The olfactory responses of mirid bugs to six plant extracts and their volatiles

Many methods for management of mirid bugs, one of the major pest species in cotton fields, have been developed in recent decades. The use of attractive plant volatiles may be a safe and effective alternative compared with chemical pesticides. Very little is known about the responses of the bugs to some specific volatiles. In this study, the attractive effects for the mirid bug Apolygus lucorum were evaluated and compared among ethanol extracts obtained from six types of plants. The results showed that A. lucorum had the highest selection response rate to the extract from Allium tuberosum seeds and 70.51% of the adults tended to choose this odour source. Among 40 identified compounds of the volatiles comprising A. tuberosum seed extracts, diethyl phthalate and methyl levulinate had significantly higher attractive effects than that of cotton leaves, but the respective selection response rates were less than 65%. We found that when methyl levulinate and diethyl phthalate were mixed in volume ratios of 3:7 and 6:4, the selection response rates of A. lucorum for the mixtures were 54.67% and 63.65%, respectively. These response rates were significantly higher than those for the two compounds presented alone. Furthermore, the selection response rates of A. lucorum based on these mixtures reached 82.75% and 75.73%, respectively when compared with cotton leaves. The two volatile mixtures also showed a significantly higher attractive effect for Adelphocoris suturalis, with respective selection response rates at 74.58% and 77.80% when compared with cotton leaves. Moreover, the two attractants showed no difference between the responses of male and female mirid bugs. We concluded that an appropriate application of the attractants described herein has the potential to develop a safe and effective approach for the monitoring and control of mirid bugs in cotton fields.

to have more than 200 types of host plants, including a number of important crop plants such as cotton, jujube and grape (Lu et al., 2010;. Recently, although luring technologies such as sex pheromone trapping and others have been developed, the commercially attractant for mirid bugs is still in its infancy stage and product categories are limited. The control of mirid bugs mainly relies on chemical pesticides. However, spraying insecticide is usually not a good choice for these pests due to their strong dispersal ability (Blackmer et al., 2004) and rapidly developed pesticide resistance (Snodgrass et al., 2009;Zhang et al., 2015;Zhen et al., 2016).
Therefore, it is imperative to seek alternatives for management of mirid bugs.
Plant organs, such as roots, stems, leaves, flowers and fruits, are known to release complex mixtures composed of small molecular weight volatiles, including alcohols, aldehydes, ketones, esters, terpenoids, nitrogen-containing compounds and aromatic compounds (Reddy & Guerrero, 2000;Shiojiri & Karban, 2008). Plants with dissimilar species and different physiological periods can release diversely distinct volatiles. And insect pests can use plant volatiles to detect the appropriate host plants as food sources or oviposition sites (Turlings & Erb, 2018). For example, Li et al. (2020) identified 13 compounds of volatiles of tobacco plant that affect electrophysiological activity of Phthorimaea operculella. Among these compounds, cis-3-Hexen-1-ol showed noteworthy attractant effect on the orientation behaviours of both male and female moths, and nonanal, decanal, decane and methyl hexadecanoate could increase the number of female oviposition. Shivaramu et al. (2017) studied the interaction of Scirtothrips dorsalis with its host plant Capsicum annum and found that volatiles of fruiting stages (FR) of C. annum were highly attractive to S. dorsalis. Furthermore, S. dorsalis were substantially attractive to infested plants than to that of un-infested FR plants. Xiu, et al. (2019a) stated that Peristenus spretus females were attracted by the two components with two concentrations (10 mg/ml and 100 mg/ml) in both Y-tube olfactometer bioassays and field experiment. These researches suggest that plant volatiles may offer a cost-effective method for monitoring, controlling and early detection of pests in the future (Turlings & Erb, 2018). However, till now, relevant studies with respect to mirid bugs, especially the plant resources with attractive activity, are limited.
In this study, the attractive effects on the mirid bug A. lucorum were evaluated and compared among ethanol extracts obtained from six types of plants, including those from the seeds of Allium tuberosum and Helianthus annuus, Abutilon theophrasti leaves, Pisum sativum seedlings, Aucklandia lappa roots and Periploca sepium root bark. Volatiles of active extracts were identified by gas chromatography-mass spectrometry (GC-MS). The efficacy of volatile and its mixtures were assessed as attractants for A. lucorum and A. suturalis.
The ultimate goal of this study was to develop a suitable trapping method that can be used for monitoring and preventing infestations of mirid bugs in the field.

