WANG Hong-min,BAl Peng-hua ,ZHANG JingZHANG Xue-minHUl QinZHENG Hai-xiaZHANG Xian-hong

1 College of Agricultural Economics and Management,Shanxi Agricultural University,Taigu 030801,P.R.China

2 College of Plant Protection,Shanxi Agricultural University,Taigu 030801,P.R.China

3 Institute of Plant Protection,Tianjin Academy of Agricultural Sciences,Tianjin 300384,P.R.China

Abstract Host-plant volatiles play an important role as cues for herbivores in search of resources,mates and oviposition sites in complex environments. Plant volatile-based attractants can be developed for pest monitoring and control. Previously,we indicated that mated female adults of Callosobruchus chinensis showed choice preference behavior toward 2-hexenal and benzaldehyde. Our objective here was to investigate the synergistic effect of host-derived attractive volatiles in attracting C.chinensis under laboratory and field conditions in Shanxi Province,China. We hypothesized that the ratio and concentration of volatiles derived from Vigna radiata play critical roles for C.chinensis in locating this host. Therefore,we collected and identified the volatiles of mungbean by using headspace collection and GC-MS. The effectiveness of different ratios and concentrations of two compounds (2-hexenal and benzaldehyde) that elicit C.chinensis searching behavior were examined in Y-tube olfactometer assays. The combination of 300 μg μL-1 2-hexenal and 180 μg μL-1 benzaldehyde loadings exhibited a synergistic effect on attracting C.chinensis (82.35%). Compared to control traps,the adults were significantly attracted to traps baited with blends,and more attraction to females than males was found for blend traps in the field experiments.Our results suggest that blends of this specific concentration and ratio of benzaldehyde and 2-hexenal can be used in traps as attractants for C.chinensis monitoring and control in the field.

Keywords:Callosobruchus chinensis,plant volatiles,synergistic effect,olfactometer,attractant

1.lntroduction

Mungbean (VignaradiataL.) is a traditional agricultural crop in China as it provides an affordable source of dietary proteins (Khinchiet al.2017). Unfortunately,pulse beetle(Callosobruchuschinensis) outbreaks in stored products cause enormous losses. This beetle is one of the most serious pests of stored beans,which has been widely distributed in many countries due to the commercial export of beans (Yanagiet al.2013; Wanget al.2018).Callosobruchuschinensisadults mate and oviposit on live mung beans in the field and are then carried over to beans placed in storage. The newly hatched larvae bore into leguminous seeds upon hatching and feed on the seed,which causes nutritive and germination losses (Chakrabortyet al.2015; Khinchiet al.2017).

Fumigants have been extensively used to control the infestation by pulse beetles in storage,but frequent applications of fumigants have posed serious problems,such as residues in food,resistance by the insects and environmental pollution (Malaikozhundan and Vinodhini 2017; Mahmud 2018). In recent years,plant oils and entomopathogenic bacteria have been used to control the larvae of stored grain pests (Khinchiet al.2017;Malaikozhundan and Vinodhini 2017),but management options for adult beetles in the field are still limited. Hence,there is an urgent need to find environmentally friendly measures for controllingC.chinensisadults in the field and protecting the stored products from infestations ofC.chinensislarvae.

An effective semiochemical lure for occurrence(presence/absence) monitoring is important in integrated pest management (IPM) (Kogan 1998). In the absence of a sex or aggregation pheromone,the development of an effective host-plant volatile lure for monitoringC.chinensisis in high demand. Host-plant volatiles play an important role in searching for resources,mates and oviposition sites in complex environments (Turlings and Erb 2018; Brancoet al.2019; Gallingeret al.2019; Guo and Wang 2019; Milonaset al.2019). Consequently,the identification of active compounds from host plants is crucial for understanding host selection behavior and olfactory function in insects.

Volatiles emitted by plants are usually diverse and complex blends (Schiestl 2010; McCormicket al.2012;Takemoto and Takabayashi 2015). The majority of insects use a mixture of volatile compounds,rather than individual components,for host recognition (Bruceet al.2005;Bruce and Pickett 2011; Chaet al.2011). For example,Karmakaret al.(2020) foundAulacophorafoveicollisLucas females responded positively to volatile blends ofSolena amplexicaulisfruits due to the synergistic action of nonanal and (E,Z)-2,6-nonadienals.Robertset al.(2019) reported similar results for vine weevil adults attracted to a mixture of seven plant volatiles,even though there were either no responses or negative responses recorded for each of these compounds when presented individually. Therefore,studying the behavioral role of blend components which occur together in host plants would be of great importance.

