A push-pull strategy to control the western flower thrips, Frankliniella occidentalis, using alarm and aggregation pheromones

Chul-Young Kim; Falguni Khan; Yonggyun Kim

doi:10.1371/journal.pone.0279646

. 2023 Feb 24;18(2):e0279646. doi: 10.1371/journal.pone.0279646

A push-pull strategy to control the western flower thrips, Frankliniella occidentalis, using alarm and aggregation pheromones

Chul-Young Kim ¹, Falguni Khan ¹, Yonggyun Kim ^1,^*

Editor: Ramzi Mansour²

PMCID: PMC9956899 PMID: 36827422

Abstract

Since the first report in 1993 in Korea, the western flower thrips, Frankliniella occidentalis, has been found in various crops throughout the country. Although more than 20 different chemical insecticides are registered to control this insect pest, its outbreaks seriously damage crop yields, especially in greenhouses. This study developed a non-chemical technique to control F. occidentalis infesting hot peppers cultivated in greenhouses. The method was based on behavioral control using an alarm pheromone (“Push”) to prevent the entry of the thrips into greenhouses and an aggregation pheromone (“Pull”) for mass trapping inside the greenhouses. The greenhouse fences were treated with a wax formulation of the alarm pheromone and a yellow CAN trap covered with sticky material containing the aggregation pheromone was constructed and deployed inside the greenhouses. Field assay demonstrated the efficacy of the push-pull tactics by reducing thrips density in flowers of the hot peppers as well as in the monitoring traps. Especially, the enhanced mass trapping to the CAN trap compared to the conventional yellow sticky trap led to significant reduction in the thrips population. This novel push-pull technique would be applicable to effectively control F. occidentalis in field conditions.

1. Introduction

The western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is one of the most devastating insect pests to many horticultural crops including more than 500 species spanning 50 plant families, especially those cultured in greenhouses [1–3]. Both the larval and adult stages cause damage to plants by directly feeding on leaves or flowers [4, 5]. Especially, adults transmit plant viruses including tomato spotted wilt virus (TSWV) [6, 7]. TSWV infections have become serious in several horticultural products in Korea, including the high-value hot pepper crop [8]. This pest, originally native to North America, has spread to more than 60 countries since the late 1970s, including Europe, Asia, and Australia [9]. It was first observed on Jeju Island in 1993 and now has been spread to most regions in Korea [10].

Different reproductive modes along with a short lifecycle allow the thrips population to rapidly increase during early and late seasons, in which female progeny develop from fertilized eggs while males develop from unfertilized eggs [11, 12]. The nutrient-based oogenesis of F. occidentalis is well-programmed in females by gonadotropic hormones including juvenile hormone, ecdysone, and prostaglandin [13, 14]. To suppress thrips outbreaks, more than 20 kinds of chemical insecticides including organophosphate, carbamate, pyrethroids, neonicotinoids, avermectin, and spinosad are registered and applied to different agricultural crops [15]. However, the control efficacy against F. occidentalis is often unsatisfactory due to its cryptic ability to avoid exposure to the chemical spray, and the development of insecticide resistance [16]. A resistant population to emamectin benzoate showed a 356-fold increased resistance by a specific mutation in the target site and an over-expression of a detoxifying enzyme compared to a susceptible strain in Korea [17]. As alternative control tactics, the use of natural enemies, microbial pathogens, and other biological control agents has been proposed under an integrated insect pest management (IPM) program against F. occidentalis [18]. In this IPM program, behavioral control using pheromones has been considered in the monitoring and mass trapping of thrips [19].

Chemical communication using pheromones plays a crucial role in mediating F. occidentalis populations [9]. A contact pheromone, 7-methyltricosane produced by male adults, acts as a territorial signal by eliciting a fight signal to other males or gives the location of a male to females [20]. Kirk and Hamilton [21] suggested the presence of male-producing sex pheromones attracting only females. The aggregation pheromone of F. occidentalis was identified as containing two components, neryl methylbutanoate (NMB) and lavandulyl acetate (LA) [22]. Kirk et al. [23] suggested another type of chemical communication in mating, an anti-aphrodisiac pheromone produced by males during mating to prevent the multiple mating of females [24]. F. occidentalis larvae release an alarm pheromone in an anal droplet containing decyl acetate (10:Ac) and dodecyl acetate (12:Ac), in which the two components are present in a relative mass ratio of 0.4:1 to 1.1:1, respectively [25]. This alarm signal promotes the vigilance of thrips as well as takeoff or refuge-seeking behaviors to survive predator attacks [26]. Moreover, the relative ratio of the two components and their total amounts vary with the hazard intensity by increasing the relative amount of 12:Ac or the total alarm pheromone concentration in the droplet, suggesting that the alarm signal is context-dependent [27].

Using aggregation and alarm pheromones, this study devised a push-pull control strategy against F. occidentalis in greenhouse conditions. Fence treatment with alarm pheromone was used to prevent the entry of thrips into the greenhouse while an aggregation pheromone was used for mass trapping inside the greenhouse. To investigate this control strategy, we tested the active pheromone compounds and determined their blend ratio in the laboratory. For field assays, both pheromones were formulated for sustainability in field conditions. Finally, the push-pull tactic was applied to a specific farm without chemical sprays and the control efficacy was compared to that of farms that followed a conventional insecticide spraying program for thrips.

2. Materials and methods

2.1. Thrips rearing

A laboratory colony of F. occidentalis that originated from a local population in Jeonju in Korea was donated by the Rural Development Administration (Jeonju, Korea). It was maintained under 25 ± 1⁰C constant temperature and a 16:8 h (L:D) photoperiod with a relative humidity of 60 ± 5%. Another laboratory colony of F. intonsa was obtained from a field collection in a hot pepper field in Geosan (N36.81 E127.78, Korea) and reared in the laboratory conditions. All thrips were fed a kidney bean (Phaseolus coccineus L.) diet according to the method described in our earlier study [12].

2.2. Pheromones

Lavandulyl acetate (LA), lavandulyl methylbutanoate (LMB), and neryl methylbutanoate (NMB) were synthesized (AD, Inc., Andong, Korea) (S1 Fig). Briefly, LA and LMB were synthesized from the precursor, lavandulol (≥ 90%, Sigma-Aldrich Korea, Seoul, Korea) in dichloromethane, which was reacted with acetyl chloride to yield LA, and reacted with isovaleryl chloride (≥ 98%, Sigma-Aldrich Korea, Seoul, Korea) to yield LMB, respectively, under the catalytic activity of trimethylamine. NMB was used in a reaction with geraniol (> 98%, Sigma-Aldrich Korea) and isovaleryl chloride under the catalytic activity of trimethylamine. All products were extracted with ethyl acetate and produced 70 ~ 75% yields. The purity of the compounds was analyzed by gas chromatography (8860 GC, Agilent, Santa Clara, CA, USA) with a DB-1 column (15 m × 0.350 mm, Agilent), an oven temperature of 280°C, and a flow rate of 1.0 mL/min, and was determined to be 98.1% for LA, 98.1% for LMB, and 99.3% for NMB. All synthesized products were in the form of a racemic mixture. Two alarm pheromone components (decyl acetate and dodecyl acetate, 98%) were purchased from Sigma-Aldrich Korea and dissolved in hexane to prepare different concentrations.

2.3. Construction of CAN trap

The CAN trap consisted of a short supporting bar (1 m), an empty can (diameter 17 cm and height 18.5 cm), and a sticky plastic bag (see Fig 2). The short supporting bar and the empty can were connected with nails and attached to another long supporting bar (1 m) at an appropriate position for the trapping height. To mix the pheromone with sticky material, 2 mg of pheromone components and 12 g of sticky material (Tanglefoot, Contech Electronics, Rochester, NY, USA) were dissolved in 6 mL of hexane. This mixture was then poured to a sticky plastic zipper bag (30 × 30 cm, HG Pack, Gunpo, Korea). After spreading the sticky mixture with a roller, the bag was used to cover the can by inside-out inversion.

2.4. Wax formulation of alarm pheromone

The alarm pheromone (a mixture of decyl acetate and dodecyl acetate in 1.5:1, g/g) was formulated in wax by the method of Kim et al. [28]. Briefly, paraffin wax (Merck, Rahway, NJ, USA) was dissolved and mixed with an emulsifier (Almax 3600, Illshinwells, Seoul, Korea), α-tocopherol (96%), and jojoba oil (see Fig 5A). After the alarm pheromone was added, the slurry was cooled to solidify.

Fig 5 — (A) Composition of the wax formulation of the repellent and installation of the wax formulations (in a small cage) in five directions around a test plant. (B) Effect of the repellent on preventing immigrating thrips for two weeks. Three asterisks above the standard deviation bars indicate a significant difference between two treatments at Type I error = 0.0001 (LSD test). (C) Determination of the effective period of the wax formulation in field conditions. Different letters above the standard deviation bars indicate a significant difference among means at Type I error = 0.05 (LSD test).

2.5. Aggregation behavior test using Y-tube olfactometer

A Y-type olfactometer (the main Y-tube length, 5 cm; two branches 2 cm long; a 45° angle between the branches; inner branch diameter, 5 cm) was placed in a dark room at 25 ± 1°C to avoid visual cue for the choice test. A flow of clean (charcoal-filtered) air at a rate of 0.6 L/min was split and passed through two glass vessels containing either an odor source or control (hexane) stimuli and entered each of the branches of the Y-tube. An air supply system (Power Air Pump, Seoul, Korea) was used for air filtration and flow rate control. Before and after the bioassay, the Y-tube was cleaned with 100% methanol. Test thrips were used three days after adult emergence. Each test used 10 ~ 30 individuals of each sex and was replicated four times by changing the source and control for replication. The response was positive when the thrips passed through the midpoint of the branches for 10 min. Otherwise, the thrips’ response was recorded as “no response.”

