Aggregation pheromones released by nymphs of the stick tea thrips (Dendrothrips minowai): identification and analysis of their potential utility for controlling thrips in tea plantations

doi:10.21203/rs.3.rs-2718753/v1

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Aggregation pheromones released by nymphs of the stick tea thrips (Dendrothrips minowai): identification and analysis of their potential utility for controlling thrips in tea plantations

https://doi.org/10.21203/rs.3.rs-2718753/v1

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BACKGROUND

The stick tea thrips, Dendrothrips minowai Priesner, is one of the most important sucking pests that seriously infest tea plants (Camellia sinensis) in China. Obvious D. minowai nymph aggregation behaviors are typically detected in tea plantations. The objectives in our study were to identify D. minowai aggregation pheromones and evaluate their potential utility for controlling thrips infestations in tea plantations.

RESULTS

Dendrothrips minowai nymphs, females, and males were significantly attracted to nymph volatiles rather than the volatiles secreted by females and males in H-tube olfactometer assays under laboratory conditions. Our gas chromatography-mass spectrometry analysis of volatiles collected from D. minowai revealed that nymphs can produce dodecyl acetate and tetradecyl acetate. Additionally, the D. minowai females and males responded positively to dodecyl acetate, tetradecyl acetate, and the mixture (dodecyl acetate : tetradecyl acetate = 1 : 1.5) according to in behavioral experiments under laboratory conditions. Moreover, both aggregation pheromones and the mixture increased the number of D. minowai adults on sticky traps by 0.9- to 2.5-times (compared with the traps containing the solvent control) in tea plantations. Most importantly, at 10 days after adding sticky traps with D. minowai aggregation pheromones to tea plantations, the number of adult thrips per 100 leaves decreased by 57–75%, which was significantly higher than those of 39% decrease induced by the solvent control.

CONCLUSION

Dodecyl acetate and tetradecyl acetate were identified as D. minowai nymph-produced aggregation pheromones that can significantly attract conspecifics under laboratory conditions. More importantly, combining the aggregation pheromones with sticky traps can significantly decrease the thrips population in tea plantations. The findings of this study on thrips pheromones may be useful for developing new methods for managing thrips that do not require potentially harmful insecticides.

Dendrothrips minowai

aggregation pheromone

electrophysiological and behavioral responses

field trapping

Pheromones are chemicals secreted by insects to induce physiological or behavioral changes of the same or different species, which made them important semiochemicals for intraspecific and interspecific communication (Yan et al., 2013; Czaczkes et al., 2015; Seybold et al., 2018). The identified insect pheromones include sex pheromones, aggregation pheromones, trail pheromones, alarm pheromones, marking pheromones, and other types (Witzgallet al., 2010; Morrison et al., 2017; Magsi et al., 2022). Aggregation pheromones, which may be released by male or female insects and/or during immature stages (eggs, nymphs/larvae, and pupae), are attractants for both males and females (Wertheim et al., 2005). There has been considerable progress in the research on the functions of aggregation pheromones released by some pests. For example, aggregation pheromones function as important food source signals that indicate appropriate feeding sites for females, males, and nymphs (Leal et al., 1995; Mizutani et al., 2008), while also mediating sexual interactions (e.g., attracting a mate) (Milne et al., 2002; Niassy et al., 2016). Additionally, the aggregation pheromones of some insects serve as kairomones that attract their natural enemies, suggesting they may be useful for pest management (Mizutani, 2006; Alim & Lim, 2011; Lim & Mainali, 2013).

