Evaluation of Selected Plant Volatiles as Attractants for the Stick Tea Thrip Dendrothrips minowai in the Laboratory and Tea Plantation

Simple Summary The stick tea thrip Dendrothrips minowai is a key pest in tea plantations in China. In recent years, plant-derived semiochemicals have attracted considerable attention as promising attractants for the management of thrips, due to their safety and low cost. In this study, compounds that have been reported to attract other thrips or emitted from tea plants were evaluated for their electroantennogram (EAG), behavioral tests and field trapping efficacy for D. minowai. The EAG relative response value of D. minowai evoked by p-anisaldehyde, 3-methyl butanal, (E)-β-ocimene, farnesene, nonanal, eugenol, (+)-α-pinene, limonene, (−)-α-pinene, and γ-terpinene was significantly higher than the other compounds. Meanwhile, p-anisaldehyde, eugenol, farnesene, methyl benzoate, 3-methyl butanal, (E)-β-ocimene, (−)-α-pinene, (+)-α-pinene, and γ-terpinene led to attraction or repellency responses of female D. minowai. In addition, trap capture numbers of female D. minowai on sticky traps baited with p-anisaldehyde, eugenol, farnesene, and 3-methyl butanal were significantly higher than the control in tea plantations. Overall, our results highlight the potential application of plant volatiles in the development of effective, eco-friendly lure formulations for use in the monitoring and management of thrips. Abstract The stick tea thrip (Dendrothrips minowai Priesner) is the main pest thrip in tea (Camellia sinensis) plantations in China, and seriously affects the quality and yield of tea. Plant-derived semiochemicals provide an alternative to pheromones as lures and these compounds possess powerful attractiveness. In this study, we selected 20 non-pheromone semiochemicals, including compounds that have been reported to attract other thrips and some volatiles emitted from tea plants as the potential attractant components for D. minowai. In electroantennogram (EAG) assays, 10 synthetic compounds (p-anisaldehyde, 3-methyl butanal, (E)-β-ocimene, farnesene, nonanal, eugenol, (+)-α-pinene, limonene, (−)-α-pinene, and γ-terpinene) elicited significant antennal responses in female D. minowai. In addition, a two-choice H-tube olfactometer bioassay showed that D. minowai displayed significant positive responses to eight compound dilutions (p-anisaldehyde, eugenol, farnesene, methyl benzoate, 3-methyl butanal, (E)-β-ocimene, (−)-α-pinene, and (+)-α-pinene) when compared with the solvent control at both 1 and 2 h. Moreover, γ-terpinene exhibited a significantly deterrent effect on D. minowai. Finally, trap catches of four compounds (p-anisaldehyde, eugenol, farnesene, and 3-methyl butanal, respectively) significantly increase in tea plantations. Among these, the maximum number of D. minowai collected by blue sticky traps baited with p-anisaldehyde was 7.7 times higher than the control. In conclusion, p-anisaldehyde, eugenol, farnesene, and 3-methyl butanal could significantly attract D. minowai in the laboratory and under field conditions, suggesting considerable potential as commercial attractants to control D. minowai populations.


Introduction
Semiochemicals are single or mixed substances emitted by one organism that stimulate a physiological or behavioral response between the same members or another species [1]. The recognition of host and non-host plants involves a complex set of physiological activities, in part mediated by semiochemicals [2]. There is no doubt that numerous semiochemical compounds released by plants could elicit significant behavioral responses of thrips [3,4]. Furthermore, semiochemicals produced by plants as well as their mimics, always provide an alternative to pheromones as lures, and sometimes can be more widely used and powerful attractants of thrips [5][6][7]. Semiochemicals produced by plants fulfill the following advantages necessary for promising lures of thrips: (a) Low toxicity to the environment, humans, and non-target beneficial arthropods, such as natural enemies; (b) volatile under normal environmental conditions; (c) stable for a long period of time; (d) the cost is relatively low compared with the established control measures; and (e) can attract more than one species of thrips [8].
The stick tea thrip Dendrothrips minowai Priesner (Thysanoptera: Thripidae) is a regional fulminant, but an easily overlooked pest of tea plants, Camellia sinensis (L.) O. Ktze. in China [20]. In particular, D. minowai have been expanded to most of the entire tea production areas and the damage degree has been intensified each year [21,22]. Both adults and nymphs of D. minowai feed on the leaflet of tea plants, causing stunted growth, color fading, and even stiffness or brittleness of the damaged leaves, which seriously affect the yield and quality of tea [23]. An array of tea plant volatiles is used by herbivores for host searching and locating, and several of these compounds have been investigated as non-pheromonal attractants to improve the capture of pests [24][25][26]. For instance, (Z)-3hexen-1-ol, (Z)-3-hexenyl acetate, and linalool are volatiles derived from tea plants and significantly attract Empoasca onukii adults [27]. However, the compounds of tea plants that can attract D. minowai are not well understood.
At present, controlling the D. minowai population is still mainly dependent on the application of pesticides, which caused pressure on the environment and risks to beneficial natural enemies [28,29]. As a result, there is an urgent need to develop a sustainable and environmentally friendly management program that provides alternatives to synthetic insecticides. Here, we specifically addressed plant volatiles, including those that have been reported to attract other thrips and some volatiles emitted from tea plants, which are effective compounds in controlling D. minowai using EAG and behavioral assays in the laboratory. Then, the efficiency of these compounds were evaluated in tea gardens, which would provide new approaches to the control of thrips.