2.6. Alarm pheromone test using dispersal behavior assay

An arena was designed using a Petri dish (10 × 3 cm, SPL Life Science, Seoul, Korea) to test the dispersion behavior of the thrips. Test thrips (20 individuals per replication) on the bean diet were put into the center of the dish. A disc (3-mm diameter filter paper) was put near the diet and 10 μL of test solution in hexane was added. After 1 min, the thrips dispersed away from the bean were counted. Hexane was used as the control. Each treatment was replicated three times.

2.7. Field test for alarm pheromone

A greenhouse (220 m²) cultivating hot peppers was used in this test. F. occidentalis was heavily infesting the peppers. In the middle of the greenhouse, test hot peppers in pots (one plant per pot) without any thrips were randomly assigned by a randomized block design with three replications. Each test plant was covered with a plastic cage (45 × 45 × 45 cm), which had large openings in five directions to allow the entry of nearby thrips. The wax-formulated pheromone (5 g) was loaded in a small pheromone cage (Green Agrotech, Kyungsan, Korea), which was hung at the opening gates of the cage. An empty pheromone cage was used as the control.

The duration of alarm pheromone efficacy was tested by harvesting the lure in the field conditions once a week. The efficacy test was conducted using the dispersion behavior assay described above.

2.8. Field test for push-pull tactics

A test greenhouse (660 m²) cultivating about 200 hot peppers was located in Gudam (GD, N36.54 E128.46), Andong, Korea. Alarm pheromones in the wax formation were applied by pasting each (5 g) to the fence around the greenhouse at 2-m intervals for pushing the thrips immigrating from outside. The alarm pheromones were re-applied every four weeks. Twenty CAN traps containing aggregation pheromone were installed inside the greenhouse for pulling the thrips. The sticky bags were replaced every week. During the assay, no chemical insecticides were applied. For reference, thrips presence was compared in two other fields cultivating hot peppers. One was a greenhouse in Hahoe (HH, about 4 km from GD) and an open field in Songchun (SC, 16 km from GD) (Fig 6A). Both fields were frequently treated with chemical insecticides including spinosad during the assay.

Fig 6 — (A) Deployment of repellents and CAN traps in a GD greenhouse. Adult occurrence in three sites. (B) Number of F. *occidentalis* in flowers. Each replication represents the total number of thrips from 10 randomly selected flowers. Each measurement was replicated three times. (C) TSWV infection rates in hot peppers and F. *occidentalis* over one month (June 13 to July 13). Each measurement represents the percentage of diseased plants out of 100 randomly chosen hosts. Each measurement was replicated three times. To determine TSWV-infected thrips, multiplex PCR analysis was performed to discriminate viruliferous thrips. In each site/collection, 30 thrips were used to analyze the viruliferous thrips for the period. An asterisk above the standard deviation bars indicates a significant difference among means at Type I error = 0.05 (LSD test). NS indicates no significant difference.

2.9. Thrips monitoring test in the field

Three yellow sticky traps (Green Agrotech) were installed inside the greenhouse for monitoring changes in the thrips population from March to July. Thrips were identified based on the morphological characters described in an earlier study [12]. Number of F. occidentalis in flowers was counted from 10 randomly selected flowers with three replications in each field. The monitoring was performed once a week for one month (June 13 ~ July 13). TSWV infection rates in the hot peppers were measured by ring-spot disease symptoms on leaves and fruits. Each experimental unit was 100 randomly chosen hosts and was replicated three times. To determine TSWV-infected thrips, multiplex polymerase chain reaction (PCR) analysis (see below) was used to discriminate viruliferous thrips. In each site/collection, 30 thrips were used to analyze the viruliferous thrips for the period.

2.10. Molecular diagnosis of TSWV using multiplex PCR

A single insect was homogenized in 35 μL of an RNA extraction solution (LGC Bioresearch Technologies, Hoddesdon, UK) in a 1.5-ml sample tube. The homogenate was incubated at 95°C for 5 min and centrifuged at 13,500 × g for 5 min to obtain supernatant. To extract RNA from hot peppers, a piece of a leaf (1 g) was homogenized in 250 μL of a lysis buffer in the Viral DNA/RNA Extraction Kit (Intron Biotechnology, Sungnam, Korea) and RNA was extracted according to the manufacturer’s instruction. PCR amplification was performed in a total volume of 20 μL containing 4 μL of template RNA, 8 μL of primers, and 8 μL of SuPrime Script RT-PCR Premix (2X) (Genetbio, Daejeon, Korea). Seven primers [12] were designed for the TSWV (5’-TGTCTAAGGTTAAGCTCACTAAGGAA-3’ and 5’-TTAAGCAAGTTCTGCAAGTATTGCCTG-3’), F. occidentalis (forward primer, 5’-GGT CGC TTC ACC GCT TCC CG-3’), F. intonsa (forward primer, 5’-GAC CAG ACT GTT CCG AGA-3’), a common reverse primer (5’-CTC TCC TGA ACW GAG GCT G-3’), and T. tabaci (5’-TCT AAA CAG AGG GAA AGG TG-3’ and 5’-AGT GTG CCA ACA AGG CAA TG-3’). Reverse transcription was performed at 50°C for 30 min. After a denaturation step at 95°C for 5 min, the subsequent PCR was proceeded according to the following temperature cycle program: 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The PCR products were electrophoresed on 2% (w/v) agarose gel containing 1 × TAE buffer and were visualized by UV light on a Gel Doc imaging system (Bio-Rad, Hercules, CA, USA). The resulting PCR product sizes were 777 bp for TSWV, 287 bp for F. occidentalis, 367 bp for F. intonsa, and 417 bp for T. tabaci [12].

2.11. Statistical analysis

Percentage data were arcsine-transformed and confirmed in normality using PROC UNIVARIATE in the SAS program [29]. Data obtained from the Y-tube or choice test were subjected to a one-way analysis of variance (ANOVA) using PROC GLM. Field assay data were assessed by two-way ANOVA. The means were compared using the least significant difference (LSD) test or Student’s t test at Type I error of 0.05. Frequency data were analyzed by Chi square test using PROC FREQ.

3. Results

3.1. Optimal pheromone composition to attract thrips

The ability of two components (LA and NMB) of the aggregation pheromone to attract thrips was tested using two thrips, F. occidentalis and F. intonsa (Fig 1). In the behavioral assays, methylbutanoate lavandulol (LMB) was used as the control (Fig 1A). LA and NMB were significantly (F = 73.50; df = 1, 6; P = 0.0001 for LA; F = 19.11; df = 1, 6; P = 0.0047 for NMB) effective in attracting adult female F. occidentalis and male F. intonsa. However, LMB did not significantly attract thrips. Then, we prepared blends containing different ratios (1:2, 1:4, and 1:7, g/g) of LA and NMB and compared their attractiveness (Fig 1B). Except for the 1:2 ratio blend, the other two blends effectively attracted both thrips species. Even though males did not show significant preferences, F. occidentalis females were significantly (F = 96.43; df = 1, 6; P < 0.0001) attracted to the 1:7 blend whereas F. intonsa females were significantly (F = 27.77; df = 1, 6; P = 0.0019) attracted to the 1:4 blend (Fig 1).

Fig 1 — (A) Screening of active components and mixture ratios in the aggregation pheromone. The pheromone components were identified as LA (lavandulyl acetate), LMB (lavandulyl methylbutanoate), and NMB (neryl methylbutanoate). (B) Choice tests of the thrips against two pheromone blends. Each response (experimental unit) used 20 adults (< 3-days-old after emergence). Each treatment was replicated four times. The detailed data are presented in S1 Fig.

3.2. Development of the yellow CAN trap containing aggregation pheromone

A yellow sticky trap (PLATE-type) has been used to monitor thrips. To enhance the trapping efficiency, a CAN-type trap covering an empty can with a sticky zipper bag (CAN-type) was designed to attract thrips from all directions because the PLATE-type attracted only to the front and back sides. When the attractiveness of the PLATE-type trap was compared to that of the CAN-type trap without any aggregation pheromone treatment, they were not significantly different based on the number of thrips per sticky unit area (Fig 2A). However, the trap color significantly (F = 5.63; df = 4, 10; P = 0.0123) influenced thrips trapping, in which attractiveness was nearly lost in black traps (Fig 2B). The addition of the aggregation pheromone to the CAN trap significantly (F = 11.34; df = 1, 4; P = 0.0028) enhanced attractiveness even in a black background (Fig 2C). With a yellow background, the addition of the aggregation pheromone increased the attractiveness of the CAN trap by almost five-fold compared to the trap without the pheromone. Based on the findings, a yellow CAN trap was constructed (Fig 2D). For the CAN trap, LA (0.25 mg) and NMB (1.75 mg) were mixed with a sticky material (12 g of sticky material in 6 mL of hexane). The mixture was then poured into a zipper bag and spread using a roller. When the sticky bag was applied to the CAN trap, the sticky inside was inverted to the outside. The height of the CAN trap was controlled by the length of the supporting bar (Fig 2E).

3.3. Trapping efficiency of CAN trap in field conditions

The trapping efficiency of the CAN trap for F. occidentalis in field conditions was compared to that of a yellow sticky plate (PLATE), which is currently used to monitor thrips including F. occidentalis (Fig 3). When we compared the trapping efficiency of these two traps for a week (June 8 ~ June 15), the CAN trap showed > 4-times higher efficiency than the PLATE-type (Fig 3A). Then, we analyzed the number of F. occidentalis collected by the two traps over five successive weeks (Fig 3B). The collection numbers were normalized by sticky unit area because of their different sizes. Except for one week in which thrips occurred at a low density, the CAN trap was much more attractive than the PLATE trap (t = 5.29; df = 24; P < 0.0001).

Fig 3 — (A) Enhanced trapping efficiency of the CAN trap containing aggregation pheromone compared to the yellow sticky plate (PLATE). CAN and PLATE traps used 20 and three replications, respectively. An asterisk above the standard deviation bars indicates a significant difference between two treatments at type I error = 0.05 (LSD test). (B) Enhanced pulling effect of the CAN trap on trapping F. *occidentalis* and B. *tabaci* in different seasons. Two and three asterisks above the standard deviation bars indicate significant differences between two treatments at Type I error = 0.01 and type I error = 0.0001 (LSD test), respectively. NS indicates no significant difference. (C) Composition of thrips trapped by the PLATE and CAN traps. The average species frequencies in CAN and PLATE traps were obtained from 20 and three replications, respectively.

