The key phytochemical cue D(+)-camphor is a promising lure for traps monitoring the new monophagous camphor tree borer Pagiophloeus tsushimanus (Coleoptera: Curculionidae)

doi:10.21203/rs.3.rs-3611212/v1

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The key phytochemical cue D(+)-camphor is a promising lure for traps monitoring the new monophagous camphor tree borer Pagiophloeus tsushimanus (Coleoptera: Curculionidae)

https://doi.org/10.21203/rs.3.rs-3611212/v1

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published 21 Aug, 2024

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The landscape plant, Cinnamomum camphora, is a broad-spectrum insect-repelling tree species, mainly due to a diversity of terpenoids, such as D(+)-camphor. Despite its formidable chemical defenses. C. camphora is easily attacked and invaded by a monophagous weevil pest, Pagiophloeus tsushimanus. Deciphering the key olfactory signal components regulating host preference could facilitate the monitoring and control strategies for this insect pest. Herein, two host volatiles, D(+)-camphor and ocimene, can induce GC-EAD/EAG reactions in both male and female adult antennae. Correspondingly, Y-tube olfactometer assays showed that the two compounds were attractive to both male and female adults. In field assays, a self-made trap device baited with 5 mg dose D(+)-camphor captured significantly more P. tsushimanus adults than isopropanol solvent controls without sexual bias. The trunk gluing trap device baited with bait can capture adults, but the number is significantly less than that of the self-made trap device and adults often fall after struggling. The cross baffle trap device never traps adults. Neither ocimene nor isopropanol solvent control could capture adults. When used in combination, ocimene cannot enhance the attraction of D(+)-camphor to both female and male adults. These results indicate that D(+)-camphor is a key active compound of P. tsushimanus adults for the host location. The combination of the host-volatile lure based on D(+)-camphor and the self-made trapping device is promising to monitor and provide an eco-friendly control strategy for this novel pest P. tsushimanus in C. camphora plantations.

Cinnamomum camphora

Pagiophloeus tsushimanus

monophagous weevil pest

D(+)-camphor

host-plant volatiles

trapping device

Some phytophagous beetles have a narrow host range and are highly specific to one or several host plant species (Xue et al. 2011; Li et al. 2017; Tang et al. 2022). For instance, both Eucryptorrhynchus brandti and E. scrobiculatus weevils are host-specific pests of Ailanthus altissima, a tree with few pests owing to its high abundance of defense compounds (Wen et al. 2021). Host chemicals play an important role in the coevolution of plants and monophagous beetles (Landolt and Phillips, 1997; Rasmann and Agrawal, 2011). Similarly, the content of D(+)-camphor in the camphor tree Cinnamomum camphora Presl (Laurales: Lauraceae) is relatively high, yet, this common insect repellent is used by a monophagous camphor tree borer Pagiophloeus tsushimanus Morimoto as a signal for host location (Chen et al. 2021; Li et al. 2021).

The camphor tree, C. camphora, is an important landscape tree species and is widely distributed in South China (Wang et al. 2012). Previous studies have shown that C. camphora has strong insect-repellent activities due to their abundant monoterpenoids, including D(+)-camphor, as main components of its essential oil, which have larvicidal and ovicidal activities against some stored product beetle pests (Nenaah et al. 2011; Guo et al. 2016; Jiang et al. 2016; Wu et al. 2020). Monoterpenoid volatile D(+)-camphor is the key component of camphor essential oil (Li et al. 2021; Li et al. 2022). Because of the insecticidal activity of these terpenoids, C. camphora plantations are less likely to be attacked or selected as a host by various herbivorous insects. However, C. camphora has been selected as the unique host plant by the new weevil pest P. tsushimanus (Chen et al. 2021; Li et al. 2021). Pagiophloeus tsushimanus was originally discovered on Tsushima Island of Kyushu in Japan and described by Morimoto (1982) (Morimoto, 1982). Since the initial report of the monophagous camphor tree borer P. tsushimanus in China in 2014, C. camphora plantations have experienced widespread damage by this destructive weevil pest in the entire municipality of Shanghai (Zhang et al. 2017; Chen et al. 2021; Li et al. 2021). Pagiophloeus tsushimanus is univoltine in Shanghai. Adults usually overwinter in grooves on the underside of branches or branch nodes, and a handful of mature larvae overwinter in tunnels (Chen et al. 2021). Adults often aggregate for feeding on the tender bark of twigs or newly emerged buds, and larvae bore into the phloem, or sometimes the cambium (Chen et al. 2021; Li et al. 2021). In addition, C. camphora plantations in adjacent areas outside Shanghai have been seriously threatened (Chen et al. 2021). Therefore, it is urgently needed to develop effective monitoring or control strategies for this pest.

