From Laboratory Screening to Greenhouse Flight Bioassay: Development of a Plant-Based Attractant for Tomicus brevipilosus

Abstract

Tomicus brevipilosus Eggers is a major forest pest affecting Pinus yunnanensis Franch and Pinus kesiya var. Langbianensis (A.Chev.) Gaussen ex Bui in Southwest China. While attractants exist for related species, this study aimed to develop a more effective, tailored attractant for T. brevipilosus. We assessed the activity of host plant volatiles using electroantennography (EAG). Female and male beetles showed strong responses to different compounds: females to β-pinene, terpinolene, (+)-3-carene, and (R)-(+)-limonene; males to (+)-3-carene, 2-isopropyl-5-methylanisole, and D(+)-camphor. An optimized blend of these compounds achieved a high selection rate (57 ± 20%) in olfactory assays. This study represents a crucial preliminary investigation. The concentrations and release systems (rubber septa and centrifuge tubes) were optimized under controlled conditions to identify the most promising candidate for future scaling, rather than for immediate large-scale application. In semi-field cage bioassays, trap catch was highest at mid-canopy height (1.5 times branch-free height), under the cage canopy, and in treatments with low-to-moderate canopy density. This work provides a foundation for developing improved monitoring and management tools for T. brevipilosus.

1. Introduction

Tomicus brevipilosus Eggers (Coleoptera: Curculionidae: Scolytinae), also known as Blastophagus khasianus Murayama [1], is a conifer bark beetle distributed across regions including Yunnan, Fujian, and Sichuan in China, Assam State in India, as well as the Palaearctic and Oriental zones of Japan and South Korea. This species primarily infests Pinus yunnanensis Franch, P. kesiya var. Langbianensis (A.Chev.) Gaussen ex Bui, P. koraiensis Siebold & Zucc, and other pine species [2,3]. In Yunnan Province, T. brevipilosus often occurs together with Tomicus yunnanensis Kirkendall & Faccoli and Tomicus minor Hartig, collectively causing significant mortality of P. yunnanensis and severe ecological and economic losses [3,4]. Their infestations seriously compromise the ecosystem services provided by P. yunnanensis forests, including water and soil conservation, carbon sequestration and oxygen release, nutrient accumulation, atmospheric purification, forest protection, and biodiversity maintenance [3,4]. Critically, effective monitoring and control tools specifically for T. brevipilosus remain underdeveloped. While research on attractants has been conducted for the sympatric species T. yunnanensis and T. minor, the olfactory preferences and optimal lure composition for T. brevipilosus are distinct and cannot be directly extrapolated, necessitating species-specific development [5,6]. Current management strategies largely draw from those used for Dendroctonus and Ips species, such as the application of Paecilomyces fungi, removal of infested wood, push-pull tactics, and trap-tree methods [7]. A common and direct application of the pest-host relationship is through the use of attractant-baited traps for monitoring and mass trapping. Currently, funnel traps and cross-vane traps baited with generic host volatiles or pheromones are sometimes employed against Tomicus spp., but their efficiency is often suboptimal and not species-specific [8]. However, these measures have so far been insufficient to curb the ongoing spread of these bark beetles.

T. brevipilosus exhibits a complex life history, including trunk invasion and shoot-feeding behaviors, which are closely regulated by chemical signals [9,10]. A field trapping experiment by Liu et al. indicated that combining myrtenol and trans-verbenol with S-(−)-α-pinene and S-(−)-β-pinene captured more adult T. brevipilosus [11]. According to existing research, semiochemicals induced by T. brevipilosus also have an attractive effect on T. yunnanensis and T. minor [12]. Concurrently, pine trees defend themselves by producing a range of resin acids and terpenoid compounds [13]. For instance, Yan et al. studied volatiles from P. yunnanensis affected by insect pests and conducted behavioral choice assays with T. yunnanensis. Their results showed that α-pinene, γ-terpinene, and 3-carene significantly enhanced the attraction of P. yunnanensis to T. yunnanensis, whereas β-pinene and 4-allylanisole acted as repellents [14].

For many bark beetles, particularly species within the Tomicus genus, host-derived volatiles often serve as the primary initial attractants, enabling them to locate susceptible trees. This reliance on kairomones (rather than solely on conspecific pheromones) provides a solid ecological basis for developing plant-based attractants, which can be highly species-specific and reduce non-target effects [15]. For example, research by Chen et al. demonstrated that in Anning County, Yunnan Province, T. brevipilosus preferred to attack and reproduce on P. yunnanensis trees already infested by T. yunnanensis and T. minor from early March to mid-April [16]. The behavior of these three beetle species is likely influenced by host plant volatiles and volatiles from other bark beetles. Utilizing host plant volatiles for trapping and killing bark beetles shows potential for prevention and control [17].

Plant volatiles play a key role in regulating host-searching and location behaviors of herbivorous insects [18]. Herbivorous insects primarily rely on their sophisticated olfactory systems to detect odors released by plants to locate suitable hosts for survival and reproduction. For instance, Liu et al. conducted GC-MS analyses on P. yunnanensis at different infestation stages and screened active compounds using Gas Chromatography-Electroantennographic Detection (GC-EAD) and Y-tube olfactometer assays. Their results revealed that the dynamic changes in Volatile Organic Compounds (VOCs) induced by Tomicus infestation guide the beetles’ behavior. Attractants likely facilitate host colonization during the early stages of pest infestation, whereas repellents may indicate declining host suitability in later stages [19]. This synergistic interaction between plant volatiles and insect pheromones enables insects to locate suitable hosts more accurately and efficiently.

Current research on T. brevipilosus remains limited, despite the importance of attractants in monitoring and controlling this pest population. Therefore, We selected 22 volatiles from P. yunnanensis and P. kesiya var. langbianensis based on previous literature [20,21]. Using electroantennography (EAG), we assessed the electrophysiological responses of T. brevipilosus to these volatiles to identify the most active compounds. These were then blended, and the most effective mixture was determined through behavioral assays. The choice of release carrier is critical for field application, as it influences the stability and persistence of the volatile emission. We compared two conventional slow-release devices—rubber septa and centrifuge tubes—by measuring residual volatiles using gas chromatography, thereby identifying the optimal carrier for each compound. Furthermore, the trapping efficiency is significantly affected by the height and location of the trap within the forest. Through simulated field trials, we determined the optimal trap height, forest position, and canopy density for effective capture. These findings offer a scientific basis for refining the trapping strategy for T. brevipilosus. Nevertheless, future studies should further investigate the behavioral traits and environmental adaptability of different populations of T. brevipilosus to enhance the stability and general applicability of the trapping methodology.