Biological Materials
Dendrothrips minowai adults were collected from tea orchards of Shaoxing royal tea village Co., Ltd., Shaoxing, China (120.71 • E, 29.94 • N). Thereafter, D. minowai were reared on tea seedling leaves (cultivar: Longjing 43) in clear glass jars in a climate chamber at 24 ± 1 • C, 60 ± 5% relative humidity, and L16:D8 photoperiod. In addition, fresh leaves were supplied daily to the thrips. To obtain groups of second adults of known age for the bioassays, D. minowai females were allowed to lay eggs on tea seedling leaves in separate glass jars for 24 h. Second generation females were used for electrophysiological recordings and behavioral bioassays.

Electrophysiological Recordings
An electroantennogram detection device, comprising an IDAC-2 interface box, CS-55 air stimulus controller (Syntech, Hilversum, The Netherlands) with 220 kV of electricity, was linked to a computer with EAG-Pro software. The head of a 1-3 day-old unmated female D. minowai individual was excised from the body using a scalpel, and connected to reference electrodes using a glass capillary. Then, the two antennae tips were immersed in a recording electrode containing electrode solutions (128 mM of NaCl, 1.9 mM of CaCl 2 , 7.6 mM of KCl, and 2.4 mM of NaHCO 3 ) (see Figure 1) [30][31][32]. Mineral oil was used as the solvent to prepare the solution (10 mg/mL) of each compound, as well as the negative control. In addition, cis-3-hexen-1-ol (100 mg/mL) was utilized as the reference response, which elicited a stable antennal response of D. minowai (based on our preliminary experiments). Thereafter, 10 µL of the odor solution was applied on a filter paper strip (5 × 50 mm) and placed inside a glass Pasteur pipette cartridge (14.5 cm long) [33]. A constant flow of clean air (charcoal and humid filtered) was provided to the antenna to remain active at a rate of 300 mL/min. The antenna was tested at 0.5 s pulses once per stimulus. Each recording trial was conducted in the following sequence: Mineral oil, reference compound, tested odor solution (10 mg/mL), reference compound, and mineral oil, applied (0.5-s pulse) at 30-s intervals. Each chemical dilution was tested on six individuals.

H-Tube Olfactometer Bioassays
A glass H-tube olfactometer (See Figure S1 for specific parameters) was used for testing the responses of the stick tea thrips to different plant volatiles. Prior to testing, the ends of the transverse tube, which were connected to two straight tubes, were sealed with gauze (200 mesh) [34]. Meanwhile, H-tube olfactometers were cleaned with ethanol and dried at 90 • C in an electric heating constant temperature drying oven (Shanghai Sumsung Laboratory Instrument Co., LTD., Shanghai, China) for 1 h. Tests were performed in a dark room, and both the temperature and relative humidity were maintained at 26 ± 1 • C and 65 ± 1%, respectively. In the tests, 20 µL mineral oil solution applied on a filter paper (diameter: 20 mm) was used as control, and 20 µL test odor dilution (10 mg/mL) applied on a filter paper (diameter: 20 mm) served as odor source. In general, D. minowai females were isolated from tea leaves into a glass jar for 2 h for acclimation and to calm down [35]. Thereafter, twenty 1-3 day-old unmated females were inoculated in the middle of the transverse tube. If the tested individuals crossed more than 5 cm of the transverse tubes, it was recorded as a positive response to the treatment or the control. If not, it was recorded as no reaction. After 1 and 2 h, we recorded the number of thrips that crossed more than and less than 5 cm of the transverse tubes, respectively. Each thrip was tested only once, and the test was repeated 10 times for each chemical. Moreover, in this study, a total of 200 D. minowai females were used. All of the bioassays were conducted between 08:00-17:00 h daytime.