Aggregation pheromones are some of the most widely studied pheromones among thrips (Hamilton et al., 2005; Li et al., 2019b; Kirk et al., 2021). The aggregation pheromones secreted by the following five thrips were recently identified: Frankliniella occidentalis (Hamilton et al., 2005), Frankliniella intonsa (Zhu et al, 2012), Thrips palmi (Akella et al., 2014), Megalurothrips usitatus (Li et al., 2019a; Liu et al, 2020), and Megalurothrips sjostedti (Niassy et al., 2019). The identified aggregation pheromones are all terpenoid esters or monoterpene alcohols, most of which are chiral compounds (Li et al., 2019b). Moreover, these thrips aggregation pheromones are all produced by males (Kirk et al., 2021). Notably, aggregation pheromones have been used for the mass trapping of thrips (Broughton et al., 2015; Kirk, 2017; Cruz-Esteban et al., 2020). More specifically, using lures containing neryl (S)-2-methylbutanoate doubled the number of trapped thrips and decreased the number of F. occidentalis adults per flower by 73% in strawberry fields in the UK (Sampson and Kirk, 2013). The aggregation pheromone of F. occidentalis had sold commercially as Thripline™ AMS (Bioline Agrosciences Ltd.; formerly known as Syngenta Bioline Ltd.) and ThriPher (Biobest Belgium N.V.) (Kirk, 2017).

The stick tea thrips (Dendrothrips minowai) is the main pest infesting tea (Camellia sinensis) plantations in China (Lyu et al., 2016; Li et al., 2020). There has recently been an increase in the occurrence of D. minowai infestations, with annual increases in the severity of the associated damage, which has resulted in serious crop losses (Luo et al., 2022; Xiu et al., 2022). However, current methods for controlling this thrips mainly rely on pesticide applications, which can seriously decrease tea quality and safety (Tewary et al., 2005; Ye et al., 2014; Nakai & Lacey, 2017). The aggregated distribution of D. minowai nymphs and females on tea leaves has been studied in detail (Zhang et al., 2019). Our analyses revealed a lack of aggregated D. minowai males in tea gardens, which differs from those of male aggregation observed for other thrips. Unfortunately, the active components of D. minowai aggregation pheromones remain unknown. In this study, behavioral assays were first conducted to characterize the responses of D. minowai nymphs, females, and males to odors released by them using a H-tube olfactometer under laboratory conditions, respectively. Furthermore, specific components were collected and identified from volatiles released by nymphs. At last, the roles of the corresponding synthetic compounds were evaluated under laboratory and field conditions. This research may be relevant for improving commercial traps using pheromones to control thrips populations in tea gardens.

2.1 Biological materials and compounds

Plants and Insects. Tea seedlings (cultivar: Longjing 43) were grown in rectangular plastic pots at the Tea Research Institute, Chinese Academy of Agricultural Sciences in Hangzhou, Zhejiang province, China (120.10°E, 30.19°N). Dendrothrips minowai adults were collected from the tea gardens of Shaoxing Royal Tea Village Co., Ltd. in Zhejiang province, China (120.71°E, 29.94°N) and then reared on cultivated tea seedlings in a climate-controlled chamber set at 24 ± 1°C with a 16-h light/8-h dark cycle and 60 ± 5% relative humidity. Nymphs and adults of the second generation were used for experiments.

Chemicals. The dodecyl acetate (95%) and tetradecyl acetate (98%) used for the electroantennogram (EAG) verification and behavioral tests were purchased from TCI (Shanghai, China) Chemical Industry Development Co., Ltd. and Alfa Aesar (China) Chemical Co., Ltd. (Shanghai, China), respectively. Paraffin oil (Sigma-Aldrich, St. Louis, MO, USA) was used as the solvent for the EAG, behavioral tests and the trapping trials.

2.2 Olfactometer bioassays

To determine the effects of D. minowai volatiles, the behavioral responses of D. minowai adults to volatiles secreted by nymphs, females, and males were tested using modified glass H-tube olfactometers (Xiu et al., 2022). To prevent thrips from escaping, both ends of the transverse tube connected to the two straight tubes were sealed with gauze (200 mesh). Before testing, the H-tube olfactometers were cleaned with ethanol and dried for 1 h at 90°C in an electric constant-temperature drying oven (Shanghai Sumsung Laboratory Instrument Co., Ltd., Shanghai, China). Treatments comprised volatiles from 200 female thrips, 200 male thrips, or 200 third instar nymphs as the olfactory cues, with air used as the control. Before the assays, the third instar nymphs and 1- to 5-day-old unmated adults were starved for 1 h. For each test, 20 individuals were transferred to the middle of the transverse tube with a soft brush and then acclimated for 10 min. At 1 and 2 h after initiating the test, the tubes were examined to determine if the thrips crossed the halfway point of the left or right arm of the transverse tube (i.e., a choice was made). Alternatively, the thrips were considered to have not made a choice if they did not cross the halfway point of the left or right arm. We recorded the total number of thrips that chose the left or right arm or neither arm (i.e., no choice). For each treatment, the tests were performed 10 times using a total of 200 D. minowai nymphs, females, and males. The tests were conducted in a darkened room set at 26 ± 1°C and 65 ± 1% relative humidity. All bioassays were conducted between 8:00 and 18:00, which coincides with the period of their activity in tea plantations (Zhang et al., 2023).