Recently,some studies have argued that the ratio and concentration of attractive volatiles are vital for host plant location and/or selection (Visser 1986; Bruceet al.2005;Zhanget al.2015; Robertset al.2019; Oseiowusuet al.2020). Websteret al.(2010) and Robertset al.(2019)reported that the concentrations of individual volatile organic compounds (VOCs) can influence the behavioral responses of vine weevil adults and black bean aphids under laboratory conditions. However,few studies have investigated the behavioral actions of volatile blends at different concentrations. Our previous work has shown that the mated female adults ofC.chinensisshowed choice preferences to 2-hexenal and benzaldehyde,and the rates of attraction were 68.89 and 64.34%,respectively,at a concentration of 100 μg μL-1(Wanget al.2017). However,the exact ratios or concentrations of the two compounds formulated into a blend for optimizing host recognition and attraction ofC.chinensishave yet to be determined,and the question as to whether there is a synergistic effect of the two components on the attraction ofC.chinensisremains to be answered.

In this study,we collected and analyzed the headspace volatiles of mungbean pods. Based on previous research,we investigated whether attraction to 2-hexenal and benzaldehyde byC.chinensisis synergistic at different ratios and concentrations in Y-tube olfactometer experiments.Trap catches of adultC.chinensiswere used to determine the most attractive ratio and concentration in the field. This study is an important step toward better understanding of the specificity of ratios and concentrations of blends of the two attractive components for gravid,mated femaleC.chinensis,and will enable a more detailed understanding of a key component of the chemical ecology for this pest with regards to host location and oviposition before its application to pest management.

2.Materials and methods

2.1.lnsects

Callosobruchuschinensiswere obtained from mungbean seeds from an experimental farmland at Shanxi Agricultural University,Shanxi Province,China,in 2015. A colony of testC.chinensiswas reared in 500-mL glass jars containing about 300 g mungbean seeds in the laboratory,and maintained at (26±1)°C,(75±5)% relative humidity (RH),and 24-h darkness. One male-female pair of newly emerged adults (0-24 h) was isolated using key sex differentiation characteristics (Raina 1970) and placed together in each Petri dish (9 cm) to mate. Observations for the adult emergence and mating period were made daily. The mated females were used in Y-tube olfactometer bioassays.

2.2.Plants and fields

Seeds of the mungbean cultivar Jinlv 1,which is commonly grown by farmers in Shanxi Province,were provided by the Horticulture Institute of the Shanxi Academy of Agricultural Sciences (Shanxi,China). Field experiments were conducted on experimental farmland at Shanxi Agricultural University (37°42´N,112°57´E),Shanxi Province,China,in 2018. The seeds were sown on 20 May 2018 in a plot (area:20 m×90 m; row spacing:30 cm×10 cm),and no fertilizers or insecticides were used.

2.3.Chemicals

HPLC-grade hexane and the identified chemicals,2-hexenal(98%) and benzaldehyde (99%),were purchased from Zhiyuan Chemical Technology Co.,Ltd.,(Tianjin,China),Sigma-Aldrich (USA),and Xiya Reagent (Shangdong,China),respectively. Each compound was diluted with HPLC-graden-hexane for behavioral tests. The solutions were kept in a freezer at -20°C until use.

2.4.Collection and analysis of volatiles

Dynamic headspace collection was used to collect volatile compounds from the mungbean pods excised from the plants at the fruiting stage. Forty pods were placed in a small glass bottle (8 cm×20 cm) into which purified air entered through a Teflon tube at a rate of 350 mL min-1.Three replications were conducted. Air was pulled out of the bottle through a trapping filter containing 200 mg of 80-100 mesh Porapak-Q adsorbent (Waters Corporation,Milford,Massachusetts,USA),and entered an activated charcoal filter on the opposite side of the bag. Before each collection,these filters were rinsed with 3 mL HPLC-grade hexane. Odor collections lasted for 4 h,and each collection was performed in the morning (n=3 odor collections) at room temperature under natural light conditions. The three repetitions represent biological repetitions. After each collection,the filters were removed and eluted with 2 mL of hexane,and the eluate was concentrated to 200 μL under a nitrogen stream and stored at -80°C until analysis.