Unexpectedly, the CAN trap was also highly efficient (t = 9.99; df = 24; P < 0.0001) in attracting the whiteflies of B. tabaci compared to the PLATE-type. Thrips collected by the CAN trap were classified into different species and the composition was compared to that of the thrips collected by the PLATE trap (Fig 3C). The CAN trap containing the F. occidentalis aggregation pheromone attracted all three thrips species of F. occidentalis, F. intonsa, and T. tabaci but it attracted more F. occidentalis than the PLATE trap without pheromone (X² = 14.03; df = 2; P = 0.0009).

3.4. Development of thrips repellent using alarm pheromone

In response to the alarm pheromone (10:Ac and 12:Ac = 1: 1.5, g/g), larvae and adults exhibited dispersal behavior within a few seconds (Fig 4A). This dispersal behavior was also observed in response to individual components, in which 12:Ac induced the behavior in both stages, whereas 10:Ac was effective only in adults (Fig 4B). Dispersal behavior to the alarm pheromone components was highly induced in adults compared to larvae. The two-component blend was more effective in inducing the dispersal behavior than single component treatments except for the comparison of 12:Ac and the blend in the adult stage. To clarify the effectiveness between the single-component (12:Ac) and the two-component blend, different doses were applied and the efficacy of inducing dispersal behavior was assessed (Fig 4C). The two-component blend was much more effective at low doses compared to the single component in both larvae and adults. In addition, the two-component blend was effective in inducing the dispersal behavior of F. intonsa (Fig 4D). However, the dispersal behavior was highly induced in F. occidentalis compared to F. intonsa at low doses. These results indicate that the alarm pheromone induced the dispersal behavior of thrips in both larvae and adults and acted as a repellent because the thrips fled the pheromone source.

The alarm pheromone-based repellent was tested in field conditions. The repellent was formulated with wax for applying by pasting (Fig 5A). To test the efficacy of the formulation, the repellents were applied to hot peppers infested with F. occidentalis in a greenhouse (Fig 5B). Test plants without any thrips were covered with a plastic cage, which had large openings in four directions to allow the entry of nearby thrips. The repellents were then applied to the openings. In the control without the repellent, thrips arrived within a day and maintained an immigrating population. However, hot peppers treated with the repellents maintained significantly (F = 192.79; df = 1, 34; P < 0.0001) low levels of thrips for at least two weeks (Fig 5C). The repellent efficacy of the wax formulation was maintained for four weeks without significant (F = 1,600.00, df = 4, 10; P = 0.0001) differences in the greenhouse conditions.

3.5. Application of the push-pull strategy to an agricultural farm cultivating hot peppers

The occurrence of F. occidentalis in three different greenhouses cultivating hot peppers in Andong was compared (Fig 6A). Push-pull control was applied to the GD greenhouse (Fig 6B), while HH and SC were negative controls and heavily treated with conventional insecticides, in which HH hot peppers were in a greenhouse, but SC peppers were in an open field. F. occidentalis adults usually occur in greenhouses in late March in Andong [12]. Our current study also found that the first adults were caught in greenhouses on March 23 in HH and April 27 in GD. In contrast, the occurrence of adults was delayed and first detected in early May in an SC open field. After small peaks in April-May, the main adult peak was observed in June-July in all three field conditions. In the main peak, HH had many more adults than GD by more than 1,000-fold. The occurrence of F. occidentalis adults in GD was lower than that in the SC open field. F. occidentalis usually occurs at low levels because the thrips prefer greenhouse conditions over open fields [30]. To further analyze the effect of the CAN trap, thrips populations in the flowers were compared (Fig 6C). The number of F. occidentalis thrips in the flowers was significantly lower in CAN trap-treated GD compared to the control farms (F = 5.89; df = 10, 30; P < 0.0001). F. occidentalis is a vector for TSWV. The TSWV infection rate was significantly different between farms (F = 8.55; df = 2, 22; P = 0.0049). Indeed, no hot peppers were infected with TSWV in CAN trap-treated GD (Fig 6D). However, the control farms had TSWV-infected plants, in which the SC site showed an infection rate of more than 30%. This was further supported by the multiplex PCR analysis. No F. occidentalis was infected with TSWV, whereas significant numbers of F. occidentalis adults were positive for TSWV in HH and SC.

4. Discussion

This study proposes a push-pull control strategy using alarm and aggregation pheromones against F. occidentalis in greenhouse conditions. To test these novel control tactics, the push-pull effects of the chemical compounds were re-evaluated. For field assays, both pheromones were formulated as wax types for the alarm pheromone and a CAN trap for the aggregation pheromone. These formulated pheromones were applied to a practical farm and the control efficacy was compared to those of other conventional farms treated with chemical insecticides. Our laboratory and field data supported the control efficacy of push-pull control tactics against F. occidentalis.

To enhance the attractiveness for F. occidentalis, two aggregation pheromone components (LA and NMB) were re-evaluated with an analog, lavandulyl methylbutanoate (LMB). LMB was identified as an aggregation pheromone component of another flower thrips, Megalurothrips sjostedti [31], and onion thrips, Thrips palmi [32]. No significant attractiveness of LMB for either F. occidentalis or F. intonsa was seen by Y-tube olfactometry. However, they were attracted to LA and NMB. The aggregation pheromones LA and NMB are shared between F. occidentalis and F. intonsa, so species-specific blend mixtures were used to prevent cross-attraction [33]. In our assay, F. occidentalis preferred a 1:7 (LA:NMB) ratio to a 1:4 ratio, whereas F. intonsa exhibited an opposite preference. Interestingly, a significant preference was observed only in the females of both species in the Y-tube olfactometer analysis in the double-choice experiment between 1:4 and 1:7 ratios, although both sexes were attracted to both ratios in the single-choice experiments. Considering that the aggregation pheromone is basically attractive to both sexes, our assay might not detect a male preference. The lower sensitivity of males may be their preference for a specific stereoisomer because our synthetic pheromone components (S1 Fig) were mixtures of stereoisomers. The LA:NMB ratio in F. occidentalis was reported to be within a range of 1:0.8 ~ 1:5 measured by solid-phase microextraction (SPME) [22]. In that study, the addition of LA to NMB gave no increase relative to NMB alone, leading to the development of a single-component commercial product. In our current study, however, the inclusion of LA in the aggregation pheromone may have had some physiological significance because unlike the 1:4 and 1:7 ratios, the 1:2 mixture ratio did not significantly attract F. occidentalis. This suggests the role of LA in the aggregation signal of F. occidentalis. Two congener species of F. occidentalis and F. intonsa share flower habitats and aggregation pheromone components, in which F. occidentalis had 1:13 (LA:NMB) ratio while F. intonsa had 1:2 ratio in a Chinese population measured by SPME [34]. The significance of these different effective mixture ratios was confirmed in field experiments where a 1:8 ratio of aggregation pheromone components caught more F. occidentalis, whereas a 1:4 ratio attracted more F. intonsa [33]. Thus, the role of LA may give species-specificity to the aggregation pheromone. Furthermore, in our current assay, LA alone significantly attracted F. occidentalis. Thus, we determined the optimal mass trapping lure to be a mixture of LA and NMB at a 1:7 mixture ratio.

An alarm pheromone consisting of 10:Ac and 12:Ac induced significant dispersion behavior in F. occidentalis. It was reported that in response to the alarm pheromone, larvae exhibited escape behavior from the source by a rapid drop from host plants, whereas exposed adults exhibited take-off behavior and avoided landing in the area [35]. Indeed, the deployment of the alarm pheromone significantly prevented the invasion of the thrips to hot peppers in the current study. These facts led us to apply the alarm pheromone to the push-pull strategy to control F. occidentalis. Other potent repellents may be developed from other sources. For example, plant odors emitted by non-preferred hosts, such as garlic (Allium sativum) and celery (Apium graveolens), showed repelling effects on F. occidentalis females [36].

To apply the pheromones to field conditions, they were formulated to meet the purpose of the push-pull strategy. Alarm pheromone components were formulated using wax for fence treatment by pasting. The wax formulation modified the methods used in other lure formulations developed using moth sex pheromone and fly lure, where their efficacy was confirmed in field conditions [28, 37]. The bioactivity of the wax formulation of the alarm pheromone lasted for at least four weeks in greenhouse conditions. Aggregation pheromone was formulated by mixing with sticky material in an attract-to-kill strategy. For this purpose, a CAN trap was devised and effectively attracted thrips by its yellow color and aggregation pheromone. The question has been raised of whether thrips from nearby plants or thrips flying over plants were collected by the CAN traps. The study findings present indirect evidence that CAN traps collected thrips flying over the plants because adult thrips in the hot pepper flowers were not evenly distributed, whereas the 20 installed CAN traps collected similar quantities of thrips. This suggests that our CAN traps attracted flying thrips. A synergistic effect of the CAN trap and another biological control method using a natural enemy was also suggested because it is known that the predator Orius laevigatus stimulates the release of an alarm pheromone [27], which induces take-off flight in flying thrips in host plants.

Interestingly, the trapping efficiency of the yellow CAN trap for the whitefly B. tabaci was enhanced without an aggregation pheromone. This suggests that the aggregation pheromone of F. occidentalis was effective in attracting B. tabaci. The two aggregation pheromone components are terpenoids. Whiteflies respond to various terpenoids including sesquiterpenes (zingiberene and curcumene) and monoterperpenes (p-cymene, α-terpinene, and α-phellandrene), although terpenoids act as repellents [38]. It is noteworthy that LMB, one of the aggregation pheromone components in thrips, is used by a sex pheromone of the mealybug, Phenacoccus madeirensis [39]. Further studies assessing the sensitivity and response of B. tabaci to the aggregation pheromone of F. occidentalis need to be conducted.