| Preparation of plant extracts
Air-dried plant parts were ground using a pulverizer, and samples of the resulting powders were soaked in anhydrous ethanol at room temperature for 15 d followed by filtering through a Buchner funnel.
The solvent content of these preparations was subsequently evaporated using a rotary evaporator in a water bath at approximately 45°C to obtain crude extracts.

| Identification of volatiles of the ethanol extracts
Five millilitres of extract was added to a glass jar (35cm high and 20 cm in diameter) containing two holes at the top. An adsorption column was inserted into one of these holes, whereas air filtered by activated carbon was introduced via the other hole. After collecting for 8 hr, the adsorption column was eluted with chromatographically pure n-hexane for GC-MS detection of samples. The internal standard was 10 -4 mol/L ethyl decanoate, and GC-MS detection was carried out using 90 μL of sample mixed with 10 μL of the internal standard.
Gas chromatography conditions. An Rtx-5 MS chromatographic column (30 m. 0.25 mm; 0.25 µm) was employed. The injector temperature was 250°C, and the carrier gas was helium without split.
The oven programme was as follows: oven temperature maintained at 40°C for 1 min, then increased to 130°C at a ramp of 4°C/min, maintained for 5 min, then increased to 250°C at a ramp of 10°C/min and maintained for 5 min.
Mass spectrometry conditions. The ion source temperature was 250°C, the scanning speed was 2,500, and the interval was 0.3 s.
To determine the main chemical components in the extracts, mass spectrograms were compared with standard spectrograms using Labsolution software.

| Response of A. lucorum to six plant extracts
Prior to experimentation, adult A. lucorum were starved for 4 hr and then used for the measurement of olfactory behaviour using a three-arm olfactometer. One arm of the olfactometer was closed with absorbent cotton, and the remaining two arms were each connected to a 250-mL glass bottle, one of which contained 30 mg of plant extract, which was passed into the air filtered by activated carbon, whereas only the air filtered by activated carbon was passed into the control group. The air flow rate was set at 2 L/min, and the air was ventilated for 5 min prior to experimentation. The experiment was repeated 9 times, with 10 adults (5 males and 5 females) being placed into the olfactometer from the centre of the top in each replicate. Thus, in total, the behaviour of 90 insects was examined.
Having commenced the experiment, all lights in the laboratory were switched off, and responses were recorded 30 min later. When the bugs had crossed half the length of an arm, this was recorded as a selective response to the odour. For each trial, the tested adults and the position of odour source were changed, and the olfactometer was also washed with ethanol after each experiment.

| The olfactory selection responses of A. lucorum between cotton leaves and main volatiles from the ethanol extracts of A. tuberosum seeds
Eleven compounds detected in the ethanol extracts of A. tuberosum seeds were selected according to their contents in the volatiles and the availability of commercial sources, and their attractive effect on A. lucorum was compared with that of cotton leaves. One arm of a three-arm olfactometer was closed with absorbent cotton, and each of the other two arms was connected to a 250-mL glass bottle, one of which contained 30 mg of volatile compound and the other contained two or three cotton leaves. The selected compounds were diethyl phthalate, methyl levulinate, heptadecanoic acid ethyl ester, hexanal, 2-methyl thiazolidine, ethyl methanesulphinate, 3,3-dimethoxy-2-butanone, 4-methyl-2-pentanol, hexanol, 10-undecenoic acid ethyl ester and 2-methyl-2-propyl-1,3-propanediol.
Other methods were the same as described in pervious subsection.