Volatiles were analyzed with an Agilent 7890A gas chromatograph coupled to a 5975C mass selective detector(GC-MS),with an HP-5MS capillary column (30 m×0.25 mm×0.25 μm,Agilent Technologies,Santa Clara,CA,USA). Helium was used as carrier gas at a flow rate of 1.3 mL min-1. Injections (1 μL) were made in splitless mode at an injector temperature of 230°C.Following injection,the column temperature was maintained at 75°C for 2 min and then increased to 280°C at 5°C min-1with a final 5-min holding time. Mass spectra were obtained using electron impact (EI,70 eV) ionization. The chemical identities of the dominant peaks present in the pod aerations were determined by comparing their mass spectra with those of the NIST library. For further identification of these compounds,GC spectra and retention times were also compared to authentic standards.

2.5.Olfactometer bioassays with synthetic blends

Dual-choice bioassays were conducted in a Y-tube olfactometer to determine the attractiveness of 2-hexenal and benzaldehyde toC.chinensisat different ratios and concentrations. The glass Y-tube olfactometer consisted of a central tube (15 cm long and 2.5 cm in diameter)and two arms (27 cm long and 2.5 cm in diameter). The arms were connected by Teflon tubes to an odor source bottle containing a 10 μL sample on filter paper strips(1 cm×2 cm). Air was purified through activated charcoal and was humidified before entering the odor source bottle.The airflow in each arm was maintained at 300 mL min-1.The glass Y-tube olfactometer was placed in an enclosed incubator with diffuse light under controlled conditions of 28-30°C and 68-75% RH. In each test,an individual mated female was introduced at the end of the Y-tube stem and was considered to have made a choice only after it crossed the decision line that was 9 cm away from the Y-junction and remained there for at least 1 min. If a beetle did not make a choice within 5 min,it was deemed unresponsive.After every two beetles were tested,the odor sources were interchanged and the positions of the odor source containers in relation to each arm were reversed to eliminate directional bias. The used Y-tubes were cleaned and dried in a 180°C oven before reuse. Bioassays were replicated at least 30 times for mated females choosing either of the arms (n=30 beetles per odor source). Each mated female was used only once.

The experiments involved the following treatments to test the attraction ofC.chinensis:1) various ratios of 2-hexenal and benzaldehyde,5:1 (100 μg μL-1+20 μg μL-1),5:2(100 μg μL-1+40 μg μL-1),5:3 (100 μg μL-1+60 μg μL-1),5:4(100 μg μL-1+80 μg μL-1),1:1 (100 μg μL-1+100 μg μL-1),4:5 (80 μg μL-1+100 μg μL-1),3:5 (60 μg μL-1+100 μg μL-1),2:5 (40 μg μL-1+100 μg μL-1) and 1:5 (20 μg μL-1+100 μg μL-1); 2) different concentrations of the mixture at the ratio of 5:3,which were prepared by mixing 50,100,200,300 or 500 μg μL-1solutions of 2-hexenal per volume with the corresponding concentration of benzaldehyde. The volume of each treatment was 10 μL and each one was compared with 10 μL of hexane.

2.6.Field attraction

The field trial was carried out in a mungbean field in Jinzhong City of Shanxi Province (37°42´N,112°57´E) from 16 August to 8 September 2018. Yellow sticky traps with rubber septa(The West Company,Phoenixville,PA,USA) were used in the experiment. The combination of 300 μg μL-12-hexenal with 180 μg μL-1benzaldehyde was dissolved in 300 μL of hexane and loaded on a rubber septum. Control septa were loaded with the same volume of hexane. All the loaded rubber septa were sealed in envelopes and air-dried overnight at 4°C before storage at -20°C.To measure the trapping efficiency of each synthetic mixture,the septa were fixed to a metal wire inserted into a hole in the center of the yellow sticky trap (25 cm×20 cm). Yellow sticky traps were hung slightly higher than the mungbean plants with at least 10 m between adjacent traps. Three replicates of each treatment were arranged in a randomized complete block design. The traps were changed every two days (except for August 27th) and the number of trapped beetles was counted. The rubber septa were replaced every 10 days.During the period of the experiment,the field was not treated with pesticides.