The push-pull strategy was effective in suppressing the frequency of TSWV-infected hot peppers. Compared to other fields such as those in HH and SC, the thrips density was low in flowers in GD treated with the push-pull strategy, as was the number in monitoring traps. TSWV-infected hot peppers exhibit characteristic disease symptoms of ring-spots on leaves and fruit [40]. Based on this disease symptom, the survey indicated no TSWV infections in hot peppers in GD in contrast to the significant numbers of disease symptoms in the other two fields. This was further supported by the multiplex PCR results, which indicated no TSWV-infected F. occidentalis in GD compared to large numbers of TSWV-positive thrips in other fields. This might be explained by a local variation in viruliferous thrips as similar variations in Thrips tabaci carrying TSWV were seen [41]. Alternatively, the suppression of thrips occurrence by the push-pull treatment might have caused the decrease in the TSWV infection rate.

The push-pull strategy has been used for effective insect pest control [42]. A push-pull control technique against F. occidentalis was proposed using UV-reflective mulch as a pushing factor to disrupt thrips colonization on host plants along with a companion plant as a pulling factor to attract the predators and trap the thrips [43]. Alternatively, the use of semiochemicals such as attractants and repellents has been considered for developing a push-pull strategy [44]. Kirk et al. [23] proposed F. occidentalis control using pheromones and other semiochemicals. This study demonstrated novel push-pull tactics using aggregation/alarm pheromones (Fig 7) to control F. occidentalis in greenhouse conditions. This control technique effectively suppressed the outbreak of F. occidentalis in a field assay and also prevented the occurrence of plant disease caused by TSWV in hot peppers. Although our current study directly evaluated the pulling efficacy of the attracting CAN trap, the pushing efficacy of the repellent fence treatment was indirectly predicted from a small-scale greenhouse experiment. Subsequent studies are needed to assess thrips occurrence outside of a test greenhouse treated with the alarm pheromone to improve the estimation of the pushing efficacy in field conditions.

Fig 7 — Arrows indicate the push and pull moving directions.

Supporting information

S1 Fig. Chemical synthesis of the aggregation pheromone components of thrips: Lavandulyl acetate (LA, 2), lavandulyl methylbutanoate (LMB, 3), and neryl methylbutanoate (NMB, 5).

LA and LMB were synthesized from the precursor, lavandulol (1) in dichloromethane, which was reacted with acetyl chloride to yield LA, and was reacted with isovaleryl chloride to yield LMB, respectively, under the catalytic activity of trimethylamine. NMB was reacted with geraniol (‘4’) and isovaleryl chloride under a catalytic activity of trimethylamine. The purity of the compounds was analyzed by gas chromatography (8860 GC, Agilent, Santa Clara, CA, USA) with a DB-1 column (15 m × 0.350 mm, Agilent) at an oven temperature of 280°C and a flow rate of 1.0 mL/min.

(DOCX)

Click here for additional data file.^{(665.8KB, docx)}

S2 Fig. Tests of two flower thrips (F. occidentalis (‘Fo’) and F. intonsa (‘Fi’)) to aggregation pheromone components using a Y-tube olfactometer.

The test pheromone components included LA (lavandulyl acetate), LMB (lavandulyl methylbutanoate), and NMB (neryl methylbutanoate). Each response (experimental unit) used 10 adults (< 3-days-old after emergence). Each treatment was replicated four times.

(DOCX)

Click here for additional data file.^{(36.2KB, docx)}

Acknowledgments

We thank Jean Lee and Juan Hong in our laboratory to provide thrips for our analysis.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was conducted with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01578901) funded by the Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Yudin LS, Cho JJ, Mitchell WC. Host range of western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), with special reference to Leucaena glauca. Environ Entomol. 1986; 15:1292–1295. [Google Scholar]
2.Schneweis DJ, Whitfield AE, Rotenberg D. Thrips developmental stage-specific transcriptome response to tomato spotted wilt virus during the virus infection cycle in Frankliniella occidentalis, the primary vector. Virology. 2017; 500:226–237. [DOI] [PubMed] [Google Scholar]
3.He Z, Guo JF, Reitz SR, Lei ZR, Wu SY. A global invasion by the thrips, Frankliniella occidentalis: current virus vector status and its management. Insect Sci. 2020; 27:626–645. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Reitz SR. Biology and ecology of the western flower thrips (Thysanoptera: Thripidae): The making of a pest. Fla Entomol. 2009; 92:7–13. [Google Scholar]
5.Shrestha A, Srinivasan R, Riley DG, Culbreath AK. Direct and indirect effects of a thrips-transmitted Tospovirus on the preference and fitness of its vector, Frankliniella fusca. Entomol Exp Appl. 2012; 145:260–271. [Google Scholar]
6.Belliure B, Janssen A, Maris PC, Peters D, Sabelis MW. Herbivore arthropods benefit from vectoring plant viruses. Ecol Lett. 2005; 8:70–79. [Google Scholar]
7.Rotenberg D, Jacobson AL, Schneweis DJ, Whitfield AE. Thrips transmission of tospoviruses. Curr Opin Virol. 2015; 15:80–89. doi: 10.1016/j.coviro.2015.08.003 [DOI] [PubMed] [Google Scholar]
8.Lee S, Lee J, Kim S, Choi H, Park J, Lee J, et al. The incidence and distribution of viral diseases in pepper by cultivation types. Res Plant Dis. 2004; 10:231–240. [Google Scholar]
9.Reitz SR, Gao Y, Kirk WDJ, Hoddle MS, Leiss KA, Funderburk JE. Invasion biology, ecology, and management of western flower thrips. Annu Rev Entomol. 2020; 65:17–37. doi: 10.1146/annurev-ento-011019-024947 [DOI] [PubMed] [Google Scholar]
10.Woo KS, Ahn SB, Lee SH, Kwon HM. First record of Frankliniella occidentalis and its distribution and host plants in Korea. Korean J Appl Entomol. 1994; 33:127. [Google Scholar]
11.Ding T, Chi H, Gökçe A, Gao Y, Zhang B. Demographic analysis of arrhenotokous parthenogenesis and bisexual reproduction of Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Sci Rep. 2018; 8:3346. doi: 10.1038/s41598-018-21689-z [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Kim C, Choi D, Kang J, Ahmed SA, Kil E, Kwon G, et al. Thrips infesting hot pepper cultured in greenhouses and variation in gene sequences encoded in TSWV. Korean J Appl Entomol. 2021; 60:387–401. [Google Scholar]
13.Choi DY, Kim Y. PGE₂ mediation of egg development in western flower thrips, Frankliniella occidentalis. Arch Insect Biochem Physiol. 2022; e21949. [DOI] [PubMed] [Google Scholar]
14.Choi DY, Kim Y. Transcriptome analysis of female western flower thrips, Frankliniella occidentalis, exhibiting neo-panoistic ovarian development. PLoS ONE 2022; 17:e0272399. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Cho S, Kyung Y, Cho S, Shin S, Jeong D, Kim S, et al. Evaluation of susceptibility of western flower thrips (Frankliniella occidentalis) and garden thrips (F. intonsa) to 51 insecticides. Korean J Appl Entomol. 2018; 57:221–231. [Google Scholar]
16.Bhuyain MMH, Lim UT. Relative susceptibility to pesticides and environmental conditions of Frankliniella intonsa and F. occidentalis (Thysanoptera: Thripidae), an underlying reason for their asymmetrical occurrence. PLoS ONE 2020; 15:e0237876. doi: 10.1371/journal.pone.0237876 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Gao Y, Yoon KA, Lee JH, Kim JH, Lee SH. Overexpression of glutamate-gated chloride channel in the integument is mainly responsible for emamectin benzoate resistance in the western flower thrips Frankliniella occidentalis. Pest Manag. Sci. 2022; doi: 10.1002/ps.7032 [DOI] [PubMed] [Google Scholar]
18.Mouden S, Sarmiento KF, Klinkhamer PG, Leiss KA. Integrated pest management in western flower thrips: past, present and future. Pest Manag Sci. 2017; 73:813–822. doi: 10.1002/ps.4531 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Sampson C, Kirk WD. Can mass trapping reduce thrips damage and is it economically viable? Management of the Western flower thrips in strawberry. PLoS ONE 2013; 8:e80787. doi: 10.1371/journal.pone.0080787 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Olaniran OA, Sudhakar AV, Drijfhout FP, Dublon IA, Hall DR, Hamilton JG, et al. A male-predominant cuticular hydrocarbon, 7-methyltricosane, is used as a contact pheromone in the western flower thrips Frankliniella occidentalis. J Chem Ecol. 2013; 39:559–568. [DOI] [PubMed] [Google Scholar]
21.Kirk WD, Hamilton JG. Evidence for a male-produced sex pheromone in the western flower thrips Frankliniella occidentalis. J Chem Ecol. 2004; 30:167–174. [DOI] [PubMed] [Google Scholar]
22.Hamilton JG, Hall DR, Kirk WD. Identification of a male-produced aggregation pheromone in the western flower thrips Frankliniella occidentalis. J Chem Ecol. 2005; 31:1369–1379. [DOI] [PubMed] [Google Scholar]
23.Kirk WDJ, de Kogel WJ, Koschier EH, Teulon DAJ. Semiochemicals for thrips and their use in pest management. Annu Rev Entomol. 2021; 66:101–119. doi: 10.1146/annurev-ento-022020-081531 [DOI] [PubMed] [Google Scholar]
24.Akinyemi AO, Kirk WDJ. Experienced males recognise and avoid mating with non-virgin females in the western flower thrips. PLoS ONE 2019; 14:e0224115. doi: 10.1371/journal.pone.0224115 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.MacDonald KM, Hamilton JG, Jacobson R, Kirk WD. Analysis of anal droplets of the western flower thrips Frankliniella occidentalis. J Chem Ecol. 2003; 29:2385–2389. [DOI] [PubMed] [Google Scholar]
26.de Bruijn PJ, Egas M, Janssen A, Sabelis MW. Pheromone-induced priming of a defensive response in Western flower thrips. J Chem Ecol. 2006; 32:1599–1603. doi: 10.1007/s10886-006-9092-1 [DOI] [PubMed] [Google Scholar]
27.de Bruijn PJ, Egas M, Sabelis MW, Groot AT. Context-dependent alarm signalling in an insect. J Evol Biol. 2016; 29:665–671. doi: 10.1111/jeb.12813 [DOI] [PubMed] [Google Scholar]
28.Kim K, Kim M, Kwon G, Kim Y. Technologies required for development of trap-based MAT control against the striped fruit fly, Bactrocera scutellata. Korean J. Appl. Entomol. 2017; 56:51–60. [Google Scholar]
29.SAS Institute Inc. SAS/STAT user’s guide, Release 6.03, Ed. Cary, NC. 1989.
30.Lee GS, Lee JH, Kang SH, Woo KS. Thrips species (Thysanoptera: Thripidae) in winter season and their vernal activities on Jeju island, Korea. J Asia Pac Entomol. 2001; 4:115–122. [Google Scholar]
31.Niassy S, Tamiru A, Hamilton JGC, Kirk WDJ, Mumm R, Sims C, et al. Characterization of male-produced aggregation pheromone of the bean flower thrips Megalurothrips sjostedti (Thysanoptera: Thripidae). J Chem Ecol. 2019; 45:348–355. doi: 10.1007/s10886-019-01054-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Akella SVS, Kirk WDJ, Lu YB, Murai T, Walters KFA, Hamilton JG. Identification of the aggregation pheromone of the melon thrips, Thrips palmi. PLoS ONE 2014; 9:e103315. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Li X, Geng S, Zhang Z, Zhang J, Li W, Huang J, et al. Species-specific aggregation pheromones contribute to coexistence in two closely related thrips species. Bull Entomol Res. 2019; 109:119–126. doi: 10.1017/S0007485318000366 [DOI] [PubMed] [Google Scholar]
34.Zhu XY, Zhang PJ, Lu YB. Isolation and identification of the aggregation pheromone released by male adults of Frankliniella intonsa (Thysanoptera: Thripidae). Acta Entomol Sin. 2012; 55:376–385. [Google Scholar]
35.Teerling CR, Pierce HD, Borden JH, Gillespie DR. Identification and bioactivity of alarm pheromone in the western flower thrips, Frankliniella occidentalis. 1993; 19:681–697. doi: 10.1007/BF00985001 [DOI] [PubMed] [Google Scholar]
36.Cao Y, Zhi J, Cong C, Margolies DC. Olfactory cues used in host selection by Frankliniella occidentalis (Thysanoptera: Thripidae) in relation to host suitability. J Insect Behav. 2014; 27:41–56. [Google Scholar]
37.Jung S, Park M, Lee S, Choi K, Hong Y, Bae S. et al. Development of wax-typed pheromone dispenser for mating disruption of the oriental fruit moth, Grapholita molesta, and its application technique. Korean J Appl Entomol. 2008; 47:255–263. [Google Scholar]
38.Bleeker PM, Diergaarde PJ, Ament K, Guerra J, Weidner M, Schütz S, et al. The role of specific tomato volatiles in tomato-whitefly interaction. Plant Physiol. 2009; 151:925–935. doi: 10.1104/pp.109.142661 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Ho HY, Su YT, Ko CH, Tsai MY. Identification and synthesis of the sex pheromone of the Madeira mealybug, Phenacoccus madeirensis Green. J Chem Ecol. 2009; 35:724–732. [DOI] [PubMed] [Google Scholar]
40.Lee JS, Cho WK, Kim MK, Kwak HR, Choi HS, Kim KH. Complete genome sequences of three tomato spotted wilt virus isolates from tomato and pepper plants in Korea and their phylogenetic relationship to other TSWV isolates. Arch Virol. 2011; 156:725–728. doi: 10.1007/s00705-011-0935-x [DOI] [PubMed] [Google Scholar]
41.Jacobson AL, Kennedy GG. Specific insect-virus interactions are responsible for variation in competency of different Thrips tabaci isolines to transmit different Tomato Spotted Wilt Virus isolates. PLoS ONE 2013; 8:e54567. doi: 10.1371/journal.pone.0054567 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Cook SM, Khan ZR, Pickett JA. The use of push-pull strategies in integrated pest management. Annu Rev Entomol. 2007; 52:375–400. doi: 10.1146/annurev.ento.52.110405.091407 [DOI] [PubMed] [Google Scholar]
43.Tyler-Julian K, Funderburk J, Srivastava M, Olson S, Adkins S. Evaluation of a push-pull system for the management of Frankliniella species (Thysanoptera: Thripidae) in tomato. Insects 2018; 9:187. doi: 10.3390/insects9040187 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Niu Y, Han S, Wu Z, Pan C, Wang M, Tang Y. et al. A push-pull strategy for controlling the tea green leafhopper (Empoasca flavescens F.) using semiochemicals from Tagetes erecta and Flemingia macrophylla. Pest Manag Sci. 2022; 78:2161–2172. doi: 10.1002/ps.6840 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0279646.r001