| The olfactory selection responses of mirid bugs between volatile mixtures and cotton leaves
One arm of the three-arm olfactometer was closed with absorbent cotton, and each of the other two arms was connected to a 250-mL glass bottle, in which was added with 30 mg of volatile mixture and the other with two or three cotton leaves to examine differences in the attractive effects of the mixture and cotton leaves on A. lucorum and A. suturalis. The gender of adults attracted by the mixtures was also recorded. Other methods were the same as described in pervious subsection.

| Data Analysis
A chi-square test was used to analyse differences among the attractive effect of the six plant extracts on A. lucorum. We also used the chi-square test to analyse the differences between the attractive effect of the 11 selected compounds from volatiles of ethanol extracts of A. tuberosum seeds and cotton leaves on A. lucorum. Selection response rate, response rate and selection coefficient were calculated as follows: Duncan's new multiple range test was used to analyse the differences among the selection response rates for diethyl phthalate, methyl levulinate and different ratios of their mixture on A. lucorum.
The chi-square test was used to analyse the differences between the attractive effect of mixtures and cotton leaves on A. lucorum and A. suturalis of two gender. All analyses were conducted using SPSS software.

| Response of A. lucorum to six plant extracts
As shown in Table 1, the selection coefficients for the extracts of A. tuberosum seeds, H. annuus seeds, and A. theophrasti leaves were greater than zero, indicating that these extracts had an attractive effect on A. lucorum. Among all extracts studied, A. tuberosum seed extracts showed the highest selection response rate, attracting the highest number A. lucorum adults. Chi-square tests revealed that the attractive effect of A. tuberosum seed extracts was significantly higher than that of the extracts of A. theophrasti leaves, P. sativum seedlings, A. lappa roots and P. sepium root bark (Table 2).

| Identification of the volatiles in A. tuberosum seed extracts
Forty compounds were identified in the volatiles of A. tuberosum seed extracts, which included alcohols, alkanes, aldehydes, alkenes and esters (Table 3). Among them, 11 compounds were selected for follow-up experiments based on their contents in the volatiles and the availability of commercial sources.

| The olfactory selection responses of A. lucorum between cotton leaves and main volatiles from the ethanol extracts of A. tuberosum seeds
As shown in Table 4, diethyl phthalate, methyl levulinate and heptadecanoic acid ethyl ester were found to have an attractive effect on A. lucorum compared with cotton leaves, although among these only diethyl phthalate and methyl levulinate had a significantly higher attractive effect than that of cotton leaves (p < .05).
However, the selection response rates of these compounds were less than 65%, and thus, we sought to examine the cases of mixtures with diethyl phthalate and methyl levulinate in an attempt to enhance the effect.

| The olfactory selection responses of A. lucorum to volatile mixtures with different mixed ratios
As shown in Table 5, the selection response rates of A. lucorum to the mixtures of methyl levulinate and diethyl phthalate did not vary regularly with the ratios of these two compounds. We found that the selection response rates were the highest with methyl levulinate:diethyl phthalate mixing ratios of 3:7 and 6:4, and they were significantly higher than those of two compounds alone.
Contrastingly, at mixed ratios of 11:9, 4:6 and 3:17, the selection response rates for the mixtures were significantly higher than that of only one compound. Accordingly, we considered methyl levulinate:diethyl phthalate volume ratios of 3:7 and 6:4 to be the optimal mixed ratios.

| The olfactory selection responses of mirid bugs between volatile mixtures and cotton leaves
At methyl levulinate:diethyl phthalate volume ratios of 3:7 and 6:4, the attractive effects of two mixtures on A. lucorum were significantly higher than that of the leaves of cotton (Figure 1). The selection response rate to these two mixtures could reach 82.75% and 75.73%, respectively. Furthermore, these two mixtures had a significantly higher attractive effect for A. suturalis, with selection response rates of 74.58% and 77.80% when compared with that of cotton leaves. The two attractants showed no difference between the responses of male and female mirid bugs (Figure 2).