2.7.Statistical analysis

SPSS version 17.0 (SPSS,Chicago,IL,USA) statistical software was used for all data analyses. The relative amounts of compounds released by mungbean pods are presented as the mean±SE. The response percentages of femaleC.chinensisin the Y-tube olfactometer were analyzed usingχ2tests. Field data were analyzed using independent-samplet-tests to compare numbers of beetles captured on blendvs.control traps,with the level of significance set to α=0.05.

3.Results

3.1.Chemical analysis

GC-MS analyses identified 31 volatile components in mungbean pod odors with their relative proportions in the collections summarized in Table 1. Mungbean pod volatiles are typically composed of green leaf volatiles (GLVs),terpenoids,aromatic compounds and fatty acid derivatives.GLVs,such as 3-hexen-1-ol,2-hexenal and 1-octen-3-ol,were detected in the volatile blends. Limonene and linalool,as common terpenoid volatiles,were also identified in the pod volatiles. Based on GC peak areas,the ratio of 2-hexenal and benzaldehyde was 5:3,which providedfurther basis for further experiments (Fig.1; Table 1).

Table 1 Mean (±SE) for the percentages (%) of volatiles released by mungbean pods

3.2.Olfactometer bioassays with synthetic blends

The synergistic effects of the two attractive compounds(2-hexenal and benzaldehyde) to mated femaleC.chinensiswere tested at different ratios and concentrations.

Optimal ratio for inducing behavioral choice of female C.chinensisIn the ratio experiment,olfactometer assays showed that mated females responded significantly to the ratio of 5:3,or 100 μg μL-12-hexenal and 60 μg μL-1benzaldehyde,with 67.74% attraction (χ2=3.90,df=1,P=0.048) (Fig.2). Interestingly,this ratio is consistent with the peak areas of the two compounds in the GC-MS of pod volatiles (Fig.1). There were no significant differences in attraction ofC.chinensisto other ratios of the mixture(P＞0.05). Therefore,the ratio of 5:3 was used in the concentration experiment.

Optimal dose for inducing behavioral choice of femaleC.chinensisIn the concentration experiment,different doses of 2-hexenal and benzaldehyde at the ratio of 5:3 were tested with mated females.Callosobruchuschinensisexhibited varying degrees of attraction for different mixture concentrations. The blends attracted 82.35% ofC.chinensisat the concentration of 300 μg μL-1+180 μg μL-1(χ2=14.24,df=1,P=0.000),followed by 100 μg μL-1+60 μg μL-1with 67.74% attraction (χ2=3.90,df=1,P=0.048) (Fig.3). No significant differences were noted between the other concentrations of the mixture in terms of attraction. The results indicated thatC.chinensiswas not attracted to the selected 5:3 ratio of the mixture at either high (500 μg μL-1+300 μg μL-1) or low (50 μg μL-1+30 μg μL-1) doses.

3.3.Field attraction assays

Fig.1 Chromatographic profiles of headspace volatiles released by mungbean pods.

Fig.2 Responses of female Callosobruchus chinensis to different ratios of 2-hexenal and benzaldehyde in the olfactometer. Control represents the solvent,hexane. Treatment represents different ratios of 2-hexenal and benzaldehyde listed on the right of the figure. Choice (%) represents the proportion of responses of female C.chinensis to control or treatment. ＊,indicates a significant difference compared with the control group as analyzed using χ2-tests (P＜0.05).

Fig.3 Responses of female Callosobruchus chinensis to different doses of 2-hexenal and benzaldehyde in the olfactometer.Control represents the solvent,hexane. Treatment represents different doses of 2-hexenal and benzaldehyde at the ratio of 5:3 listed on the right of the figure. Choice (%) represents the proportion of responses of female C.chinensis to control or treatment.＊,indicates a significant difference compared with the control group as analyzed using χ2-tests (P＜0.05); ＊＊＊,indicates P＜0.001.

In the field,moreC.chinensisadults were found in the traps that were baited with the lure containing 300 μg μL-12-hexenal and 180 μg μL-1benzaldehyde than in the traps baited with empty lures during the investigation period (P＜0.05) (Fig.4). The average number of trappedC.chinensispeaked at 143.3/trap for the blends and 25.0/trap for the control traps.