2.3 Collection and identification of thrips volatiles

Thrips volatiles were collected for 2 h via solid phase microextraction (Hamilton et al., 2005; Zhang et al., 2011). Specifically, 200 third instar nymphs were transferred to a 1.5 mL transparent glass vial (Agilent Technologies, Palo Alto, CA, USA) using a soft brush. Before collecting the thrips volatiles, the blank fiber was heated in the GC inlet for 30 min to prevent contamination. Next, a cleaned fiber (50/30 µm DVB/CAR/PDMS) (57348-U; Supelco, Poole, UK) was introduced into the 1.5 mL transparent glass vial to absorb the volatiles secreted by the nymphs (Niassy et al., 2019; Liu et al., 2020). The glass vial were illuminated from above by a 100 W tungsten filament lamp to induce patrolling behavior at 25 ± 1°C (Kirk and Hamilton, 2004; Akella et al., 2014). The volatiles collected from the empty 1.5 mL transparent glass vial served as the blank control. Volatiles were collected from three biological replicates.

The collected thrips volatiles and the blank control were analyzed using a GC-MS system (7890A GC and 5973 MS) that included an HP-5MS column (30 m × 0.25 mm internal diameter × 0.25 µm film thickness) (Agilent Technologies). The GC conditions were as follows: injection temperature of 250°C; detector temperature of 280°C; column temperature program of 40°C for 5 min, increase to 160°C at 2°C/min, increase to 250°C at 30°C/min, and maintain for 5 min. The MS analysis was performed under the following conditions: the ionization mode was set at EI 70 eV, with helium used as the carrier gas at an average flow rate of 1 mL/min; source moderate transmission and line temperatures were both set at 250°C; scan range of 35–650 amu; scanning speed of 1,000 amu/s. The volatiles specific to nymphs, which were revealed during comparisons with the volatiles from the females, males, and the blank control, were identified by comparing their mass spectra with those of authenticated samples in the NIST 2005 database. They were further confirmed by injecting commercially available synthetic chemical standards. The ratio of two aggregation pheromones was calculated by comparing their peak areas.

2.4 EAG trials

The EAG method was used to test the responses of D. minowai female and male adults to odors. The head of 1- to 5-day-old virgin D. minowai female and male adults was cut off using a scalpel. Then, the head was fixed to a reference electrode using a glass capillary, which filled with a solution consisting of 128 mM NaCl, 1.9 mM CaCl₂, 7.6 mM KCl, and 2.4 mM NaHCO₃. Both antenna tips were placed in a recording electrode filled with the solution (Luo et al., 2017). The detached antennae were mounted between the fork-shaped metal electrodes of the EAG probe (PRG-3 EAG Combi-probe; Syntech, Hilversum, The Netherlands) using an electrically conductive gel (Spectra 360 electrode gel; Parker Laboratories Inc.). The electrode at the distal end of the antennae was connected to a signal acquisition system (IDAC-2; Syntech), which was controlled using the EAG software (version 2.6) (Syntech). The prepared antennae were exposed to the main air stream (charcoal-filtered and humidified air at 200 mL/min) through an L-shaped glass tube (13 mm inner diameter and 17 mm outer diameter) (approximately 5 mm from the antennae). A filter paper strip (0.5 cm × 5 cm) containing an odorant solution was inserted in a glass Pasteur pipette (150 mm long; Volac®) to produce an odor cartridge, which was fixed to a small hole (3 mm diameter) on the other side of the L-shaped glass tube. An air stimulus controller (CS-55; Syntech) was used to generate air flow (300 mL/min for 0.5 s) that passed the odor cartridge before entering the main air stream. Both dodecyl acetate and tetradecyl acetate were dissolved in paraffin oil and then serially diluted (0.001, 0.01, 0.1, 1, and 10 µg/µL), after which a 10 µL aliquot was added to a filter paper strip (0.5 cm × 5 cm) that was placed inside a glass Pasteur pipette (Magsi et al., 2022). The odorants were used for the subsequent EAG assay, with paraffin oil serving as the blank control. A continuous flow of clean air produced by the CS-55 stimulus controller and passed through charcoal and distilled water was provided to the antennae at a rate of 300 mL/min. Each recording trial was conducted using the following series of stimuli: paraffin oil, odorant (0.001, 0.01, 0.1, 1, and 10 µg/µL), and paraffin oil. For each stimulus, the treatment duration was 0.5 s with intervals of 30 s. For both dodecyl acetate and tetradecyl acetate, four individuals per sex were tested.