Decision Letter 0

Ramzi Mansour

29 Sep 2022

PONE-D-22-23616A push-pull strategy to control the western flower thrips, Frankliniella occidentalis, using alarm and aggregation pheromonesPLOS ONE

Dear Dr. Kim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 13 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ramzi Mansour

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

3. Thank you for stating in your Funding Statement:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This work was conducted with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01578901) funded by the Rural Development Administration, Republic of Korea."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

6. We note that Figure 6A in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 6A to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an ""Other"" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I read the manuscript by Yonggyun Kim et al. with interest. The paper aims to develop a non-chemical technique to control F. occidentalis infesting hot peppers cultivated in greenhouses in Korea. The method was based on behavioral control using an alarm pheromone to prevent the entry of the thrips into greenhouses and an aggregation pheromone for mass trapping inside the greenhouses. This technique effectively suppressed thrips populations in field conditions and also prevented the occurrence of plant disease caused by TSWV in hot peppers. In my opinion, I think is a nice piece of work and original, however there are some improvements to be made especially in materials and Methods section.

Introduction

I believe that you need a much strong justification for studying the western flower thrips, Frankliniella occidentalis as pest I did not find any strong justification for using F. occidentalis compared to other insects such as aphids for example!

Materials and methods

The overall level of details for the Materials and Methods is appropriate, however there are some informations should be add in the text:

Line 98 page 5: Please precise if it was the same laboratory conditions described below.

Line 103-105 page 5: please give the purity of the molecules: Lavandulyl acetate (LA), lavandulyl methylbutanoate (LMB), and neryl methylbutanoate (NMB).

Line 108-110 page 6: Purity of molecules are missing here.

Line 114 page 6: add the purity of the alarm pheromone components

Line 115: please correct Signal-Aldrich >> Sigma-Aldrich

Line 125: I did not find any justification for using hexane as solvent please explain.

Line 132: Give the purity of α-tocopherol.

Line 138: explain the use of a dark room

Line 143: here, normally the Y-tube should be cleaned with methanol after each replication not only before and after the bio assay.

Line 143: give the sex of individuals: only females? Please justify

Line 182: information about pesticides used in fields are missing here. Please add the active molecules of these insecticides.

Line 206-211: add references about used primers.

Line 218: reference is missing.

Line 220: Do you check the normality of values before using the one-way analysis of variance (ANOVA) ? please add the information.

Reviewer #2: The manuscript does not provide a proper paragraph of Conclusions which must be drawn appropriately based on the data presented in the text. The data obtained in the study support the conclusions. All the experiments, trials have been conducted rigorously , with appropriate control and replication.So that also the descriptions of Material and Methods must be precse and complete. Although the manuscript has been presented in a correct and standard English, it might be useful to ask for a revision of the text by a mother language lecturer.

Reviewer #3: The article in review is titled “A push-pull strategy to control the western flower thrips, Frankliniella occidentalis, using alarm and aggregation pheromones”. This article presents a novel push-pull system for the management of Frankliniella occidentalis thrips using an interesting and new combination of pheromones and traps. The experimental design was simple and straightforward, and acceptable. The article is mostly well-written and has a strong introduction supporting the importance of the experiment. There are many components to this research involving different scientific techniques and methods, which are interesting but may have contributed to a few issues which need to be addressed before the publication process can progress.

There are some areas in the methods section which need clarification, and it seems as though the results section contains much of the information which fits better into the methods section. There are major problems with the reporting of the statistical results and the description of the statistical analyses used. The statistical values reported do not match the tests that were reported to be used in the methods section. Additionally, the discussion section could use some expanding to include discussion of the study’s limitations, future directions, and more summarizing of the results highlighting the importance for the field of study. More details on suggested areas for improvement can be found below.

Overall, I believe that this is an interesting and important study for the field of integrated pest management, but it requires significant revisions before it can be considered for publication. Specifically, there are some major issues involving the choice and reporting of statistical analyses that require extra attention, and the discussion section is weak.

Areas for improvement

Major

1.Results section appears to be missing the F-values, and degrees of freedom in the reporting of the results of the ANOVA. There appear to be sections of the results section that might actually belong in the methods section, for example the details about the trap types and configuration.

2.The results reported for the trap comparison test use a Chi-squared value rather than an F-value which would be expected from the ANOVA that was purported to be used to analyze the results. The statistics reported in the results section do not match the statistical tests that were delineated in the methods section. Lines 304-306 only a P-value is reported, what statistical test was used here and what was the associated value? Additionally, were the data tested to confirm that they meet the assumptions of the statistical tests chosen for analysis? Did the data transformation correct for normality?

3. In the discussion section there is a lack of discussion about any potential shortcomings/weaknesses of the study and the discussion section should have more time spent discussing future directions.

Minor

1.The abstract could use more information regarding the results of the study.

2.In the methods section under thrips rearing, it is stated that the thrips were fed a kidney bean diet for five days after germination. This could use some clarification as I believe the authors meant after hatching.

3.Y-tube methods (line 137-146) could use more explanation. It says that thrips were used three days after emergence, is that referring to emergence from the egg (hatching), or emergence as adults from the pupal stage? Additionally, the methods state that each test used 10-30 individuals, were these individuals tested separately (one at a time in the olfactometer) or were they all places into the olfactometer at the same time, please clarify.