2. Materials and Methods

2.1. Test Insects

The test insects were collected from the Mengxian Town Forest Farm in Ning’ er County, Pu’ er City, Yunnan Province. Bark beetles infesting the shoots of P. kesiya var. langbianensis were selected, and the affected shoots were excised and transported to the laboratory. Adult T. brevipilosus were subsequently identified based on key morphological characteristics, including the presence or absence of mycangia in the second groove of the elytral declivity, the pattern of punctures, the length of mycangial hairs, the shape of the abdominal tergite, and the orientation of bristles. The intact pine shoots were stored at 4 °C for no longer than one month. The identified adults were maintained in insect-rearing containers with wood sections of Simao pine, under controlled conditions of temperature (24 ± 1) °C and relative humidity 65% ± 5%.

2.2. Experimental Materials

Compound selection: Based on previous literature [22,23,24,25], twenty-two volatile compounds from the shoots and trunks of P. yunnanensis and P. kesiya var. langbianensis were selected as experimental reagents due to their notable attractancy to Tomicus spp. These compounds are listed in

Table 1. The 22 plant volatiles used in this study.

No.	Compound	CAS	Purity	Source
1	β-Caryophyllene	87-44-5	>80% (GC)	Macklin
2	Camphene	79-92-5	96% (GC)	Macklin
3	(+)-3-Carene	498-15-7	≥90% (GC)	Macklin
4	γ-Terpinene	99-85-4	95%(GC)	Macklin
5	β-Pinene	18172-67-3	≥95% (GC)	Macklin
6	Myrcene	123-35-3	≥90.0% (GC)	Macklin
7	α-Pinene	80-56-8	98% (GC)	Macklin
8	(+)-α-Pinene	7785-70-8	98% (GC)	Macklin
9	Sabinene	3387-41-5	98% (GC)	Macklin
10	β-Phellandrene	555-10-2	85% (GC)	Macklin
11	Terpinolene	586-62-9	85% (GC)	Macklin
12	D(+)-Camphor	464-49-3	≥96% (GC)	Macklin
13	(—)-Myrtenol	515-00-4	95% (GC)	Macklin
14	α-Caryophyllene	6753-98-6	93% (GC)	Macklin
15	2-Isopropyl-5-methylanisole	1076-56-8	≥96% (GC)	Macklin
16	(R)-(+)-Limonene	5989-27-5	≥99% (GC)	Macklin
17	α-Terpineol	10482-56-1	98% (GC)	Macklin
18	Bornyl acetate	76-49-3	≥97% (GC)	Macklin
19	(+)-Longifolene	475-20-7	90% (GC)	Macklin
20	trans-β-Farnesene	18794-84-8	95% (GC)	Macklin
21	2,6-Di-tert-butyl-4-methylphenol	128-37-0	>99.0% (GC)	Macklin
22	(S)-cis-Verbenol	18881-04-4	≥95% (GC)	Macklin

.

Test materials: The materials and equipment used in the study included: 1.5 mL centrifuge tubes; rubber septa; an atmospheric sampler; activated charcoal tubes (outer diameter: 6 mm, length: 75 mm); Pasteur pipettes; odorless Teflon tubing; amber glass vials; the adsorbent Paropak Q (Waters Associates Inc., Milford, MA, USA; 50–80 mesh); and n-hexane (chromatographic grade). Additionally, three 120-mesh insect cages (2 m × 2 m × 2 m) were employed, along with magnifying glasses, sticky traps, plastic bottles, scissors, scalpels, heavy-duty string, and resealable plastic bags.

2.3. Selective Responses of T. Brevipilosus to Plant Volatiles

2.3.1. Preparation of Monomer Compound Volatile Solution

Based on the results of preliminary experiments, the six selected plant volatiles—(+)-3-carene, β-pinene, terpinolene, D(+)-camphor, 2-isopropyl-5-methyl anisole, and (R)-(+)-limonene—were individually mixed and diluted with liquid paraffin to prepare three concentration levels (1, 10, and 100 μg/μL). Pure liquid paraffin was used as the control.

2.3.2. Preparation of Mixed Plant Compound Solution

The six volatiles showing the highest EAG activity were mixed at their respective optimal concentrations using liquid paraffin as the solvent, resulting in four test mixtures. A two-choice olfactory assay was then conducted to evaluate the behavioral responses of T. brevipilosus to these mixtures, with pure liquid paraffin serving as the control.

2.3.3. Electroantennogram (EAG)

In this experiment, the electroantennogram (EAG) responses of male and female T. brevipilosus adults to plant volatiles were measured using an IDAC-2 electroantennograph (Syntech, Lindau, Germany), with a continuous moisturized air stream directed at the fixed antennae at a flow rate of 400 mL/min. Adult heads were excised with a fine scalpel, and both ends of an isolated antenna were carefully attached to the electrodes of a PR probe using Spectra^® 360 conductive gel, taking care not to submerge the antenna in the gel, to ensure proper electrical contact.

A 10 μL aliquot of each test volatile solution was applied evenly to a filter paper strip (4.0 cm × 1.0 cm), which was then inserted into a Pasteur pipette. During stimulation, the puff duration was set to 0.5 s, with an interval of at least 30 s between consecutive stimulations. A 1-min interval was maintained when switching between different concentrations of the same compound. For each antenna, all concentrations of a single compound were tested sequentially from low to high. Each treatment (volatile and concentration) was replicated five times for each sex, resulting in 10 replicates per treatment in total.

To minimize the influence of individual variation and compensate for the decline in antennal viability over time during the experiment, the EAG response to each test substance was normalized relative to the antenna’s response to the liquid paraffin control (100% liquid paraffin). The number of replicates for the control group was consistent with that of the experimental groups.

2.3.4. Behavioral Experiment

The behavioral responses of T. brevipilosus to the mixed volatile compounds were evaluated using a Y-tube olfactometer under controlled environmental conditions (temperature: 25 °C; relative humidity: 45–55%). Each arm of the olfactometer was connected to a spherical drying tube, which in turn was connected to a vacuum pump to ensure a stable and continuous air stream during the experiment. The base of the olfactometer’s main stem was plugged with absorbent cotton to prevent the insects from escaping.

A filter paper strip (3.0 cm × 1.0 cm) impregnated with 10 μL of the test sample was placed in one drying tube to serve as the odor source. The contralateral drying tube contained a filter paper strip with an equivalent amount of liquid paraffin as the control. For each test, ten T. brevipilosus adults were introduced at the entrance of the olfactometer’s main stem. The entire apparatus was covered with black polyethylene film to eliminate any potential bias from the insects’ phototactic behavior. The vacuum pump was activated, and the airflow in the main stem was adjusted to 200 L/h.