2.5 Behavioral responses of D. minowai to synthetic components

Behavioral responses of D. minowai to dodecyl acetate and tetradecyl acetate were also evaluated using H-tube olfactometers. Briefly, 20 µL aliquots of chemical stimuli (10 µg/µL dodecyl acetate and tetradecyl acetate diluents) and a blank control (paraffin oil as the solvent) were added to separate filter discs (diameter: 50 mm) fitted to the bottom of the straight tube of olfactometers as the odor source. The olfactometers, methods, and protocols were as described above. For each component, the tests were performed 10 times using 200 females and males, respectively.

2.6 Trapping experiments

Field trials were conducted in the tea plantations of Shaoxing Royal Tea Village Co., Ltd. in Zhejiang province, China (120.71°E, 29.94°N) from 31 May to 27 June 2022 to evaluate the biological effects of dodecyl acetate and tetradecyl acetate. The rubber septa (Zhangzhou Ingeer Agricultural Science and Technology Co., Ltd., Fujian province, China) containing synthetic aggregation pheromones [100 µL dodecyl acetate diluent (100 µg/µL), 100 µL tetradecyl acetate diluent (100 µg/µL), and a mixture containing 40 µL dodecyl acetate diluent and 60 µL tetradecyl acetate diluent based on the ratio of the two components thrips released] were attached to the blue sticky traps made of PVC (10 cm × 25 cm; Hangzhou Yihao Agricultural Technology Co., Ltd., Zhejiang province, China), suspending on poles with the base approximately 10 cm above tea plants and 6 m separating each trap. In addition, rubber septa loaded with 100 µL hexane were used as the control. For each treatment (dodecyl acetate, tetradecyl acetate, mixture of dodecyl acetate and tetradecyl acetate, and solvent control), four replicates were analyzed. The sticky traps containing different lures were randomly assigned to different parts of the tea garden. Before the trapping trials, sticky traps without any lures were placed in the tea garden for 3 days to confirm that the thrips populations were uniformly distributed in the different treatment areas (Cai et al. 2017; Xiu et al. 2022). The number of D. minowai adults on the sticky traps was recorded every 3 days, after which the sticky traps were replaced, with the rubber septa attached to the new sticky traps. New lures were added after 12 days. To avoid spatial effects, the traps were randomly repositioned each time after the number of adults was recorded. The traps with compounds were compared with the control traps in terms of the number of trapped thrips.

2.7 Evaluation of the control effect

Four sites were selected for field control assays, with each site (30 m long) containing 10 rows of tea plants. The relative densities of D. minowai adults were determined during the initial surveys (27 June 2022). Sites were separated by 20 m and located in the tea gardens of Shaoxing Royal Tea Village Co., Ltd. The sites were randomly selected for treatments with dodecyl acetate, tetradecyl acetate, the mixture, or hexane. Each treatment involved three replicates. The rubber septa, dosages of synthetic aggregation pheromones in the rubber septa, and the blue sticky traps were the same as those used for trapping trials, but unlike the trapping trials, the traps were separated by 2 m. At 3, 7, and 10 days after adding the sticky traps with lures, the population quantities were monitored. The traps were replaced for each survey, but the lures were retained and attached to the new traps.