During the entire experimental period,the total numbers of female and male adults in the traps with the blend were greater compared to the controls (female:525.7±17.38,F=3.54,df=4,P＜0.0001; male:317.3±46.3,F=0.61,df=4,P＜0.0001) (Fig.5). In addition,more female adults were attracted to traps baited with blends,but there was no difference in attraction to femalesvs.males for control traps (Fig.5).

4.Discussion

Plant volatiles serve as important cues to insects searching for resources,mates and oviposition sites (Jumaet al.2016;Sunet al.2016a; Webster and Cardé 2016; Xu and Turlings 2018). In this study,a large number of green leaf volatiles(GLVs) was identified from mungbean pods,including 3-hexen-1-ol,2-hexenal and 1-octen-3-ol. GLVs are constitutively released from healthy plants and herbivoreinduced plants,which can provide important signals in plantherbivore interactions (Holopainen 2004; Sunet al.2010;Scalaet al.2013). For example,2-hexenal is reportedly emitted from a group of plants,such as peach shoots(Prunuspersica) (Piñero and Dorn 2007),soybean (Glycine max) (O'Neillet al.2010),maize (Zeamays) (Ardanuyet al.2016),cotton (Gossypiumhirsutum) (Guo and Wang 2019)and tobacco (Nicotianatabacum) (Guo and Wang 2019).Notably,the aromatic compound benzaldehyde identified from our study has also been found in other legumes crops,such as cowpea (Vignaunguiculata) (Babuet al.2003),soybean (Glycinemax) (Boueet al.2003) and khesari(Lathyrussativus) (Adhikaryet al.2015). In addition,the synergistic effect of benzaldehyde combined with other plant volatiles has been reported in a number of insects,includingCydiamolesta(Nataleet al.2003),Manducasexta(Riffellet al.2009) andSitonahumeralis(Lohonyaiet al.2019).

Fig.4 Callosobruchus chinensis adults caught in blend traps and controls traps. Blend traps represent the yellow sticky with a rubber septum loaded with 300 μg μL-1 2-hexenal and 180 μg μL-1 benzaldehyde. Control traps represent the yellow sticky with a rubber septum loaded with hexane. Data are mean±SE from 16 Aug.to 8 Sep.in 2018.

Fig.5 Total number (±SE) of Callosobruchus chinensis adults in the field traps. Blend traps represent the yellow sticky with a rubber septum loaded with 300 μg μL-1 2-hexenal and 180 μg μL-1 benzaldehyde. Control traps represent the yellow sticky with a rubber septum loaded with hexane. ＊＊＊,indicates significant difference between blend traps and control traps(P＜0.001).

It is well documented that insects respond to specific blends of VOCs to recognize host plants and not just one single compound,and that some specific components in volatile blends are biologically more important than others with respect to plant-insect interactions (Lightet al.2001;Reddy and Guerrero 2004; Gregget al.2010; Gebreziher and Nakamuta 2016). For example,earlier studies found a synergistic effect of a combination of green leaf volatiles and aromatics for female oriental fruit mothCydiamolesta(Nataleet al.2003; Pinero and Dorn 2007). Therefore,studying the behavioral role of the blend proportions and complexity of host plants would be of great importance.Based on the results of a previous study (Wanget al.2017),we observed the ratio of 5:3,100 μg μL-12-hexenal and 60 μg μL-1benzaldehyde,attracted moreC.chinensis females than other ratios,suggesting that this ratio of the volatile blends based on the GC-MS peak areas for pod volatiles is important for herbivore attraction (Tasinet al.2010; Adhikaryet al.2015). Similarly,several studies have shown that altering the ratios between attractive volatile blend components can influence the behavioral responses of herbivores (Visser and Avé 1986; Bruceet al.2005;Bruce and Pickett 2011; Magalhãeset al.2018; Karmakaret al.2020).