After a 10-min acclimatization period, the insects’ choices were recorded. An insect was considered to have chosen the odor source if it moved more than halfway into the corresponding arm or entered the attached drying tube. Conversely, movement beyond the midpoint of the control arm or into its drying tube was recorded as a choice for the control. A lack of movement beyond these points within the observation period was noted as no response.

Each bioassay was performed using a cohort of 30 field-collected adults. In all the behavioral measurements, the number of adult T. brevipilosus used for each treatment was 10. The same individuals were used for one complete round of testing across all samples but were discarded afterward to avoid habituation. Each treatment was replicated five times. Calculate according to Formulas (1)–(3).

$$\mathrm{S}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\%)={\displaystyle \frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s} \mathrm{s}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{o}\mathrm{d}\mathrm{o}\mathrm{r} \mathrm{s}\mathrm{o}\mathrm{u}\mathrm{r}\mathrm{c}\mathrm{e}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}}} \times 100$$

(1)

$$\mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{e} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\%)={\displaystyle \frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s} \mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}}} \times 100$$

(2)

$$\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{s}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\%)={\displaystyle \frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s} \mathrm{s}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}}} \times 100$$

(3)

2.4. Selection of Slow-Release Carriers for Attractant Application in Forest

2.4.1. Preparation of Sustained-Release Materials

A gas chromatography (GC) system with flame ionization detection (FID) was established for quantification using the external standard method. Based on pre-experiments that estimated the residual amount in rubber septa (0.1–50 µg/g) and the release rate from the liquid paraffin-centrifuge tube system (0.5–200 µg/d), a calibration curve was prepared using n-hexane standard solutions at five concentrations: 1, 10, 50, 100, and 200 µg/mL. The GC analysis followed a modified method from Zhang [19]. Separation was achieved on a DB-5MS capillary column (30 m × 0.25 mm × 0.25 µm) with helium (99.999%) as the carrier gas at a flow rate of 1.2 mL/min. The injector and detector temperatures were both set at 280 °C. The oven temperature program was: 50 °C held for 2 min, ramped to 180 °C at 10 °C/min and held for 5 min, then ramped to 280 °C at 15 °C/min and held for 10 min. The injection volume was 2 µL. Each standard concentration was analyzed in triplicate. The standard curves for all seven compounds, constructed by plotting peak area against concentration, showed excellent linearity with correlation coefficients (R²) greater than 0.99, confirming the method’s suitability for quantitative analysis (

Table 2. Calibration formula of standard curve for content of 7 compounds and peak area.

Compound	Standard Concentration Determination of Concentration (μg/mL)	Calibration Equation	Correlation Coefficient (R2)
β-Pinene	1, 10, 50, 100, 200	y = 227.11x − 1259.9	0.993
(+)-3-Carene	1, 10, 50, 100, 200	y = 812.15x − 3178.2	0.9908
2-Isopropyl-5-methylanisole	1, 10, 50, 100, 200	y = 727.26x − 2083	0.9904
Terpinolene	1, 10, 50, 100, 200	y = 1135.8x − 6476.7	0.9921
(R)-(+)-Limonene	1, 10, 50, 100, 200	y = 1174.2x − 1317.3	0.9904
(S)-cis-Verbenol	1, 10, 50, 100, 200	y = 1643x − 1488.6	0.9939
D(+)-Camphor	1, 10, 50, 100, 200	y = 1490.4x − 1981.1	0.9916

).

2.4.2. Preparation of the Rubber Septum Sustained-Release Dispenser

Identical rubber septa were prepared by puncturing each one with 10–15 small holes using tweezers. This perforation facilitated solvent absorption and promoted a more uniform distribution of the volatile compounds within the septum. Subsequently, 100 μL of the mixed solution was precisely measured and applied into the central groove of each septum. The septa were then allowed to soak for 24 h to ensure complete absorption of the solution before use.

2.4.3. Preparation and Field Deployment of Sustained-Release Dispensers

The centrifuge tube dispensers were prepared by pipetting 1 mL of the mixed solution (diluted in liquid paraffin) into 1.5 mL centrifuge tubes. The lids were tightly closed and further sealed with parafilm to prevent evaporation or leakage. A pre-made hole (1 mm in diameter) in each lid served as a release vent for the volatile compounds. Both the completed rubber septum and centrifuge tube dispensers were deployed by suspension in an open outdoor area with good ventilation to facilitate compound diffusion. A distance of 20 m was maintained between individual dispensers to avoid cross-interference, with three replicates set up for each treatment type.

2.4.4. Determination of Release Rates

Following the preparation and outdoor deployment of the rubber septum and centrifuge tube dispensers, specific time points (0, 1, 2, 3, 4, 7, 10, 13, 16, 19, 22, 25, and 28 days) were selected for sampling. At each interval, the residual volatile compounds in the dispensers were collected and analyzed to determine their release profiles over time.

For the rubber septa, the residual amount of volatiles was determined using an organic solvent extraction method. Each retrieved septum was cut into small pieces (2–4 mm³) with scissors. The pieces were transferred into a 4.5 mL amber glass vial to protect the contents from light. Then, 2.0 mL of n-hexane was accurately added to the vial, which was immediately sealed. Extraction was allowed to proceed for 24 h to ensure complete dissolution of the residual compounds into the solvent.

The extracted solution was analyzed by gas chromatography (GC) with an injection volume of 2 µL. The peak area for each compound was recorded and converted into its residual mass (µg) using the pre-established standard curve equations. Finally, the embedding rate, residual rate, and release rate of the volatiles from the rubber septa were calculated using Equations (4)–(7) to comprehensively evaluate the performance of this sustained-release system.

$$\mathrm{Embedding} \mathrm{rate} (\%)={\displaystyle \frac{\mathrm{Residual} \mathrm{amount} \mathrm{on} \mathrm{Day} 0}{\mathrm{Theoretical} \mathrm{content} \mathrm{of} \mathrm{volatile}}} \times 100$$

(4)

$$\mathrm{Residual} \mathrm{rate} (\%)={\displaystyle \frac{\mathrm{Residual} \mathrm{amount} \mathrm{on} \mathrm{a} \mathrm{specific} \mathrm{day}}{\mathrm{Theoretical} \mathrm{content} \mathrm{of} \mathrm{volatile}}} \times 100$$

(5)

$$\begin{array}{cc}\mathrm{Release} \mathrm{rate} \mathrm{for} \mathrm{the} \mathrm{first} 7 \mathrm{days} (\% {\mathrm{day}}^{-1})& =\mathrm{Residual} \mathrm{amount} \mathrm{on} \mathrm{the} \mathrm{previous} \mathrm{day}\\ & - \mathrm{Residual} \mathrm{amount} \mathrm{on} \mathrm{the} \mathrm{current} \mathrm{day}\end{array}$$

(6)

$$\mathrm{Release} \mathrm{rate} \mathrm{after} 7 \mathrm{days} (\% {\mathrm{day}}^{-1})={\displaystyle \frac{\mathrm{Residual} \mathrm{amount} 3 \mathrm{days} \mathrm{prior} - \mathrm{Residual} \mathrm{amount} \mathrm{on} \mathrm{the} \mathrm{current} \mathrm{day}}{3}}$$

(7)

2.5. Simulated Field Experiment for Trap Placement Selection

Three large vertical insect-rearing cages were arranged in a row with 1 m spacing between them inside a greenhouse at the Houshan Botanical Garden of the Yunnan Academy of Forestry and Grassland Science. P. yunnanensis seedlings were placed inside each cage. A small hole was made precisely at the center of the top of each cage (which was sealed with tape after setup), through which a trap containing the lure was suspended. A white porcelain pan filled with soapy water was positioned directly beneath each trap to capture insects.