2.8 Statistical analysis

All data were checked for normality and equality of variances prior to the statistical analysis. Datasets that did not fit the assumption of homogeneity of variance for the analysis were log-transformed [log₁₀(x + 1)]. For the H-tube olfactometer bioassays, the t-test was performed to determine the significance of the difference in the number of thrips between the treatment and control (P < 0.05). Unresponsive individuals were not included in the analysis, but they were listed at the end of the data bars. The EAG amplitudes (mean ± SE) were control-adjusted and visualized (average value and concentration-dependent response curve). The differences in the EAG responses among the diluents were compared by a one-way ANOVA followed by Tukey’s HSD post hoc analysis. For the field trapping trials and the evaluation of the control effect, data were compared by a one-way ANOVA followed by Duncan’s multiple range test (P < 0.05). Graphs were drawn using GraphPad Prism 7.0 and OriginPro 2021. The SAS 9.1 program was used for statistical analyses.

3.1 Odorants produced by D. minowai nymphs attracted conspecifics

In the H-tube olfactory bioassays, the volatiles from 200 nymphs significantly attracted D. minowai nymphs, females, and males at both 1 and 2 h (nymphs: 1 h: F_{1, 18} = 42.88, P < 0.001; 2 h: F_{1, 18} = 78.80, P < 0.001; females: 1 h: F_{1, 18} = 81.85, P < 0.001; 2 h: F_{1, 18} = 80.00, P < 0.001; males: 1 h: F_{1, 18} = 99.80, P < 0.001; 2 h: F_{1, 18} = 79.79, P < 0.001) (Fig. 1A). However, the volatiles from the D. minowai females did not attract conspecifics (nymphs: 1 h: F_{1, 18} = 0.43, P = 0.521; 2 h: F_{1, 18} = 0.507, P = 0.486; females: 1 h: F_{1, 18} = 0.25, P = 0.627; 2 h: F_{1, 18} = 1.28, P = 0.272; males: 1 h: F_{1, 18} = 1.39, P = 0.254; 2 h: F_{1, 18} = 2.75, P = 0.114) (Fig. 1B). In addition, the volatiles from D. minowai males did not attract nymphs or females (nymphs: 1 h: F_{1, 18} = 2.69, P = 0.118; 2 h: F_{1, 18} = 1.22, P = 0.284; females: 1 h: F_{1, 18} = 3.27, P = 0.087; 2 h: F_{1, 18} = 1.95, P = 0.180), but they did significantly repel other males (1 h: F_{1, 18} = 56.13, P < 0.001; 2 h: F_{1, 18} = 97.59, P < 0.001) (Fig. 1C).

3.2 Two specific compounds released by D. minowai nymphs were identified via the GC-MS analysis

The detailed comparison of TIC chromatograms revealed two specific peaks in the GC profiles of D. minowai nymphs. Screening the NIST library using the corresponding mass spectra as queries suggested the specific compounds were dodecyl acetate (55.03 min) and tetradecyl acetate (65.49 min) (Fig. 2). We further validated these compounds by comparing their retention times and mass spectra with those of the standards analyzed under the same conditions. The retention times of the natural compounds were identical to those of the synthetic dodecyl acetate and tetradecyl acetate (Fig. S1). Moreover, the mass spectra were similar (Fig. S2). On the basis of the peak areas, the ratio of dodecyl acetate to tetradecyl acetate was approximately 1:1.5 (Table S1).

3.3 Dodecyl acetate and tetradecyl acetate elicited significant EAG responses

Compared with the effects of paraffin oil, the D. minowai females and males exhibited concentration-dependent EAG responses to the aggregation pheromones at five concentrations (0.001, 0.01, 0.1, 1, and 10 µg/µL) (dodecyl acetate: female: F_{5, 18} = 58.41, P < 0.001; male: F_{5, 18} = 21.97, P < 0.001; tetradecyl acetate: female: F_{5, 18} = 22.07, P < 0.001; male: F_{5, 18} = 30.43, P < 0.001) (Fig. 3). Additionally, there were significant differences in the EAG amplitudes for the two components at different concentrations (Fig. 3A, B). Specifically, for D. minowai females and males, EAG amplitudes for dodecyl acetate were the highest at 1 µg/µL (Fig. 3A), whereas the EAG amplitudes for tetradecyl acetate peaked at 10 µg/µL (Fig. 3B).