| D ISCUSS I ON
In this article, we tested olfactory response of A. lucorum to the ethanol extracts from six plants and found that the extract obtained from A. tuberosum seeds had the best attractive effect on A. lucorum.
In its volatiles, methyl levulinate and diethyl phthalate had significant attractive effects on male and female adults of A. lucorum and A. suturalis when mixed with certain ratios. We hope our study could provide the basis and valuable guide for monitoring and controlling mirid bugs in cotton fields in the future. lucorum ; Bradysia odoriphaga produced strong electrophysiological and behavioural responses to some disulphides (Yang et al., 2019 In this study, both A. suturalis and A. lucorum showed olfactory preference to this mixed attractant suggesting the similarity of olfactory response between the two species of mirid bugs (Wang et al., 2018).

TA B L E 1 Attractive effects of six plant extracts on Apolygus lucorum
Some compounds have a wide spectrum of attraction based on this kind of olfactory similarity. For example, (E)-3-hexen-1-ol showed at-

tractive effects on both genders of Phthorimaea operculella and female
Anagrus nilaparvatae (Li et al., 2020;Mao et al., 2018); nonanal elicited a significant electrophysiological response and olfactory preference of Harmonia axyridis, meanwhile electroantennogram responses of male Hyphantria cunea also be detected when stimulated by nonanal (Tang et al., 2012;Xiu, et al., 2019b). In addition, Orseolia oryzivora showed electrophysiologically activity on (E)-caryophyllene, and these compounds were also marginally attractive to female Anopheles gambiae (Meza et al., 2020;Ogah et al., 2017). In this article, we only tested olfactory preference of two mirid bugs species to the mixed attractant; thus, the spectrum of attraction still needs to be explored to make sure if it has a potential to control some other mirid bugs like Lygus pratensis or even some pests not belong to Miridae in the future research.
In summary, we identified two attractants (methyl levulinate and diethyl phthalate) in extracts of Allium tuberosum seeds, for *and ** represent a significant difference (p < .05) and extremely significant difference (p < .01), respectively, between the attractive effects of two plant extracts (Chi-square test).

TA B L E 2
Chi-square analysis of the attractive effects of six plant extracts on Apolygus lucorum which there is currently almost no literature regarding their attractive effects on mirid bugs, and determined the two most effective ratios of mixtures of these two compounds for attracting the adults of A. lucorum and A. suturalis. Moreover, we found that the two mixtures showed no difference with respect to the responses of adult males and females, and thus believe that they TA B L E 3 Identification of compounds in volatile of Allium tuberosum seed extracts based on gas chromatography-mass spectrometry analysis The mixed ratios in the table are volume ratios of methyl levulinate to diethyl phthalate. The data presented in the table are means ± standard errors. Different uppercase and lowercase letters indicate extremely significant (p < .01) and significant (p < .05) differences between different treatments for the same mixed ratio (DMRT).

TA B L E 5
The selection reaction rate of Apolygus lucorum to methyl levulinate (M), diethyl phthalate (D) and their mixtures currently unable to predict the active range of these attractants or determine the most appropriate method for their application in the field (Cai et al., 2017). Moreover, we believe that there may be other components with attractive effects that might warrant assessment in the future, given that the volatiles of A. tuberosum seed extracts contained 40 compounds. Nowadays, some commercially attractant like Miride (Shenzhen Bioglobal Bioagriculture Technology Co., Ltd) has been pretested in field. Even though the commercially attractant for mirid bugs is still in its infancy stage and product categories are limited compared with Lepidoptera pests, we hope our study could provide the basis and valuable guide for monitoring and controlling mirid bugs in cotton fields in the future.

CO N FLI C T O F I NTE R E S T S TATE M E NT
We declare that there are no conflicts of interest relating to the submission of this manuscript, and that the manuscript has been approved for publication by all authors.