For each replicate, a total of 10 T. brevipilosus adults were released gently onto the pine seedlings within a cage, with the number per seedling randomized. The experiment was conducted over 7-day cycles. At the end of each cycle, all captured insects were collected. The recovered beetles were then carefully sorted and identified.

2.5.1. Experiment on Trap Height

A self-made trap was positioned at the center of each insect-rearing cage, with eight potted host plants (P. yunnanensis) arranged equidistantly around it to ensure uniform spatial distribution within the cage. The trap’s hanging height was set as a relative value. Specifically, we first measured the height from the soil surface to the top of the branch-free trunk section for each seedling (denoted as h). The trap was then suspended at 0.5, 1.0, 1.5, 2.0, and 2.5 times this height (Figure 1). The lure consisted of 1 mL of the selected compound (identified in Section 2) dispensed via a centrifuge tube slow-release device. The experiment included five height treatments, each with three replicates, resulting in a total of 15 experimental units. The optimal trap hanging height was determined based on capture efficiency. After each 7-day trial, all captured adult T. brevipilosus were collected and counted, and the lure was replaced.

2.5.2. Experiment on Trap Placement Within the Forest

Potted P. yunnanensis seedlings were arranged to simulate three distinct forest positions: forest gap, understory, and forest edge. The trap was suspended at the optimal height determined in the previous experiment (Section 2.5.1). The lure consisted of 1 mL of the selected compound (identified in Chapter 2) delivered via a centrifuge tube-based slow-release dispenser. The experimental design included three placement treatments, each with three replicates, resulting in a total of nine experimental units. The most effective trap location was determined based on capture rates. After each trial cycle, all captured adult T. brevipilosus were collected and counted, and the lure was replaced.

2.5.3. Experiment on Canopy Density Selection

The key variable in this experiment was canopy density, defined as the ratio of the vertical projection area of the tree crowns to the total forest floor area (a dimensionless quantity ranging from 0 to 1), which is a key indicator of light conditions within the forest. We simulated four distinct canopy density grades by using shade nets and adjusting the arrangement of potted P. yunnanensis seedlings: high (0.9–1.0), moderate (0.6–0.7), low (0.5–0.6), and very low (0.3–0.4). Traps were suspended at the previously determined optimal height (Section 2.5.1), using 1 mL of the selected compound in a centrifuge tube as the lure. The experimental design included four canopy density treatments with three replicates each, totaling 12 experimental units. The most effective canopy density for trap placement was evaluated based on capture success. After each trial, all captured adult T. brevipilosus were collected and the lures were replaced.

2.6. Data Analysis

Electroantennogram (EAG) data and behavioral assay data were processed and analyzed using Microsoft Excel 2010 and IBM SPSS Statistics 21.0. A t-test was applied to analyze the behavioral choice data. The relative EAG response value was calculated using the following formula: Relative EAG Response = (Mean EAG Response to Plant Volatile)/[(Mean Pre-test Control EAG Response + Mean Post-test Control EAG Response)/2] × 100%.

Data organization and statistical analyses were performed using Excel 2010 and SPSS 21.0. A paired-sample t-test was used to compare the effects of the two sustained-release materials (rubber septum vs. centrifuge tube) on the compound embedding rate and final residual rate. For comparisons of the embedding rate, release rate, and final residual rate among different sustained-release materials, a Tukey’s test was employed for post-hoc multiple comparisons. The significance level was set at α = 0.05. A chi-square (χ²) test was used to assess the difference in release rates for the same compound between the two sustained-release formulations.

3. Results

3.1. Electrophysiological and Behavioral Responses of T. Brevipilosus to Plant Volatiles

3.1.1. EAG Responses of T. Brevipilosus Adults to 22 Plant Volatiles

Female adults of T. brevipilosus exhibited significantly higher relative EAG responses to β-pinene, terpinolene, (+)-3-carene, and (R)-(+)-limonene compared to the other 18 compounds tested, with all relative values exceeding 40% (

Table 3. EAG responses of male and female adults to 22 plant volatiles.

Volatile Number	Plant Volatiles	Relative Value of Female Adults (%)	Relative Value of Male Adults (%)
1	β-Pinene	65 ± 22 a	11 ± 5 cd
2	β-Phellandrene	37 ± 12 bcde	16 ± 2 cd
3	Sabinene	29 ± 5 cdefg	17 ± 5 bc
4	Bornyl acetate	15 ± 1 efg	18 ± 11 bc
5	2-Isopropyl-5-methylanisole	33 ± 6 bcdef	42 ± 8 a
6	Terpinolene	48 ± 5 abcd	20 ± 2 bc
7	(+)-3-Carene	57 ± 4 ab	47 ± 8 a
8	Myrcene	14 ± 4 efg	18 ± 1 bc
9	(−)-Myrtenol	25 ± 4 defg	18 ± 6.76 bc
10	D(+)-Camphor	13 ± 4 efg	44 ± 9 a
11	(+)-α-Pinene	12 ± 0 efg	15 ± 0 cd
12	γ-Terpinene	12 ± 2 efg	12 ± 5 cd
13	β-Caryophyllene	10 ± 3 fg	16 ± 5 cd
14	α-Pinene	12 ± 4 efg	11 ± 1 cd
15	(S)-cis-Verbenol	15 ± 9 efg	27 ± 9 b
16	(+)-Longifolene	9 ± 4 fg	6 ± 2 d
17	trans-β-Farnesene	12 ± 2 fg	10 ± 1 cd
18	(R)-(+)-Limonene	53 ± 48 abc	12 ± 4 cd
19	α-Caryophyllene	7 ± 2 g	9 ± 2 cd
20	α-Terpineol	8 ± 3 fg	14 ± 2 cd
21	Camphene	16 ± 9 efg	12 ± 5 cd
22	2,6-Di-tert-butyl-4-methylphenol	13 ± 4 efg	10 ± 6 cd

Note: Different letters among treatments indicate significantly different means.