3.4 Dodecyl acetate, tetradecyl acetate, and the mixture attracted D. minowai females and males

The responses of female and male D. minowai to aggregation pheromones and the blank control significantly differed (Fig. 4a, b). Specifically, more thrips were attracted to dodecyl acetate, tetradecyl acetate, or the mixture than those of the control. This was observed for both females (dodecyl acetate: 1 h: F_{1, 18} = 141.06, P < 0.001; 2 h: F_{1, 18} = 153.39, P < 0.001; tetradecyl acetate: 1 h: F_{1, 18} = 81.68, P < 0.001; 2 h: F_{1, 18} = 52.57, P < 0.001; mixture: 1 h: F_{1, 18} = 193.29, P < 0.001; 2 h: F_{1, 18} = 233.73, P < 0.001) (Fig. 4a) and males (dodecyl acetate: 1 h: F_{1, 18} = 158.76, P < 0.001; 2 h: F_{1, 18} = 212.33, P < 0.001; tetradecyl acetate: 1 h: F_{1, 18} = 161.05, P < 0.001; 2 h: F_{1, 18} = 180.99, P < 0.001; mixture: 1 h: F_{1, 18} = 200.08, P < 0.001; 2 h: F_{1, 18} = 95.06, P < 0.001) (Fig. 4b).

3.5 The addition of lures with pheromones increased the number of D. minowai adults in traps

Before the lures were attached to the sticky traps, there were no differences in the number of D. minowai adults on sticky traps (F_{3, 12} = 2.13, P = 0.150) (Fig. 5). In contrast, the sticky traps with lures containing dodecyl acetate, tetradecyl acetate, or the mixture caught more D. minowai adults than the control sticky traps with lures containing hexane. The lures were renewed once during the trapping trials. Notably, compared with the control, the lures that combined dodecyl acetate and tetradecyl acetate (1:1.5) consistently trapped more D. minowai adults (6 June 2022: F_{1, 6} = 6.82, P = 0.040; 9 June 2022: F_{1, 6} = 30.88, P = 0.0014; 13 June 2022: F_{1, 6} = 24.74, P = 0.003; 16 June 2022: F_{1, 6} = 17.08, P = 0.0061; 19 June 2022: F_{1, 6} = 7.76, P = 0.032; 23 June 2022: F_{1, 6} = 282.04, P < 0.001; 27 June 2022: F_{1, 6} = 20.34, P = 0.004). Furthermore, the sticky traps with lures containing dodecyl acetate and tetradecyl acetate captured 1.8-, 2.4-, 2.5-, 1.6-, 1.9-, 2.1-, and 2.4-times more thrips than the control traps on 6, 9, 13, 16, 19, 23, and 27 June 2022, respectively (Fig. 5).

3.6 Decreased densities of D. minowai adults in pheromone-treated areas

At the beginning of the field assays, there were no major differences in the number of D. minowai adults per 100 leaves among the treatment sites (dodecyl acetate: 203.3 ± 10.26; tetradecyl acetate: 196.0 ± 12.12; mixture: 209.3 ± 18.01; control: 214.3 ± 36.47) (F_{3, 8} = 0.91, P = 0.478) (Fig. 8). At 10 days after the addition of sticky traps with lures containing aggregation pheromones, the number of D. minowai adults per 100 leaves was significantly lower for the pheromone-treated sites (dodecyl acetate: 84.7 ± 5.51; tetradecyl acetate: 84.7 ± 2.52; mixture: 52.3 ± 4.16) than for the control site (135.7 ± 7.09) (F_{3, 8} = 136.56, P < 0.001). Thus, the number of adult thrips per 100 leaves decreased by 58%, 57%, and 75% for the traps with dodecyl acetate, tetradecyl acetate, and the mixture, respectively, which was greater than the decrease induced by the control trap (i.e., 39%) (Table 1).