H-Tube Olfactometer Bioassays
A glass H-tube olfactometer (See Figure S1 for specific parameters) was used for testing the responses of the stick tea thrips to different plant volatiles. Prior to testing, the ends of the transverse tube, which were connected to two straight tubes, were sealed with gauze (200 mesh) [Error! Reference source not found.]. Meanwhile, H-tube olfactometers were cleaned with ethanol and dried at 90 °C in an electric heating constant temperature drying oven (Shanghai Sumsung Laboratory Instrument Co., LTD., Shanghai, China) for 1 h. Tests were performed in a dark room, and both the temperature and relative humidity were maintained at 26 ± 1 °C and 65 ± 1%, respectively. In the tests, 20 μL mineral oil solution applied on a filter paper (diameter: 20 mm) was used as control, and 20 μL test odor dilution (10 mg/mL) applied on a filter paper (diameter: 20 mm) served as odor source. In general, D. minowai females were isolated from tea leaves into a glass jar for 2 h for acclimation and to calm down [Error! Reference source not found.]. Thereafter, twenty 1-3 day-old unmated females were inoculated in the middle of the transverse tube. If the tested individuals crossed more than 5 cm of the transverse tubes, it was recorded as a positive response to the treatment or the control. If not, it was recorded as no reaction. After 1 and 2 h, we recorded the number of thrips that crossed more than and less than 5 cm of the transverse tubes, respectively. Each thrip was tested only once, and the test was repeated 10 times for each chemical. Moreover, in this study, a total of 200 D. minowai females were used. All of the bioassays were conducted between 08:00-17:00 h daytime.

Field Trapping Trials
To determine the attractiveness of active compounds produced in the laboratory to D. minowai, with both higher electrophysiological response value and positive behavioral response, field trapping trials were conducted in two tea gardens at Hangzhou Fuhaitang Tea Ecological Technology Co., Ltd. (120.03° E, 30.13° N) and Shaoxing Royal Tea Village Co., Ltd. (Shaoxing, China) in September 2020 and 2021. The cultivar in both tea plantations was clone Longjing 43. A red rubber septa (Zhangzhou Ingeer Agricultural Science and Technology Co. Ltd., Zhangzhou, Fujian Province, China) (117.74° E, 24.52° N) comprising 100 μL of compound dilution (concentration: 100 mg/mL, 10 mg active compound)

Field Trapping Trials
To determine the attractiveness of active compounds produced in the laboratory to D. minowai, with both higher electrophysiological response value and positive behavioral response, field trapping trials were conducted in two tea gardens at Hangzhou Fuhaitang Tea Ecological Technology Co., Ltd. (120.03 • E, 30.13 • N) and Shaoxing Royal Tea Village Co., Ltd. (Shaoxing, China) in September 2020 and 2021. The cultivar in both tea plantations was clone Longjing 43. A red rubber septa (Zhangzhou Ingeer Agricultural Science and Technology Co. Ltd., Zhangzhou, Fujian Province, China) (117.74 • E, 24.52 • N) comprising 100 µL of compound dilution (concentration: 100 mg/mL, 10 mg active compound) or solvent (hexane) solution was placed on a blue sticky trap. Each compound dilution was replicated 6 times, for a total of 48 blue sticky traps. These traps were placed with 6 m spacing between traps to minimize the interference between lures. Prior to the trapping trials, blue sticky traps without any lures were placed at the trap sites for 2 days to confirm that the thrip populations were at approximately equal population densities in the different treatment areas (Hangzhou: 30 August 2020; Shaoxing: 31 August 2021) [36]. The trap check was conducted after 2 days of experiment, and trap catches were recorded. Meanwhile, the traps were replaced on each sampling day, but the lures were not replaced during the experiment. Each volatile treatment was replicated 3 times in 2020 and 2021.