). The strongest response was observed for β-pinene (65 ± 22%), followed by (+)-3-carene (57 ± 4%), (R)-(+)-limonene (53 ± 48%), and terpinolene (48 ± 5%). Additionally, the relative responses to β-phellandrene (37 ± 12%) and 2-isopropyl-5-methylanisole (33 ± 6%) were significantly higher than those to the remaining 16 compounds, with values above 30%. For the other 16 compounds, the relative EAG responses of females were all below 30%.

Male adults showed significantly higher relative EAG responses to (+)-3-carene, 2-isopropyl-5-methylanisole, and D(+)-camphor compared to the other 19 compounds, with all values above 40% (

Table 4. The female adults of T. brevipilosus responded to EAG at different concentrations of six compounds.

Compound/Concentration	1 μg/μL	10 μg/μL	100 μg/μL
β-Pinene	16 ± 4 c	65 ± 16 a	37 ± 9 b
Terpinolene	30 ± 8 c	65 ± 8 a	49 ± 8 b
2-Isopropyl-5-methylanisole	11 ± 2 b	15 ± 3 a	15 ± 4 a
D(+)-Camphor	13 ± 8 ab	18 ± 6 a	9 ± 4 b
(R)-(+)-Limonene	49 ± 12 b	46 ± 3 b	73 ± 10 a
(+)-3-Carene	57 ± 10 a	68 ± 10 a	24 ± 3 b

Note: Different letters among treatments indicate significantly different means.

). The strongest response in males was to (+)-3-carene (47 ± 8%), followed by D(+)-camphor (44 ± 9%) and 2-isopropyl-5-methylanisole (42 ± 8%). Among the remaining compounds, the response to (S)-cis-verbenol (27 ± 9%) was significantly higher than that to the other 18 compounds.

Significant sexual dimorphism in EAG responses was observed for several compounds. The relative responses of females to β-pinene, β-phellandrene, terpinolene, and (R)-(+)-limonene were significantly higher than those of males. Conversely, the response to D(+)-camphor was significantly greater in males than in females. No significant differences between sexes were detected for the remaining 17 compounds. Notably, both male and female adults showed high relative responses to 2-isopropyl-5-methylanisole and (+)-3-carene.

Based on these results, six compounds that elicited relative EAG responses greater than 40% in at least one sex—β-pinene, terpinolene, (+)-3-carene, (R)-(+)-limonene, 2-isopropyl-5-methylanisole, and D(+)-camphor—were selected for further dose-response experiments at concentrations of 1, 10, and 100 μg/μL.

3.1.2. EAG Responses of Female Adults to Different Concentrations of Six Plant Volatiles

The relative EAG responses of female T. brevipilosus adults to the six selected volatiles varied with concentration (Table 4). For β-pinene and terpinolene, the response at 10 μg/μL was significantly higher than at 1 μg/μL or 100 μg/μL. In the case of (+)-3-carene, the responses at 1 μg/μL and 10 μg/μL were significantly greater than at 100 μg/μL. Conversely, for (R)-(+)-limonene, the response at 100 μg/μL was significantly higher than at the two lower concentrations. No significant differences were observed among the three concentrations for 2-isopropyl-5-methylanisole or D(+)-camphor, although the response tended to be slightly higher at 10 μg/μL. In summary, with the exception of (R)-(+)-limonene, which elicited the strongest response at 100 μg/μL, the highest relative EAG response for the other five compounds was observed at 10 μg/μL.

3.1.3. EAG Responses of Male Adults to Different Concentrations of Six Plant Volatiles

The relative EAG responses of male T. brevipilosus adults to the six volatiles showed distinct concentration-dependent patterns (

Table 5. EAG responses of male adults to different concentrations of 6 compounds.

Compound/Concentration	1 μg/μL	10 μg/μL	100 μg/μL
β-Pinene	7 ± 4 b	16 ± 4 a	10 ± 4 b
Terpinolene	17 ± 3 b	27 ± 7 a	30 ± 4 a
2-Isopropyl-5-methylanisole	84 ± 15 a	62 ± 12 b	29 ± 6 c
D(+)-Camphor	51 ± 12 a	54 ± 11 a	25 ± 8 b
(R)-(+)-Limonene	25 ± 6 b	20 ± 4 b	32 ± 6 a
(+)-3-Carene	28 ± 5 b	74 ± 14 a	18 ± 4 b

Note: Different letters among treatments indicate significantly different means.

). The response to β-pinene and (+)-3-carene was significantly higher at 10 μg/μL than at 1 μg/μL or 100 μg/μL. For terpinolene, responses at 10 μg/μL and 100 μg/μL were significantly greater than at 1 μg/μL. In contrast, 2-isopropyl-5-methylanisole elicited a significantly stronger response at 1 μg/μL compared to the two higher concentrations. Similarly, for D(+)-camphor, the responses at 1 μg/μL and 10 μg/μL were significantly higher than at 100 μg/μL. The response to (R)-(+)-limonene was significantly greater at 100 μg/μL than at the lower concentrations.

Based on the EAG results, the most effective concentrations for eliciting strong responses from female and male adults were selected for behavioral assays. The selected compounds and concentrations were as follows: for females, 10 μg/μL β-pinene, 10 μg/μL terpinolene, 100 μg/μL (R)-(+)-limonene, and a combination of 1 μg/μL and 10 μg/μL (+)-3-carene; for males, 1 μg/μL 2-isopropyl-5-methylanisole and a combination of 1 μg/μL and 10 μg/μL D(+)-camphor. These were blended into a single lure mixture for subsequent indoor behavioral experiments.

3.1.4. Behavioral Responses of T. Brevipilosus Adults to Four Mixed Plant Volatiles

The olfactory behavioral responses of T. brevipilosus to four mixed volatile blends were evaluated using a Y-tube olfactometer. The response data were calculated using the relevant formulas, and the detailed results are presented in

Table 6. Olfactory responses of the adults to 4 mixed compounds.