4.Line 189-190 discussing the sampling of flowers states: “in flowers in 10 randomly selected flowers was counted in three fields three times a week for 190 one month (June 13 � July 13) once in a week”. This statement is confusing, was the sampling conducted three times a week or one time per week?

5.Line 222- should be arcsine perhaps?

6.In discussing the results of the field study of the push and pull components, it is stated that the Frankliniella occidentalis numbers are lower in the experimental greenhouse, but what were the results for each life stage and sex? Was it only the total number of all life stages and males and females that were reduced, or was there a difference in the response of the different life stages (larvae versus adults) or sexes?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: PONE-D-22-23616.docx

Click here for additional data file.^{(557.7KB, docx)}

PLoS One. 2023 Feb 24;18(2):e0279646. doi: 10.1371/journal.pone.0279646.r002

Author response to Decision Letter 0

11 Oct 2022

[Reviewer #1]

Comment #1-1: Introduction. I believe that you need a much strong justification for studying the western flower thrips, Frankliniella occidentalis as pest I did not find any strong justification for using F. occidentalis compared to other insects such as aphids for example!

Response: The thrips as a serious pest is explained by direct feeding damage and indirect TSWV transmission. We did not find the direct analysis on the statistical data of the relative damage compared to aphids. Thus we did not add the further information.

Comment #1-2: Materials and methods. The overall level of details for the Materials and Methods is appropriate, however there are some informations should be add in the text:

Line 98 page 5: Please precise if it was the same laboratory conditions described below.

Response: It is clarified as follows: “…. reared in the laboratory conditions.”

Comment #1-3: Line 103-105 page 5: please give the purity of the molecules: Lavandulyl acetate (LA), lavandulyl methylbutanoate (LMB), and neryl methylbutanoate (NMB).

Response: Added

Comment #1-4: Line 108-110 page 6: Purity of molecules are missing here.

Response: Added

Comment #1-5: Line 114 page 6: add the purity of the alarm pheromone components

Response: added

Comment #1-6: Line 115: please correct Signal-Aldrich >> Sigma-Aldrich

Response: Corrected as suggested

Comment #1-7: Line 125: I did not find any justification for using hexane as solvent please explain.

Response: It is rephrased as follows: “To mix the pheromone with sticky material, 2 mg of pheromone components and 12 g of sticky material (Tanglefoot, Contech Electronics, Rochester, NY, USA) were dissolved in 6 mL of hexane. This mixture was then poured to a sticky plastic zipper bag (30 � 30 cm, HG Pack, Gunpo, Korea).”

Comment #1-8: Line 132: Give the purity of α-tocopherol.

Response: Added

Comment #1-9: Line 138: explain the use of a dark room

Response: Added as follows: “to avoid visual cue for the choice test”

Comment #1-10: Line 143: here, normally the Y-tube should be cleaned with methanol after each replication not only before and after the bio assay.

Response: As you see, we wrote the cleaning steps.

Comment #1-11: Line 143: give the sex of individuals: only females? Please justify

Response: Sex was discriminated. This information is added as follows: “Each test used 10 � 30 individuals of each sex and was replicated four times by changing the source and control for replication.”

Comment #1-12: Line 182: information about pesticides used in fields are missing here. Please add the active molecules of these insecticides.

Response: Added as follows: “Both fields were frequently treated with chemical insecticides including spinosad during the assay.”

Comment #1-13: Line 206-211: add references about used primers.

Response: Added

Comment #1-14: Line 218: reference is missing.

Response: Added

Comment #1-15: Line 220: Do you check the normality of values before using the one-way analysis of variance (ANOVA) ? please add the information.

Response: Added as follows “Percentage data were arsine-transformed and confirmed in normality using PROC UNIVARIATE in the SAS program [29].”

[Reviewer #2]

Comment #2-1: The manuscript does not provide a proper paragraph of Conclusions which must be drawn appropriately based on the data presented in the text. The data obtained in the study support the conclusions. All the experiments, trials have been conducted rigorously , with appropriate control and replication. So that also the descriptions of Material and Methods must be precse and complete. Although the manuscript has been presented in a correct and standard English, it might be useful to ask for a revision of the text by a mother language lecturer.

Response:

(1) At the end of Discussion, the conclusion is described as follows: “This control technique effectively suppressed the outbreak of F. occidentalis in a field assay and also prevented the occurrence of plant disease caused by TSWV in hot peppers. Although our current study directly evaluated the pulling efficacy of the attracting CAN trap, the pushing efficacy of the repellent fence treatment was indirectly predicted from a small-scale greenhouse experiment. Subsequent studies are needed to assess thrips occurrence outside of a test greenhouse treated with the alarm pheromone to improve the estimation of the pushing efficacy in field conditions.” This also explains a limitation of this technology in field conditions.

(2) Some details are added in M&M. These are marked in red color.

(3) Text is cleaned by a English-editing company, Harrisco, co.

[Reviewer #3]

Comment #3-1: Results section appears to be missing the F-values, and degrees of freedom in the reporting of the results of the ANOVA. There appear to be sections of the results section that might actually belong in the methods section, for example the details about the trap types and configuration.

Response: All F values are added. The trap was newly designed. Thus in M&M, the structure and detailed configuration were explained. In Results, the background rationale to construct the trap was explained.

Comment #3-2: The results reported for the trap comparison test use a Chi-squared value rather than an F-value which would be expected from the ANOVA that was purported to be used to analyze the results. The statistics reported in the results section do not match the statistical tests that were delineated in the methods section. Lines 304-306 only a P-value is reported, what statistical test was used here and what was the associated value? Additionally, were the data tested to confirm that they meet the assumptions of the statistical tests chosen for analysis? Did the data transformation correct for normality?

Response: All the percent data were transformed by arcsine and tested by PROC UNIVARIATE for normality. This information is added. All the F test values are added.

Comment #3-3: In the discussion section there is a lack of discussion about any potential shortcomings/weaknesses of the study and the discussion section should have more time spent discussing future directions.

Response: At the end of Discussion, the information is described as follows: “This control technique effectively suppressed the outbreak of F. occidentalis in a field assay and also prevented the occurrence of plant disease caused by TSWV in hot peppers. Although our current study directly evaluated the pulling efficacy of the attracting CAN trap, the pushing efficacy of the repellent fence treatment was indirectly predicted from a small-scale greenhouse experiment. Subsequent studies are needed to assess thrips occurrence outside of a test greenhouse treated with the alarm pheromone to improve the estimation of the pushing efficacy in field conditions.” This also explains a limitation of this technology in field conditions.

Comment #3-4: The abstract could use more information regarding the results of the study.

Response: The additional information is added as follows: “Field assay demonstrated the efficacy of the push-pull tactics by reducing thrips density in flowers of the hot peppers as well as in the monitoring traps. Especially, the enhanced mass trapping to the CAN trap compared to the conventional yellow sticky trap led to significant reduction in the thrips population. This novel push-pull technique would be applicable to effectively control F. occidentalis in field conditions.”

Comment #3-5: In the methods section under thrips rearing, it is stated that the thrips were fed a kidney bean diet for five days after germination. This could use some clarification as I believe the authors meant after hatching.

Response: Corrected as follows: “All thrips were fed a kidney bean (Phaseolus coccineus L.) diet according to the method described in our earlier study [12].”

Comment #3-6: Y-tube methods (line 137-146) could use more explanation. It says that thrips were used three days after emergence, is that referring to emergence from the egg (hatching), or emergence as adults from the pupal stage? Additionally, the methods state that each test used 10-30 individuals, were these individuals tested separately (one at a time in the olfactometer) or were they all places into the olfactometer at the same time, please clarify.

Response: Some uncertainty is rephrased as follows: “A Y-type olfactometer (the main Y-tube length, 5 cm; two branches 2 cm long; a 45o angle between the branches; inner branch diameter, 5 cm) was placed in a dark room at 25 ± 1oC to avoid visual cue for the choice test. A flow of clean (charcoal-filtered) air at a rate of 0.6 L/min was split and passed through two glass vessels containing either an odor source or control (hexane) stimuli and entered each of the branches of the Y-tube. An air supply system (Power Air Pump, Seoul, Korea) was used for air filtration and flow rate control. Before and after the bioassay, the Y-tube was cleaned with 100% methanol. Test thrips were used three days after adult emergence. Each test used 10 � 30 individuals of each sex and was replicated four times by changing the source and control for replication.”

Comment #3-7: Line 189-190 discussing the sampling of flowers states: “in flowers in 10 randomly selected flowers was counted in three fields three times a week for 190 one month (June 13 � July 13) once in a week”. This statement is confusing, was the sampling conducted three times a week or one time per week?

Response: This is rephrased as follows: “Number of F. occidentalis in flowers was counted from 10 randomly selected flowers with three replications in each field. The monitoring was performed once a week for one month (June 13 � July 13).”

Comment #3-8: Line 222- should be arcsine perhaps?

Response: Corrected as suggested

Comment #3-9: In discussing the results of the field study of the push and pull components, it is stated that the Frankliniella occidentalis numbers are lower in the experimental greenhouse, but what were the results for each life stage and sex? Was it only the total number of all life stages and males and females that were reduced, or was there a difference in the response of the different life stages (larvae versus adults) or sexes?

