component ofE.pseudoconspersain China.It may likely not have been identified because of its low abundance in the sex pheromone gland.

The ratio and dose of components directly affect the attractiveness of synthetic sex pheromones (Maet al.2016;Yanet al.2019).Our field tests showed that (R)-10Me14Me-15:iBu and 14Me-15:iBu have the highest catch numbers at doses of 0.75 mg per septum and 0.1 mg per septum,respectively.Our speculation that an efficient formula containing 0.75 mg (R)-10Me14Me-15:iBu and 0.1 mg 14Me-15:iBu has a higher attractiveness was successfully proved by the field tests.The efficient sex pheromone formula captured more than twice the number of male moths captured by commercial sex pheromone products.This formula will improve the control efficiency of sex pheromones inE.pseudoconspersa.Further research on the application of the efficient formula in mass trapping and mating disruption is required to achieve pheromonebasedE.pseudoconspersacontrol.

5.Conclusion

Only 10Me14Me-15:iBu has been identified from the Chinese biotype ofE.pseudoconspersa.Accordingly,the racemate of 10Me14Me-15:iBu has been used for commercial lure production.However,it cannot meet the demands of effective pest control.The present study focused on EAG activities and field attractiveness of chiral enantiomers of 10Me14Me-15:iBu and the minor sex pheromone component of the Japanese population,14Me-15:iBu.It demonstrated that (R)-10Me14Me-15:iBu is the major sex pheromone component ofE.pseudoconspersa,and 14Me-15:iBu can elicit the EAG activity of male antennae and significantly increase male moth catch numbers.Therefore,14Me-15:iBu might be the minor sex pheromone component.Furthermore,an efficient sex pheromone formula containing 0.75 mg (R)-10Me14Me-15:iBu and 0.1 mg 14Me-15:iBu has significantly higher attractiveness than commercial pheromone products.These findings will improve the control efficiency of sex pheromones inE.pseudoconspersa.

Acknowledgements

This study was funded by the National Key R&D Program of China (2021YFD1601100),the Key Research and Development Program of Zhejiang Province,China(2019C02033),the National Natural Science Foundation of China (31701795),and the earmarked fund for the China Agriculture Research System (CARS-19).

Declaration of competing interest

The authors declare that they have no conflict of interest.