Statistical Analyses
All of the data were checked for normality and equality of variances prior to statistical analysis. Datasets that did not fit assumptions were square-root (sqrt) transformed to meet the requirements of equal variance and normality. The EAG relative value was calculated based on the following: EAG amplitude values of tested stimulus-the average EAG amplitude values of the control)/(the average EAG amplitude values of the reference compound-the average EAG amplitude values of the control) × 100%. Comparisons of recorded EAG values between different compounds were performed by one-way ANOVA following Tukey's HSD test (p < 0.05). In the behavioral trial, the t-test was applied to assess the number of thrip individuals between the treatment and control (p < 0.05). In the field evaluation, we performed a general linear model (GLM) analysis to assess the effect of various active compounds on thrips. Dunnett's one-tailed t-test was controlled for Type 1 experiment-wise error for comparisons of each compound against control (a Duncan test with α = 0.05). The graph of EAG relative value was performed using the GraphPad Prism 7.0. The graph of behavioral response and the field evaluation were performed using OriginPro 2021. All of the analyses were conducted using SAS 9.4.

EAG Response of D. minowai Adults to Single Compound
To comprehensively evaluate the electrophysiological activity, EAG responses of D. minowai to 20 compounds were tested. For a specific concentration (10 mg/mL), 10 volatiles elicited significant antennal responses to female D. minowai, with the mean relative values (±SEM) approximately equal to 1.00 (p-anisaldehyde: 1.48 ± 0.046; 3-methyl butanal: 1.43 ± 0.040; (E)-β-ocimene: 1.31 ± 0.121; farnesene: 1.25 ± 0.093; nonanal: 1.17 ± 0.059; eugenol: 1.15 ± 0.040; (+)-α-pinene: 1.11 ± 0.057; limonene: 1.08 ± 0.072; (−)-α-pinene: 0.96 ± 0.061; γ-terpinene: 0.95 ± 0.036). Whereas 10 other compounds elicited weak antennal responses. Among these, p-anisaldehyde elicited the strongest EAG response to D. minowai (p < 0.001) (Figure 2). cal analysis. Datasets that did not fit assumptions were square-root (sqrt) transformed to meet the requirements of equal variance and normality. The EAG relative value was calculated based on the following: EAG amplitude values of tested stimulus-the average EAG amplitude values of the control)/(the average EAG amplitude values of the reference compound-the average EAG amplitude values of the control) × 100%. Comparisons of recorded EAG values between different compounds were performed by one-way ANOVA following Tukey's HSD test (p < 0.05). In the behavioral trial, the t-test was applied to assess the number of thrip individuals between the treatment and control (p < 0.05). In the field evaluation, we performed a general linear model (GLM) analysis to assess the effect of various active compounds on thrips. Dunnett's one-tailed t-test was controlled for Type 1 experiment-wise error for comparisons of each compound against control (a Duncan test with α = 0.05). The graph of EAG relative value was performed using the GraphPad Prism 7.0. The graph of behavioral response and the field evaluation were performed using OriginPro 2021. All of the analyses were conducted using SAS 9.4.

EAG Response of D. minowai Adults to Single Compound
To comprehensively evaluate the electrophysiological activity, EAG responses of D. minowai to 20 compounds were tested.

Discussion
Plants constantly emit complex blends of volatile odors, which provide cues for herbivores [Error! Reference source not found.]. The application of plant volatiles to attract and kill pests is an important part of integrated pest management (IPM) strategies [Error! Reference source not found.]. In the future, the electrophysiological and behavioral response experiments in the laboratory are an important basis to test and utilize these volatile odors for trapping in field [Error! Reference source not found.]. We screened seven electrophysiological and behavioral active volatiles of D. minowai using EAG detection and H-tube olfactometer bioassays. Furthermore, four compounds significantly increase the trap captures of D. minowai in tea garden, and the trapping effect was relatively better than those of other thrips. This work enriched the research on plant-derived semiochemicals for use in attracting and killing non-flowering thrips, which will promote the development of the commercial attractant products.
Generally, the behavioral response of flower-inhabiting thrips has been studied extensively, indicating that flower-inhabiting thrips respond to pyridine and benzene [Er-