Response: We did not examine larval density because we assessed the efficacy mostly based on the trapping adults on the yellow sticky trap. However, the figure 6C suggests the total number of larvae and adults. Thus the reduction by the push-pull can be explained by the total reduction not only by adults.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(18.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0279646.r003

Decision Letter 1

Ramzi Mansour

8 Dec 2022

PONE-D-22-23616R1A push-pull strategy to control the western flower thrips, Frankliniella occidentalis, using alarm and aggregation pheromonesPLOS ONE

Dear Dr. Kim,

Please submit your revised manuscript by Jan 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Ramzi Mansour

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: all corrections were done. I feel that this manuscript A push-pull strategy to control the western flower thrips, Frankliniella occidentalis,using alarm and aggregation pheromones is now acceptable for publication

Reviewer #2: Dear Authors,

I read carefully the revised text of your mns PONE -D-22-23616, titled " A push-pull strategy to control the Western Flower Thrips, Frankliniella occidentalis,using alarm and aggregation pheromones". Even I read the replies you provided to each reviewers. I appreciated the effort done in order to justify and preserve your previous "draft", or to accept and change some parts as suggested by reviewers. In particular, I am satisfied for the adding of several new data to minor/major issues recorded in M& M and Results Sections. Such as detailed records regarding some texts , i.e. in subprgrs. 2.4, 2.5, 2.9 that more improved the data presentation , technical clarification , i.e. 2.3, and statistical analysis , i.e. 2.11. Details were added also to Results Section , i.e. F and P values in 3.1, 3.2 and 3.4 subparagraphs.

The new version of Discussion Section has more clearly recorded the main steps obtained in the technical part of this study , such as the control efficacy of push-pull tactics against Frankliniella occidentalis . Also the actractiveness of the aggregation pheromone compounds has been critically outlined for the other thrips species considered. Finally, the future perspectives of the novel push-pull tactics in the control of pest thrips might be proposed for open field applications.The well presented comments let me satisfied of the new version of the text, even without a separated Conclusions Section. Here, I attached the pdf of the revised version of the text that I consider suitable for publication.

Reviewer #3: Section 3-3 in the results section still reports statistical values (t-test and chi-squared) that are not mentioned in the methods section for statistical analyses performed. Are these the post-hoc analyses, or were they just forgotten in the methods section?

At the end of section 3-3 there is an F-value reported of 1600.00. This is an exceptionally high F-value, is that the accurate F-value or is the decimal point in the wrong place, for example should it actually be 160 or 16? If it is indeed 1600, that is very impressive!

All other comments have been addressed, the manuscript looks great.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Kara Tyler-Julian

**********

Attachment

Submitted filename: PONE-D-22-23616_R1 (3).pdf

Click here for additional data file.^{(3.3MB, pdf)}

PLoS One. 2023 Feb 24;18(2):e0279646. doi: 10.1371/journal.pone.0279646.r004

Author response to Decision Letter 1

8 Dec 2022

[Reviewer #1]

Comment: All corrections were done. I feel that this manuscript A push-pull strategy to control the western flower thrips, Frankliniella occidentalis,using alarm and aggregation pheromones is now acceptable for publication

Response: Thank you for a nice review!

[Reviewer #2]

Comment: I read carefully the revised text of your mns PONE -D-22-23616, titled " A push-pull strategy to control the Western Flower Thrips, Frankliniella occidentalis,using alarm and aggregation pheromones". Even I read the replies you provided to each reviewers. I appreciated the effort done in order to justify and preserve your previous "draft", or to accept and change some parts as suggested by reviewers. In particular, I am satisfied for the adding of several new data to minor/major issues recorded in M& M and Results Sections. Such as detailed records regarding some texts , i.e. in subprgrs. 2.4, 2.5, 2.9 that more improved the data presentation , technical clarification , i.e. 2.3, and statistical analysis , i.e. 2.11. Details were added also to Results Section , i.e. F and P values in 3.1, 3.2 and 3.4 subparagraphs. The new version of Discussion Section has more clearly recorded the main steps obtained in the technical part of this study, such as the control efficacy of push-pull tactics against Frankliniella occidentalis. Also the actractiveness of the aggregation pheromone compounds has been critically outlined for the other thrips species considered. Finally, the future perspectives of the novel push-pull tactics in the control of pest thrips might be proposed for open field applications. The well presented comments let me satisfied of the new version of the text, even without a separated Conclusions Section. Here, I attached the pdf of the revised version of the text that I consider suitable for publication.

Response: Thank you for a nice review!

[Reviewer #3]

Comment: Section 3-3 in the results section still reports statistical values (t-test and chi-squared) that are not mentioned in the methods section for statistical analyses performed. Are these the post-hoc analyses, or were they just forgotten in the methods section?

Response: The stat method is now added to the M&M.

Comment: At the end of section 3-3 there is an F-value reported of 1600.00. This is an exceptionally high F-value, is that the accurate F-value or is the decimal point in the wrong place, for example should it actually be 160 or 16? If it is indeed 1600, that is very impressive!

Response: We calculated the F value again and confirmed.

Comment: All other comments have been addressed, the manuscript looks great.

Response: Thank you for a nice review!

Attachment

Submitted filename: Response to Reviewers comments.docx

Click here for additional data file.^{(13.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0279646.r005

Decision Letter 2

Ramzi Mansour

12 Dec 2022

A push-pull strategy to control the western flower thrips, Frankliniella occidentalis, using alarm and aggregation pheromones

PONE-D-22-23616R2

Dear Dr. Kim,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ramzi Mansour

Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0279646.r006

Acceptance letter

Ramzi Mansour

16 Dec 2022

PONE-D-22-23616R2

A push-pull strategy to control the western flower thrips, Frankliniella occidentalis, using alarm and aggregation pheromones

Dear Dr. Kim:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Ramzi Mansour

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Chemical synthesis of the aggregation pheromone components of thrips: Lavandulyl acetate (LA, 2), lavandulyl methylbutanoate (LMB, 3), and neryl methylbutanoate (NMB, 5).

(DOCX)

Click here for additional data file.^{(665.8KB, docx)}

S2 Fig. Tests of two flower thrips (F. occidentalis (‘Fo’) and F. intonsa (‘Fi’)) to aggregation pheromone components using a Y-tube olfactometer.

(DOCX)

Click here for additional data file.^{(36.2KB, docx)}

Attachment

Submitted filename: PONE-D-22-23616.docx

Click here for additional data file.^{(557.7KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(18.8KB, docx)}

Attachment

Submitted filename: PONE-D-22-23616_R1 (3).pdf

Click here for additional data file.^{(3.3MB, pdf)}

Attachment

Submitted filename: Response to Reviewers comments.docx

Click here for additional data file.^{(13.3KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0279646.ref001] 1.Yudin LS, Cho JJ, Mitchell WC. Host range of western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), with special reference to Leucaena glauca. Environ Entomol. 1986; 15:1292–1295. [Google Scholar]

[pone.0279646.ref002] 2.Schneweis DJ, Whitfield AE, Rotenberg D. Thrips developmental stage-specific transcriptome response to tomato spotted wilt virus during the virus infection cycle in Frankliniella occidentalis, the primary vector. Virology. 2017; 500:226–237. [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref003] 3.He Z, Guo JF, Reitz SR, Lei ZR, Wu SY. A global invasion by the thrips, Frankliniella occidentalis: current virus vector status and its management. Insect Sci. 2020; 27:626–645. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref004] 4.Reitz SR. Biology and ecology of the western flower thrips (Thysanoptera: Thripidae): The making of a pest. Fla Entomol. 2009; 92:7–13. [Google Scholar]

[pone.0279646.ref005] 5.Shrestha A, Srinivasan R, Riley DG, Culbreath AK. Direct and indirect effects of a thrips-transmitted Tospovirus on the preference and fitness of its vector, Frankliniella fusca. Entomol Exp Appl. 2012; 145:260–271. [Google Scholar]

[pone.0279646.ref006] 6.Belliure B, Janssen A, Maris PC, Peters D, Sabelis MW. Herbivore arthropods benefit from vectoring plant viruses. Ecol Lett. 2005; 8:70–79. [Google Scholar]

[pone.0279646.ref007] 7.Rotenberg D, Jacobson AL, Schneweis DJ, Whitfield AE. Thrips transmission of tospoviruses. Curr Opin Virol. 2015; 15:80–89. doi: 10.1016/j.coviro.2015.08.003 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref008] 8.Lee S, Lee J, Kim S, Choi H, Park J, Lee J, et al. The incidence and distribution of viral diseases in pepper by cultivation types. Res Plant Dis. 2004; 10:231–240. [Google Scholar]

[pone.0279646.ref009] 9.Reitz SR, Gao Y, Kirk WDJ, Hoddle MS, Leiss KA, Funderburk JE. Invasion biology, ecology, and management of western flower thrips. Annu Rev Entomol. 2020; 65:17–37. doi: 10.1146/annurev-ento-011019-024947 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref010] 10.Woo KS, Ahn SB, Lee SH, Kwon HM. First record of Frankliniella occidentalis and its distribution and host plants in Korea. Korean J Appl Entomol. 1994; 33:127. [Google Scholar]

[pone.0279646.ref011] 11.Ding T, Chi H, Gökçe A, Gao Y, Zhang B. Demographic analysis of arrhenotokous parthenogenesis and bisexual reproduction of Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Sci Rep. 2018; 8:3346. doi: 10.1038/s41598-018-21689-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref012] 12.Kim C, Choi D, Kang J, Ahmed SA, Kil E, Kwon G, et al. Thrips infesting hot pepper cultured in greenhouses and variation in gene sequences encoded in TSWV. Korean J Appl Entomol. 2021; 60:387–401. [Google Scholar]

[pone.0279646.ref013] 13.Choi DY, Kim Y. PGE₂ mediation of egg development in western flower thrips, Frankliniella occidentalis. Arch Insect Biochem Physiol. 2022; e21949. [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref014] 14.Choi DY, Kim Y. Transcriptome analysis of female western flower thrips, Frankliniella occidentalis, exhibiting neo-panoistic ovarian development. PLoS ONE 2022; 17:e0272399. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref015] 15.Cho S, Kyung Y, Cho S, Shin S, Jeong D, Kim S, et al. Evaluation of susceptibility of western flower thrips (Frankliniella occidentalis) and garden thrips (F. intonsa) to 51 insecticides. Korean J Appl Entomol. 2018; 57:221–231. [Google Scholar]