Composite Number of Mixed Compounds	Compounds	Selection Rate (%)	Reactivity (%)	Control (%)
1	10 μg/μL β-Pinene + 10 μg/μL Terpinolene + 1 μg/μL 2-Isopropyl-5-methylanisole + 1 μg/μL D(+)-Camphor + 100 μg/μL (R)-(+)-Limonene + 1 μg/μL (+)-3-Carene	40 ± 25 b	67 ± 33 a	17 ± 15 a
2	10 μg/μL β-Pinene + 10 μg/μL Terpinolene + 1 μg/μL 2-Isopropyl-5-methylanisole + 1 μg/μL D(+)-Camphor + 100 μg/μL (R)-(+)-Limonene + 10 μg/μL (+)-3-Carene	57 ± 20 a	67 ± 16 a	67 ± 10 a
3	10 μg/μL β-Pinene + 10 μg/μL Terpinolene + 1 μg/μL 2-Isopropyl-5-methylanisole + 10 μg/μL D(+)-Camphor + 100 μg/μL (R)-(+)-Limonene + 1 μg/μL (+)-3-Carene	27 ± 16 c	50 ± 28 a	17 ± 15 a
4	10 μg/μL β-Pinene + 10 μg/μL Terpinolene + 1 μg/μL 2-Isopropyl-5-methylanisole + 10 μg/μL D(+)-Camphor + 100 μg/μL (R)-(+)-Limonene + 10 μg/μL (+)-3-Carene	30 ± 11 bc	57 ± 27 a	27 ± 24 a

Note: Different letters among treatments indicate significantly different means.

.

The selection rate for Mixture 2, composed of 10 μg/μL β-pinene, 10 μg/μL terpinolene, 1 μg/μL 2-isopropyl-5-methylanisole, 1 μg/μL D(+)-camphor, 100 μg/μL (R)-(+)-limonene, and 10 μg/μL (+)-3-carene blended at a 1:1 volume ratio, was significantly higher than those of the other three mixtures. The overall order of selection rates was: Mixture 2 > Mixture 1 > Mixture 4 > Mixture 3. No significant differences were observed in the response rates or the control selection rates among the four mixtures.

Since T. brevipilosus often co-infests with T. yunnanensis and T. minor, and (S)-cis-verbenol has been reported as an effective attractant for the latter two species [17], (S)-cis-verbenol was incorporated into the mixed compound for subsequent experiments.

3.2. Selection of a Sustained-Release Carrier for Field Application of the Attractant

3.2.1. Embedding Rate in Different Sustained-Release Materials

The embedding rates of the individual compounds varied significantly between the two carrier materials (Figure 2). The embedding rates of D(+)-camphor and terpinolene were significantly higher in the centrifuge tubes than in the rubber septa. In contrast, the embedding rates of (S)-cis-verbenol and (R)-(+)-limonene were significantly higher in the rubber septa. No significant differences were observed between the two carriers for β-pinene, (+)-3-carene, or 2-isopropyl-5-methylanisole.

When comparing compounds within the same carrier, no significant differences in embedding rate were found among D(+)-camphor, 2-isopropyl-5-methylanisole, and β-pinene in the centrifuge tubes. Similarly, the embedding rates of (+)-3-carene and (R)-(+)-limonene were not significantly different from each other in this carrier. Significant differences were detected among all other pairwise comparisons within the centrifuge tubes.

For the mixture containing all seven compounds, the embedding rate was significantly higher in the centrifuge tubes (83 ± 3%) than in the rubber septa (71 ± 1%).

3.2.2. Final Residual Rate of Compounds in Different Sustained-Release Materials

The final residual rates of the compounds differed significantly between the two carrier materials (Figure 3). The residual rates of β-pinene, 2-isopropyl-5-methylanisole, and (R)-(+)-limonene were significantly higher in the centrifuge tubes than in the rubber septa. Conversely, the residual rates of terpinolene and D(+)-camphor were significantly higher in the rubber septa. No significant differences between the two carriers were observed for (+)-3-carene or (S)-cis-verbenol.

For the mixture containing all seven compounds, the final residual rate did not differ significantly between the centrifuge tubes (12 ± 4%) and the rubber septa (10 ± 3%).

3.2.3. Release Rates in Different Sustained-Release Materials

Release Rate of β-Pinene

The release rate of β-pinene peaked on the first day in both carriers, with values of 282.47 ± 3.79 µg/d for the rubber septum and 223.13 ± 2.56 µg/d for the centrifuge tube (Figure 4). For the rubber septum, the release rate decreased rapidly during the first seven days, dropping to 22.67 ± 0.17 µg/d, and then declined more gradually, reaching a negligible level of 1.77 ± 0.11 µg/d by day 25. In contrast, the release rate from the centrifuge tube remained relatively high for the first 10 days before stabilizing, with the minimum rate (2.47 ± 0.58 µg/d) observed on day 28. Throughout the 28-day period, the release rate from the centrifuge tube was consistently higher than that from the rubber septum on all days except the first, indicating a more sustained and effective release profile for β-pinene in the centrifuge tube system.

Release Rate of (+)-3-Carene

The release rate of (+)-3-carene on day 1 was significantly higher from the centrifuge tube (354.25 ± 12.56 µg/d) than from the rubber septum (184.05 ± 14.08 µg/d), with both carriers showing their maximum release rate on the first day (Figure 5).

In the rubber septum, the release rate decreased rapidly over the first four days, from the initial peak to 12.33 ± 0.56 µg/d, followed by a more gradual decline. By day 10, the rate had fallen to 2.28 ± 0.27 µg/d, after which it persisted at a very low level. In contrast, the release profile from the centrifuge tube was more variable: the rate dropped sharply to 167.55 ± 13.71 µg/d on day 2, increased again to 240.35 ± 21.09 µg/d on day 3, and then declined rapidly. From day 16 onward (25.82 ± 1.17 µg/d), the release stabilized at a slow, gradual rate.

Throughout the release period, the average release rate from the centrifuge tube was significantly higher than that from the rubber septum. However, the release from the centrifuge tube was notably less stable.

Release Rate of 2-Isopropyl-5-Methylanisole

On the first day, there was no significant difference in the release rate of 2-isopropyl-5-methylanisole between the centrifuge tube (122.56 ± 8.76 µg/d) and the rubber septum (101.45 ± 4.18 µg/d). The maximum release rate from the rubber septum occurred on day 1, whereas for the centrifuge tube, it was observed on day 2 (177.67 ± 9.89 µg/d).

In the centrifuge tube, the release rate peaked on day 2 and then decreased rapidly to 43.69 ± 1.07 µg/d by day 4. After day 4, the decline stabilized, gradually decreasing until day 19 (10.15 ± 0.69 µg/d), after which it persisted at a very low level. In the rubber septum, the release rate decreased rapidly from its day-1 peak to 18.56 ± 1.79 µg/d by day 4, stabilized until day 16, and reached a negligible rate of 1.82 ± 0.02 µg/d (Figure 6).

During the initial 4 days, the release rate from the centrifuge tube was significantly higher than that from the rubber septum. After day 4, however, the difference between the two carriers was not significant. Although the overall release rate was lower in the rubber septum, it provided a more stable release profile over time.