Table 1

*Dendrothrips minowai* population dynamics determined using sticky traps containing synthetic aggregation pheromones and the solvent control in a tea garden at Shaoxing from 27 June to 7 July 2022
Treatments	No. of thrips before traps placed	3 days after traps placed		7 days after traps placed		10 days after traps placed
Treatments	No. of thrips before traps placed	No.of thrips	Reduced percent (%)	No.of thrips	Reduced percent (%)	No.of thrips	Reduced percent (%)
Dodecyl acetate	203.3 ± 10.26 a	156.7 ± 17.01 a	23.09 ± 4.692	122.3 ± 7.09 a	39.84 ± 1.311	84.7 ± 5.51 a	58.38 ± 0.723
Tetradecyl acetate	196.0 ± 12.12 a	132.3 ± 7.51 a	32.47 ± 0.526	111.0 ± 1.73 a	43.23 ± 3.513	84.7 ± 2.52 a	56.64 ± 3.916
Mixtures	209.3 ± 18.01 a	142.7 ± 14.19 a	31.11 ± 12.871	66.7 ± 10.79 b	68.26 ± 2.739	52.3 ± 4.16 b	74.98 ± 0.689
Solvent control	214.3 ± 36.47 a	209.3 ± 15.57 b	5.66 ± 9.638	166.7 ± 10.6 c	24.65 ± 10.170	135.7 ± 7.09 c	38.68 ± 7.922
Note: Data listed are mean ± SEM. A one-way ANOVA followed by Duncan’s multiple range test were performed to analyze the data. Different letters indicate significant differences in the number of thrips between treatments.

In earlier studies, researchers identified five thrips species that form male aggregations, while also isolating the corresponding aggregation pheromones secreted by the males (Milne et al., 2002, Niassy et al., 2016). However, D. minowai nymphs and females form aggregations on tea leaves, but the males do not. A recent study indicated the D. minowai nymphs aggregate more than the females in tea plantations (Zhang et al., 2023). In addition, the aggregation pheromones may be secreted by both sexes and/or during the immature stages (eggs, nymphs/larvae, and pupae) (Wertheim et al., 2005). We hypothesized that the aggregation pheromones of D. minowai may be secreted by nymphs or females. Thus, we conducted behavioral assays using nymphs and females as the sources of odorants under laboratory conditions. We determined that D. minowai nymphs, females, and males were all attracted to the volatiles produced by nymphs, but not to the volatiles secreted by females and males. Accordingly, we analyzed the volatiles from nymphs and identified two specific components that attracted D. minowai adults under laboratory and field conditions. The results of this study were inconsistent with the findings of earlier research on other thrips. Hence, the data presented herein may be relevant for designing new pest management strategies for controlling thrips in tea gardens.

Alarm pheromones are another type of pheromone detected in the anal droplets of thrips nymphs (Teerling et al., 1993a; MacDonald et al., 2003; Suzuki et al., 2004). For example, F. occidentalis anal droplets contain the alarm pheromones decyl acetate (10Ac) and dodecyl acetate (12Ac) (Teerling et al., 1993b; MacDonald et al., 2003). These alarm pheromones repel walking nymphs and adults from the host plants, while also decreasing the oviposition rate of adult females (MacDonald, 2002). In the current study, we identified dodecyl acetate as one of the compounds secreted by D. minowai nymphs (Fig. 2, 3, 4). However, dodecyl acetate significantly attracted D. minowai adults (Fig. 6), which is in contrast to the defensive effect of dodecyl acetate on F. occidentalis adults. We speculated that dodecyl acetate is not an alarm pheromone for D. minowai. Earlier research showed aggregation pheromones can attract both males and females (Zhang et al., 2011; Krik et al., 2021). Therefore, dodecyl acetate was defined as an aggregation pheromone. Although some components of the anal droplets or anal secretions of several species have been identified, relatively few have been functionally characterized (Krik et al., 2021). For example, tetradecyl acetate has been detected in the anal secretions of Leeuwenia pasanii, Xylaplothrips inquilinus, and four Liothrips species, but their functions remain unknown (Suzuki et al., 2004). In the present study, tetradecyl acetate was designated as an aggregation pheromone because it was observed to attract D. minowai adults.