[pone.0279646.ref016] 16.Bhuyain MMH, Lim UT. Relative susceptibility to pesticides and environmental conditions of Frankliniella intonsa and F. occidentalis (Thysanoptera: Thripidae), an underlying reason for their asymmetrical occurrence. PLoS ONE 2020; 15:e0237876. doi: 10.1371/journal.pone.0237876 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref017] 17.Gao Y, Yoon KA, Lee JH, Kim JH, Lee SH. Overexpression of glutamate-gated chloride channel in the integument is mainly responsible for emamectin benzoate resistance in the western flower thrips Frankliniella occidentalis. Pest Manag. Sci. 2022; doi: 10.1002/ps.7032 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref018] 18.Mouden S, Sarmiento KF, Klinkhamer PG, Leiss KA. Integrated pest management in western flower thrips: past, present and future. Pest Manag Sci. 2017; 73:813–822. doi: 10.1002/ps.4531 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref019] 19.Sampson C, Kirk WD. Can mass trapping reduce thrips damage and is it economically viable? Management of the Western flower thrips in strawberry. PLoS ONE 2013; 8:e80787. doi: 10.1371/journal.pone.0080787 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref020] 20.Olaniran OA, Sudhakar AV, Drijfhout FP, Dublon IA, Hall DR, Hamilton JG, et al. A male-predominant cuticular hydrocarbon, 7-methyltricosane, is used as a contact pheromone in the western flower thrips Frankliniella occidentalis. J Chem Ecol. 2013; 39:559–568. [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref021] 21.Kirk WD, Hamilton JG. Evidence for a male-produced sex pheromone in the western flower thrips Frankliniella occidentalis. J Chem Ecol. 2004; 30:167–174. [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref022] 22.Hamilton JG, Hall DR, Kirk WD. Identification of a male-produced aggregation pheromone in the western flower thrips Frankliniella occidentalis. J Chem Ecol. 2005; 31:1369–1379. [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref023] 23.Kirk WDJ, de Kogel WJ, Koschier EH, Teulon DAJ. Semiochemicals for thrips and their use in pest management. Annu Rev Entomol. 2021; 66:101–119. doi: 10.1146/annurev-ento-022020-081531 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref024] 24.Akinyemi AO, Kirk WDJ. Experienced males recognise and avoid mating with non-virgin females in the western flower thrips. PLoS ONE 2019; 14:e0224115. doi: 10.1371/journal.pone.0224115 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref025] 25.MacDonald KM, Hamilton JG, Jacobson R, Kirk WD. Analysis of anal droplets of the western flower thrips Frankliniella occidentalis. J Chem Ecol. 2003; 29:2385–2389. [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref026] 26.de Bruijn PJ, Egas M, Janssen A, Sabelis MW. Pheromone-induced priming of a defensive response in Western flower thrips. J Chem Ecol. 2006; 32:1599–1603. doi: 10.1007/s10886-006-9092-1 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref027] 27.de Bruijn PJ, Egas M, Sabelis MW, Groot AT. Context-dependent alarm signalling in an insect. J Evol Biol. 2016; 29:665–671. doi: 10.1111/jeb.12813 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref028] 28.Kim K, Kim M, Kwon G, Kim Y. Technologies required for development of trap-based MAT control against the striped fruit fly, Bactrocera scutellata. Korean J. Appl. Entomol. 2017; 56:51–60. [Google Scholar]

[pone.0279646.ref029] 29.SAS Institute Inc. SAS/STAT user’s guide, Release 6.03, Ed. Cary, NC. 1989.

[pone.0279646.ref030] 30.Lee GS, Lee JH, Kang SH, Woo KS. Thrips species (Thysanoptera: Thripidae) in winter season and their vernal activities on Jeju island, Korea. J Asia Pac Entomol. 2001; 4:115–122. [Google Scholar]

[pone.0279646.ref031] 31.Niassy S, Tamiru A, Hamilton JGC, Kirk WDJ, Mumm R, Sims C, et al. Characterization of male-produced aggregation pheromone of the bean flower thrips Megalurothrips sjostedti (Thysanoptera: Thripidae). J Chem Ecol. 2019; 45:348–355. doi: 10.1007/s10886-019-01054-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref032] 32.Akella SVS, Kirk WDJ, Lu YB, Murai T, Walters KFA, Hamilton JG. Identification of the aggregation pheromone of the melon thrips, Thrips palmi. PLoS ONE 2014; 9:e103315. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref033] 33.Li X, Geng S, Zhang Z, Zhang J, Li W, Huang J, et al. Species-specific aggregation pheromones contribute to coexistence in two closely related thrips species. Bull Entomol Res. 2019; 109:119–126. doi: 10.1017/S0007485318000366 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref034] 34.Zhu XY, Zhang PJ, Lu YB. Isolation and identification of the aggregation pheromone released by male adults of Frankliniella intonsa (Thysanoptera: Thripidae). Acta Entomol Sin. 2012; 55:376–385. [Google Scholar]

[pone.0279646.ref035] 35.Teerling CR, Pierce HD, Borden JH, Gillespie DR. Identification and bioactivity of alarm pheromone in the western flower thrips, Frankliniella occidentalis. 1993; 19:681–697. doi: 10.1007/BF00985001 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref036] 36.Cao Y, Zhi J, Cong C, Margolies DC. Olfactory cues used in host selection by Frankliniella occidentalis (Thysanoptera: Thripidae) in relation to host suitability. J Insect Behav. 2014; 27:41–56. [Google Scholar]

[pone.0279646.ref037] 37.Jung S, Park M, Lee S, Choi K, Hong Y, Bae S. et al. Development of wax-typed pheromone dispenser for mating disruption of the oriental fruit moth, Grapholita molesta, and its application technique. Korean J Appl Entomol. 2008; 47:255–263. [Google Scholar]

[pone.0279646.ref038] 38.Bleeker PM, Diergaarde PJ, Ament K, Guerra J, Weidner M, Schütz S, et al. The role of specific tomato volatiles in tomato-whitefly interaction. Plant Physiol. 2009; 151:925–935. doi: 10.1104/pp.109.142661 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref039] 39.Ho HY, Su YT, Ko CH, Tsai MY. Identification and synthesis of the sex pheromone of the Madeira mealybug, Phenacoccus madeirensis Green. J Chem Ecol. 2009; 35:724–732. [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref040] 40.Lee JS, Cho WK, Kim MK, Kwak HR, Choi HS, Kim KH. Complete genome sequences of three tomato spotted wilt virus isolates from tomato and pepper plants in Korea and their phylogenetic relationship to other TSWV isolates. Arch Virol. 2011; 156:725–728. doi: 10.1007/s00705-011-0935-x [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref041] 41.Jacobson AL, Kennedy GG. Specific insect-virus interactions are responsible for variation in competency of different Thrips tabaci isolines to transmit different Tomato Spotted Wilt Virus isolates. PLoS ONE 2013; 8:e54567. doi: 10.1371/journal.pone.0054567 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref042] 42.Cook SM, Khan ZR, Pickett JA. The use of push-pull strategies in integrated pest management. Annu Rev Entomol. 2007; 52:375–400. doi: 10.1146/annurev.ento.52.110405.091407 [DOI] [PubMed] [Google Scholar]

[pone.0279646.ref043] 43.Tyler-Julian K, Funderburk J, Srivastava M, Olson S, Adkins S. Evaluation of a push-pull system for the management of Frankliniella species (Thysanoptera: Thripidae) in tomato. Insects 2018; 9:187. doi: 10.3390/insects9040187 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0279646.ref044] 44.Niu Y, Han S, Wu Z, Pan C, Wang M, Tang Y. et al. A push-pull strategy for controlling the tea green leafhopper (Empoasca flavescens F.) using semiochemicals from Tagetes erecta and Flemingia macrophylla. Pest Manag Sci. 2022; 78:2161–2172. doi: 10.1002/ps.6840 [DOI] [PubMed] [Google Scholar]

PERMALINK

A push-pull strategy to control the western flower thrips, Frankliniella occidentalis, using alarm and aggregation pheromones

Chul-Young Kim

Falguni Khan

Yonggyun Kim

Roles

Abstract

1. Introduction

2. Materials and methods

2.1. Thrips rearing

2.2. Pheromones

2.3. Construction of CAN trap

Fig 2. Development of a yellow CAN trap (CAN) to attract F. occidentalis using the aggregation pheromone.

2.4. Wax formulation of alarm pheromone

Fig 5. Field assay of alarm pheromone-based repellent efficacy against F. occidentalis in a greenhouse cultivating hot peppers.

2.5. Aggregation behavior test using Y-tube olfactometer

2.6. Alarm pheromone test using dispersal behavior assay

2.7. Field test for alarm pheromone

2.8. Field test for push-pull tactics

Fig 6. Application of push-pull strategy to F. occidentalis infesting hot peppers (200 plants) in a greenhouse (660 m2).

2.9. Thrips monitoring test in the field

2.10. Molecular diagnosis of TSWV using multiplex PCR

2.11. Statistical analysis

3. Results

3.1. Optimal pheromone composition to attract thrips

Fig 1. Differential preference analysis of two flower thrips (F. occidentalis and F. intonsa) for aggregation pheromone using a Y-tube olfactometer.

3.2. Development of the yellow CAN trap containing aggregation pheromone

3.3. Trapping efficiency of CAN trap in field conditions

Fig 3. Field efficacy of a yellow CAN trap containing aggregation pheromone.

3.4. Development of thrips repellent using alarm pheromone

Fig 4. Development of a thrips repellent using an alarm pheromone of F. occidentalis.

3.5. Application of the push-pull strategy to an agricultural farm cultivating hot peppers

4. Discussion

Fig 7. A working model of the push-pull strategy against F. occidentalis using repellent (sound signal logo) and CAN trap (yellow can) in a greenhouse cultivating hot peppers.

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ramzi Mansour

Roles

Author response to Decision Letter 0

Decision Letter 1

Ramzi Mansour

Roles

Author response to Decision Letter 1

Decision Letter 2

Ramzi Mansour

Roles

Acceptance letter

Ramzi Mansour

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Fig 6. Application of push-pull strategy to F. occidentalis infesting hot peppers (200 plants) in a greenhouse (660 m²).