Discussion
Plants constantly emit complex blends of volatile odors, which provide cues for herbivores [37]. The application of plant volatiles to attract and kill pests is an important part of integrated pest management (IPM) strategies [38]. In the future, the electrophysiological and behavioral response experiments in the laboratory are an important basis to test and utilize these volatile odors for trapping in field [4]. We screened seven electrophysiological and behavioral active volatiles of D. minowai using EAG detection and H-tube olfactometer bioassays. Furthermore, four compounds significantly increase the trap captures of D. minowai in tea garden, and the trapping effect was relatively better than those of other thrips. This work enriched the research on plant-derived semiochemicals for use in attracting and killing non-flowering thrips, which will promote the development of the commercial attractant products.
Generally, the behavioral response of flower-inhabiting thrips has been studied extensively, indicating that flower-inhabiting thrips respond to pyridine and benzene [4,10], whereas the non-flowering thrips do not respond to these dilutions [39]. For example, methyl isonicotinate (MI) and p-anisaldehyde elicit stronger behavioral responses to more than 10 flower-inhabiting species of thrips [4,14,40]. Similar to previous reports, we found that non-flower-inhabiting D. minowai do not respond to MI and 4-acetylpyridine dilutions in this study. However, contrary to previous reports, it was demonstrated that D. minowai exhibited a highly significant preference to p-anisaldehyde and methyl benzoate dilutions in the present study. In addition, γ-terpinene emitted from tea plants had significant avoidance effects on D. minowai in the laboratory, which showed that the repellent properties of γ-terpinene to D. minowai would be helpful in the development of push-pull strategy.
At present, there are few reports regarding the electrophysiological trials of thrips. It is hypothesized that the difficulty of electrophysiological trials may be due to the small antenna of thrips. Coupled gas chromatography-electroantennogram detection (GC-EAD) showed that one component of male Megalurothrips usitatus specific odor namely (2E, 6E)farnesyl acetate elicited a significant electrophysiological response to M. usitatus [41]. In this study, we screened 11 synthetic compounds, including p-anisaldehyde, eugenol, farnesene, limonene, methyl isonicotinate, 3-methyl butanal, nonanal, (E)-β-ocimene, (−)-α-pinene, (+)-α-pinene, and γ-terpinene, that elicited a significant antennal response to female D. minowai. These results determine that D. minowai rely on more than one component to search for host plants, and multiple components work together in locating host plants. This phenomenon has been studied extensively in other herbivores. For example, three Adelphocoris species responded to seven compounds in EAG trials from 11 host plants [31].
Research on plant-derived semiochemicals for thrips could promote the development of efficient and novel measures as alternatives to synthetic insecticides [4,29]. Generally, non-pheromone semiochemicals can trap both sexes. However, in our trials, few male D. minowai were found to be attracted to traps. This could be due to the fact that male D. minowai fly fast, their behaviors are likely to be more complex, and they rely on many plant cues (color, odor, shape, etc.) [42]. In our trials, the maximum number of female D. minowai collected by blue sticky traps baited with p-anisaldehyde was 7.7 times higher than hexane. The trapping effect of p-anisaldehyde on D. minowai was relatively better than the other thrips, which suggested that p-anisaldehyde has a great application prospect. For example, significant 2.4-3.9 × increases in Frankliniella occidentalis numbers in traps with MI when compared with those without MI were found during the whole season in a nectarine orchard [6], and the number of F. occidentalis on the sticky cards baited with the commercial non-pheromone semiochemical product Lurem-TR could be increased by 4 times [15]. Occasionally, a specific proportion of compound mixture is more attractive than single volatiles [43][44][45]. For instance, traps with both ethyl nicotinate and ethyl isonicotinate lures caught 2-3 times higher numbers of adult New Zealand flower thrips than traps with ethyl isonicotinate only [46]. Binyameen et al. [47] observed a significant additive trapping effect for the response of onion thrips (Thrips tabaci) to eugenol and ethyl isonicotinate combined. Therefore, a certain fieldwork and laboratory evaluation is required on an urgent basis in the near future, which would provide theoretical reference for the development of D. minowai attractants.

Conclusions
In conclusion, we screened seven electrophysiological and behavioral active nonpheromone semiochemicals of D. minowai using EAG detection and H-tube olfactometer bioassays. In the fields, we demonstrated that the positive effect of p-anisaldehyde, eugenol, farnesene, and 3-methyl butanal as attractants of D. minowai caused increased efficiency in catching traps. In particular, the maximum number of D. minowai trapped by blue sticky traps baited with p-anisaldehyde was 7.7 times higher than the control. In the future, this