Thrips aggregation pheromones have been added to colored sticky traps to evaluate their biological effects in the field (Cruz-Esteban et al., 2020; Mfuti et al., 2021). In a previous investigation involving a rose greenhouse, 1.9- to 4.2-times more F. occidentalis insects were caught by sticky traps containing Thripline™ AMS (male sex aggregation pheromone) than by unbaited traps over a 2-week period (Broughton et al., 2015). In addition, compared with unbaited traps in a blackberry field, the use of blue and yellow traps containing the aggregation pheromone neryl (S)-2-methylbutanoate increased the number of captured F. occidentalis insects by 2.5- to 3-times (Cruz-Esteban et al., 2020). Consistent with the results of these previous studies, 0.9- to 2.5-times more D. minowai adults were caught by sticky traps baited with dodecyl acetate, tetradecyl acetate, and the mixture than by the solvent-containing control traps. Moreover, researchers had tested whether mass trapping can decrease thrips-induced damages. The mass trapping of F. occidentalis in a strawberry field using blue sticky traps baited with neryl (S)-2-methylbutanoate reportedly decreases the number of adult pests per flower by 73% as well as fruit bronzing by 68% (Sampson & Kirk, 2013). Similarly, we found that the mass trapping of D. minowai using blue sticky traps containing dodecyl acetate, tetradecyl acetate, and the mixture respectively decreased the number of D. minowai adults per 100 leaves by 58%, 57%, and 75% in tea gardens.

Some studies revealed differences in the efficacy of the major and minor aggregation pheromones for trapping F. occidentalis. Specifically, field trials involving two aggregation pheromones showed that the major compound alone caught the most insects, with no evidence of the synergism between aggregation pheromones in Spain (Hamilton et al., 2005; Sampson, 2014). However, synergistic effects on F. intonsa were detected when the minor aggregation pheromone (R)-lavandulyl acetate was added to the main pheromone components (Geng et al., 2017). In the present study, mixing dodecyl acetate with tetradecyl acetate (1:1.5) increased the number of trapped insects (Fig. 7, Table 1). Moreover, the addition of both compounds to the traps resulted in the fewest D. minowai adults per 100 leaves on days 3, 7, and 10 of the trapping trials. In addition to increasing the number of trapped insects, the aggregation pheromones have other effects. For example, they can enhance walking activities, influence mating behaviors, and attract predators (Olaniran, 2012; Walsh et al., 2016; Vaello et al., 2017). Although some thrips aggregation pheromones have been identified, synthesized, and explored, their functions have not been comprehensively determined, unlike the corresponding pheromones in other pests (Kirk et al., 2021). Accordingly, functionally characterizing thrips aggregation pheromones is critical for optimizing their utility for controlling insect pests.

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (32001918) and the China Agriculture Research System (CARS-19).

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Aggregation pheromones released by nymphs of the stick tea thrips (Dendrothrips minowai): identification and analysis of their potential utility for controlling thrips in tea plantations

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Abstract

BACKGROUND

RESULTS

CONCLUSION

Figures

1. Introduction

2. Materials And Methods

2.1 Biological materials and compounds

2.2 Olfactometer bioassays

2.3 Collection and identification of thrips volatiles

2.4 EAG trials

2.5 Behavioral responses of D. minowai to synthetic components

2.6 Trapping experiments

2.7 Evaluation of the control effect

2.8 Statistical analysis

3. Results

3.1 Odorants produced by D. minowai nymphs attracted conspecifics

3.2 Two specific compounds released by D. minowai nymphs were identified via the GC-MS analysis

3.3 Dodecyl acetate and tetradecyl acetate elicited significant EAG responses

3.4 Dodecyl acetate, tetradecyl acetate, and the mixture attracted D. minowai females and males

3.5 The addition of lures with pheromones increased the number of D. minowai adults in traps

3.6 Decreased densities of D. minowai adults in pheromone-treated areas

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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