In terms of the concentrations of VOCs that elicit a positive behavioral response,responses ofC.chinensis to different concentrations of the two synthetic volatiles were tested. In the current study,we found thatC.chinensis was not attracted to the mixture at high or low concentrations,whereas it was strongly attracted to the mixture at an intermediate concentration (except for 200 μg μL-1+120 μg μL-1). Similar results were also reported using single compounds for attracting mated femaleC.chinensis,which showed a significant choice preference for the intermediate concentration (100 μg μL-1) (Wanget al.2017). Often when testing compounds at concentrations higher than those found in nature,a misleading behavioral response may be recorded,as demonstrated in electrophysiological experiments (Andersonet al.1995; Luet al.2017;Oseiowusuet al.2020).

Many factors,such as the concentration of the blend,background odors,temperature,humidity,and surrounding plants,will affect the differences in the attractiveness of blends to insects in the laboratory and in the field (Knudsenet al.2008; Knudsen and Tasin 2015; Sunet al.2016b; Caiet al.2017; Xiuet al.2019b). We consistently found that yellow sticky traps baited with 300 μg μL-12-hexenal and 180 μg μL-1benzaldehyde captured a higher number ofC.chinensis than control traps in the field. Insects are known to locate and select their host plants by an integration of a series of visual,chemical and gustatory stimuli. Yellow color is a broad-spectrum attractant for many insect species (Tanget al.2016),which is illustrated by theC.chinensis adults captured in this study in blank control yellow sticky traps.In addition to visual cues,host plant volatiles conclusively increased the attraction ofC.chinensis adults to traps due to behaviors associated with host plant location,as illustrated by our results showing that yellow sticky traps baited with host plant volatiles captured higher numbers ofC.chinensis than control traps in the field.

The optimal ratio and concentration of the attractive blends was screened using mated females in the laboratory,which resulted in more attraction to females than males for blend traps in the field. These findings also indicated different roles of the same VOCs in the chemical ecology of males and females (Germinaraet al.2019). Similar results have also been found in other insects,probably because females (even virgin females) use host-plant volatiles to locate oviposition sites (Landolt and Phillips 1997; Nakamutaet al.1997; Sweeneyet al.2010). Also,this phenomenon may be due to sexual differences in the number of antennal olfactory neurons tuned to a certain compound in the olfaction system (Ragusoet al.1996). For example,femaleTetropiumfuscumhad twice as many ORNs that responded to host volatiles compared with males (Mackayet al.2015).

Synthetic attractants based on plant volatiles could be used to monitor and trap insect pests,as they are known to effectively attract both sexes of herbivorous insect pests and provide management tools to improve pest control.Several studies have shown that host-derived volatiles were successfully deployed in the management of agricultural and forestry pests,such asEctropisobliqua,Empoasca onukiiandAdelphocorisspecies (Sunet al.2016b; Caiet al.2017; Xiuet al.2019a). Our study will support the potential usefulness of the optimal combinations of 2-hexenal and benzaldehyde to lureC.chinensisadults in crop fields,as they were found to capture a large number of adults,and consequently fewer eggs were laid on mung beans and fewer larvae could be found in crop storage. Further research to develop a safe and highly effective method for monitoring and controllingC.chinensisshould focus on how to enhance the efficiency of the attractive blends in field conditions,such as trap design and trap placement. In addition,emitters of blends should be tested and adapted to optimize their efficacy under field conditions.

5.Conclusion

Green leaf volatiles of 2-hexenal and aromatic benzaldehyde are natural components forC.chinensis in host recognition.The optimal ratio and concentration (300 μg μL-12-hexenal and 180 μg μL-1benzaldehyde) of the attractive blends were identified in laboratory and field experiments. We provide further evidence that the ratio and concentration of attractive volatiles derived from natural host plant volatiles are vital components for host-plant recognition cues. From this perspective,this study enhances our knowledge of the interaction of effective attractive constituents in complex odor blends and contributes to the development of an efficient monitoring system involving plant volatiles forC.chinensis.

Acknowledgements

We are very grateful to Associate Prof.Wang Bing from Institute of Plant Protection,Chinese Academy of Agricultural Sciences,for the revision of the manuscript. This research was funded by the Shanxi Provincial Key Research and Development Project,China (201803D221004-8),the earmarked fund for China Agriculture Research System(CARS-08-G10),the Research and Demonstration of Key Green Technology for High Quality of Coarse Cereals in Northwest Shanxi (201703D211002-8) and the Shanxi Province Science Foundation for Youths(201801D221305).