Release Rate of Terpinolene

On day 1, the release rate of terpinolene from the rubber septum (351.79 ± 18.67 µg/d) was significantly higher than that from the centrifuge tube (254.91 ± 12.27 µg/d). The maximum release rate from the rubber septum occurred on day 1, while for the centrifuge tube, it was observed on day 2 (297.53 ± 21.81 µg/d).

In the centrifuge tube, the release rate peaked on day 2, sharply decreased to 103.93 ± 5.38 µg/d by day 3, and further declined to 51.96 ± 8.57 µg/d on day 4. After day 4, the release rate gradually stabilized, decreasing slowly until day 19 (8.76 ± 1.91 µg/d), after which it persisted at a low level. In the rubber septum, the release rate decreased rapidly after the day-1 peak, falling to 22.16 ± 4.29 µg/d by day 7, followed by minor fluctuations until it reached 7.73 ± 0.52 µg/d on day 19 (Figure 7).

Throughout the release period, the release rates from the two carriers showed no significant difference on all days except for day 1 and day 2. On day 2, the release rate from the centrifuge tube was significantly higher than that from the rubber septum.

Release Rate of (R)-(+)-Limonene

The maximum release rate of (R)-(+)-limonene occurred on day 1 for both the rubber septum (427.23 ± 23.79 µg/d) and the centrifuge tube (362.96 ± 17.43 µg/d). The release rate from the centrifuge tube was significantly higher than that from the rubber septum on days 2 and 3.

In the centrifuge tube, the release rate remained relatively stable on days 2 and 3, then decreased rapidly to 98.53 ± 5.97 µg/d on day 4. It stabilized again from day 4 to day 7, gradually declined to 52.79 ± 3.48 µg/d by day 10, and then decreased slowly and steadily, reaching 11.36 ± 1.13 µg/d on day 22. In the rubber septum, the release rate dropped rapidly after the day-1 peak to 133.48 ± 11.17 µg/d on day 2, declined further to 111.59 ± 9.89 µg/d on day 3, but increased to 156.93 ± 13.36 µg/d on day 4. After day 4, the rate gradually decreased, falling to 16.67 ± 1.97 µg/d by day 13, after which it persisted at a low level (Figure 8).

Overall, the release profile from the centrifuge tube was relatively stable, whereas the release from the rubber septum showed considerable fluctuation during the first 10 days.

Release Rate of (S)-Cis-Verbenol

On day 1, there was no significant difference in the release rate of (S)-cis-verbenol between the centrifuge tube (210.94 ± 15.36 µg/d) and the rubber septum (204.73 ± 12.27 µg/d), with both carriers reaching their maximum release rate on this day.

In the centrifuge tube, the release rate decreased to 152.88 ± 9.71 µg/d on day 2, increased to 161.25 ± 8.31 µg/d on day 3, and then remained relatively stable, fluctuating around 100 µg/d from day 4 to day 19. After day 19, it declined rapidly, reaching a minimum of 24.42 ± 1.68 µg/d on day 28. In the rubber septum, the release rate dropped sharply after the first day to 48.36 ± 3.16 µg/d on day 2, followed by a gradual decline, falling to 5.47 ± 0.03 µg/d by day 10, after which it persisted at a negligible level (Figure 9).

Throughout the release period, the centrifuge tube provided a higher and more stable release rate for (S)-cis-verbenol. In contrast, the release rate from the rubber septum remained below 50 µg/d after day 1, was generally low, and approached zero by day 13.

Release Rate of D(+)-Camphor

On day 1, the release rate of D(+)-camphor from the centrifuge tube (101.17 ± 10.39 µg/d) was significantly higher than that from the rubber septum (67.79 ± 6.42 µg/d). The release rate peaked on day 2 for both carriers, with the rate from the centrifuge tube (146.47 ± 15.45 µg/d) being significantly higher than that from the rubber septum (91.16 ± 6.37 µg/d).

In the centrifuge tube, the release rate increased rapidly from day 1 to day 2, then decreased sharply to 48.59 ± 2.18 µg/d on day 3. It continued to decline gradually until day 13 (7.29 ± 0.93 µg/d), after which it persisted at a low level. A similar pattern was observed in the rubber septum: the rate increased from day 1 to day 2, then dropped rapidly to 49.81 ± 4.59 µg/d on day 3, and gradually decreased until day 19, reaching a near-zero rate of 0.79 ± 0.01 µg/d (Figure 10).

Throughout the release period, the release rate from the centrifuge tube was significantly higher than that from the rubber septum on days 1 and 2. On all other days, no significant differences were observed between the two carriers.

Release Rate of the Mixed Compound

On day 1, the release rate of the mixed compound reached its maximum and was similar between the two carriers, with values of 1719.83 ± 95.27 µg/d for the rubber septum and 1675.47 ± 72.16 µg/d for the centrifuge tube.

In the centrifuge tube, the release rate decreased rapidly after day 1, reaching 571.35 ± 26.79 µg/d by day 4. From day 4 to day 16, it declined at a steady rate to 142.19 ± 7.51 µg/d, after which it decreased slowly, reaching 45.61 ± 3.41 µg/d on day 28. In the rubber septum, the rate dropped sharply after day 1 to 779.47 ± 43.91 µg/d on day 2, then declined gradually and uniformly until day 13 (47.73 ± 4.17 µg/d). After day 13, it persisted at a very low rate until the end of the observation period on day 28 (Figure 11).

Throughout the release period, the release rate from the centrifuge tube was higher than that from the rubber septum on all days except day 1, when the rate was slightly higher for the rubber septum. The mixed compound in the centrifuge tube maintained a measurable release rate until day 25, whereas in the rubber septum, the rate dropped to a very low level after day 13. This indicates a more prolonged release duration for the mixed compound when dispensed from the centrifuge tube.

3.3. Simulated Field Deployment: Trap Placement Selection

Analysis of the trap hanging height indicated that the trapping rate at 1.5 times the branch-free height (1.5H) (70 ± 10%) was significantly higher than at 0.5H, 1H, 2H, and 2.5H. The trapping rate at 2.5H (13 ± 6%) was also significantly different from those at 0.5H (33 ± 6%), 1H (33 ± 6%), and 2H (37 ± 15%). Therefore, the optimal trap hanging height for T. brevipilosus is 1.5 times the branch-free height (Figure 12). The test insects were a mixed group of different ages.

Analysis of the trap location within the forest showed that the trapping rate under the forest canopy (47 ± 12%) was significantly higher than in forest gaps (19 ± 2%) or at the forest edge (67 ± 6%), with the edge location yielding the lowest rate. Consequently, trap placement under the forest canopy resulted in the best trapping efficacy, while placement in forest gaps was the least effective (Figure 13). The test insects were a mixed group of different ages.

Evaluation of the effect of canopy density revealed that the trapping rate in stands with low canopy density (73 ± 6%) was significantly higher than in those with high, moderate, or very low density. The trapping rate in moderately dense stands (40 ± 10%) was also significantly higher than in stands with high or very low canopy density. In summary, traps deployed in stands with low to moderate canopy density achieved the best capture results for T. brevipilosus (Figure 14). The test insects were a mixed group of different ages.

4. Discussions

Our study systematically developed a plant-based attractant for T. brevipilosus by integrating electrophysiological screening, behavioral assays, and release system optimization. The distinct EAG responses between sexes reveal a sophisticated olfactory recognition mechanism in this species. Females showed strong antennal sensitivity to common host terpenes (β-pinene, terpinolene, (+)-3-carene, and (R)-(+)-limonene), which aligns with their role in primary host location [26,27]. Interestingly, males exhibited unique responsiveness to D(+)-camphor and 2-isopropyl-5-methylanisole—compounds less frequently reported as key kairomones for other Tomicus species [28,29]. This sexual dimorphism may reflect divergent ecological functions: female preference for widespread host terpenes likely facilitates oviposition site selection, while male sensitivity to more specific compounds could be linked to mate-seeking behavior or pheromone synergy.

Notably, we observed decreasing EAG responses with increasing concentrations for several compounds, particularly 2-isopropyl-5-methylanisole and (+)-3-carene. This nonlinear dose-response relationship suggests potential sensory adaptation or peripheral interaction at higher concentrations, a phenomenon documented in other bark beetle species but rarely explored in Tomicus [28,30]. The optimal active concentrations (1–10 μg/μL for most compounds) provide crucial guidance for practical formulation development.

When contextualizing our findings within global Tomicus research, important patterns emerge. The effectiveness of β-pinene and (+)-3-carene aligns with studies on European T. piniperda and North American conifer beetles, confirming their role as universal host recognition cues [21,31]. However, the significant male-specific response to D(+)-camphor represents a distinctive feature of T. brevipilosus, potentially reflecting adaptations to local host chemotypes or unique chemical communication channels. This species-specific profile underscores the limitation of directly applying lure formulations developed for other Tomicus species in Yunnan’s ecosystems [26,29].

Our comprehensive evaluation of release systems revealed that the centrifuge-tube design offered superior performance for most compounds, particularly by maintaining stable release rates over 28 days. This simple yet effective system represents a practical advancement over conventional rubber septa, which showed rapid decline in release rates for key compounds like β-pinene and (+)-3-carene. The centrifuge tube’s transparency, low cost, and modifiable design present distinct advantages for commercial adaptation and field deployment compared to specialized polymeric dispensers.

Environmental mediation of volatile dispersion deserves particular emphasis. Our findings that traps placed under canopy cover in low-to-moderate density stands yielded highest captures reflect the complex interaction between volatile dispersion and microclimate. Dense canopy likely creates a semi-enclosed space that accumulates volatiles, while completely open areas may experience rapid dilution of chemical signals [30]. This has direct implications for field application: optimal trapping efficiency requires careful matching of release rates to local vegetation structure and atmospheric conditions. A previous study on Xyleborus affinis Eichhoff reported optimal trapping at heights of 1–2.5 m, with efficacy declining significantly beyond this range [32], further supporting the importance of spatial optimization in trap deployment.

Monoterpene Hydrocarbons generally possess lower molecular weights and higher vapor pressures compared to their oxygenated counterparts. This explains their characteristically high initial release rates (“burst release”) observed on the first day in both carriers (Figure 4, Figure 5, Figure 6 and Figure 8). For instance, β-pinene and (+)-3-carene, both bicyclic monoterpenes, exhibited the most pronounced burst effect. However, their subsequent release profiles diverged based on carrier interaction. The rapid decline in release rate from the rubber septa suggests either higher affinity or faster diffusion through the rubber matrix, leading to quick depletion. In contrast, the centrifuge tubes, acting as a simple reservoir, provided a more sustained release for most monoterpene hydrocarbons, maintaining higher rates after the initial burst. The anomalous peak for (+)-3-carene in the centrifuge tube on day 3 (Figure 6) may be attributed to experimental artifacts such as temperature fluctuations or the compound’s specific crystallization/dissolution dynamics within the reservoir.

Oxygenated Monoterpenes, featuring polar functional groups (e.g., hydroxyl in (S)-cis-verbenol, ketone in D(+)-camphor, ether in 2-isopropyl-5-methylanisole), typically exhibit higher boiling points and lower vapor pressures. This translates to generally lower and more stable release rates. Their interaction with the polar components of the rubber septa is likely stronger, affecting their release kinetics more significantly than for hydrocarbons. This is particularly evident for (S)-cis-verbenol (Figure 10), whose release was drastically higher and more prolonged from the centrifuge tube, suggesting strong retention within the rubber septum that impeded its release. The release of D(+)-camphor and 2-isopropyl-5-methylanisole was less stable than that of (S)-cis-verbenol from the centrifuge tube, possibly due to their specific solid-state properties or solubility.

A technical limitation of this study is the potential influence of minor, highly active impurities on EAG responses. While the chemical analysis in our current study focused on the major identified constituents, and thus cannot retrospectively address this issue, we acknowledge this common challenge in semiochemical research. Future studies could employ more advanced purification techniques and multidimensional gas chromatography to rule out the influence of potent trace components. Furthermore, future work could apply more technically demanding methods to standardize release rates based on compound properties [33], enabling more quantitative prediction and comparison of release kinetics across different compounds and dispenser systems.

5. Conclusions

This study establishes a comprehensive foundation for monitoring and managing T. brevipilosus through plant-based semiochemicals. We identified a highly attractive blend (10 μg/μL β-pinene, 10 μg/μL terpinolene, 1 μg/μL 2-isopropyl-5-methylanisole, 1 μg/μL D(+)-camphor, 100 μg/μL (R)-(+)-limonene, and 10 μg/μL (+)-3-carene) that achieved 56.67% selection in behavioral assays. The centrifuge tube emerged as the optimal release system for this blend, providing stable emission over extended periods.

Our findings demonstrate that successful attraction requires integrating multiple components: (1) a species-specific volatile combination accounting for sexual dimorphism, (2) an appropriate release system maintaining optimal emission rates, and (3) strategic trap placement considering environmental effects on volatile dispersion.

Several limitations warrant acknowledgment. The controlled conditions of our semi-field experiments, while necessary for mechanistic understanding, may not fully represent the complexity of natural forests. The absence of long-term field validation and economic analysis requires addressing before large-scale implementation. Future research should focus on multi-season field trials across different forest types, potential synergies with pheromones, and refining release technologies for operational deployment.