The larvae of wood-boring beetles live beneath the bark during the whole damage period. Because the larvae’s destructive activities are concealed, it leads to difficulties in effective pest control. Semiochemicals play a key role in mate, host, and oviposition location in plant-insect interactions (Hanks, 1999; Karlsson et al. 2009). Volatiles that mediate insect behavior have received much attention. Plant volatiles are important olfactory cues in the host selection of phytophagous beetles (Pham et al. 2020). For example, Cyrtotrachelus buqueti is the main oligotrophic pest in bamboo forests, and the volatiles of bamboo play a decisive role in its host location (Tang et al. 2022). Host plant-derived volatiles of Ailanthus altissima play an important role in the host-oriented behavioral response of two weevils Eucryptorrhynchus scrobiculatus E. brandti (Wen et al. 2021). Traps baited with volatile attractants are an economical and valuable measure for pest monitoring and control (Crook et al. 2014). In general, polyphagous insects distinguish between host and non-host plants via the quantity of volatile components, while monophagous insects identify host plants by the quality of volatiles (Rajapakse et al. 2010). For many specialized insects, the active components in their host plant volatiles have been identified. For instance, the volatile (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) released from the host plant Alternanthera philoxeroides (Mart.) Griseb showed significantly higher attraction to adults of Agasicles hygrophila Selman & Vogt (Li et al. 2017). Isothiocyanates released by cruciferous plants are harmful to common herbivorous insects, but they can strongly attract adults to feed or stimulate the oviposition of specialized insect females Plutella xylostella Linnaeus (Liu et al. 2020). Therefore, it is more advantageous to develop attractants with strong attraction and simple components by using the strong tendency of monophagous insects to utilize host-specific odor compounds for host location.

The biological characteristics of pests are the core basis for developing effective control strategies (Akbar et al. 2017). In our previous study, an observed phenomenon during the first indoor and field observations is that P. tsushimanus has a special preference for C. camphora compared to other Lauraceae trees (Li et al. 2021). However, it is difficult to establish a stable population of closely related species (Li et al. 2019). At present, the mechanism of host identification and the location of this pest, and key phytochemicals in the host selection process are still unclear. Therefore, the key phytochemicals driving the host location of this insect pest were identified using the combination of chemical analysis and behavioral assays. This study aims to determine the effective compounds for the development of attractants, further promoting eco-friendly control strategies for this pest. In addition, it can deepen our understanding of the interspecific chemical communication in P. tsushimanus, especially regarding the mechanisms of host identification and location.

2.1 Insects

Pagiophloeus tsushimanus pupae were collected from trunks of C. camphora trees from the Maogang (30°56′6.15″ N, 121°12′32.76″ E), Songjiang District, Shanghai, China in 2020 and 2021, which were placed in the wells of petri dish (six round wells with 2 cm in diameter) with wet cotton balls until adults’ emergence. Then Next, the newly emerged adults were immediately collected, and their sex was distinguished by differences in their external genitalia. Ten males or females were reared in each plastic container (60 cm long, 30 cm wide, 20 cm high) with 10 small circular holes (diameter 1 mm) for ventilation (Chen et al. 2021). Adults were kept in an incubator (26 ± 1 ℃, 60 ± 5% relative humidity, 14:10 light photoperiod), and fed with the fresh two-year-old twigs of camphor trees.

2.2 Headspace aeration of host volatiles

Two-year-old twigs of camphor tree (15 cm length) were utilized for analysis of headspace volatiles. The air outlet was fitted with a volatile trap, composed of a glass tube and Porapak Q adsorbent (200 mg; Sigma-Aldrich, St. Louis, MO, USA) secured in the tube by glass wool plugs. Charcoal-filtered air was pulled (350 ml/min) through a collection container (500 mL conical flask) and host-produced volatiles were collected for 24 h using an atmospheric air collection pump (Beijing Municipal Institute of Labour Protection, Beijing, China). The Porapak Q and traps were cleaned using Soxhlet-extracted dichloromethane before use. Trapped volatiles were eluted from each adsorbent tube with 1 mL dichloromethane and loaded into chromatographic sample vials. Volatile extracts were concentrated to 200 µL by a nitrogen blower. Each sample vial was sealed until used in experiments. Six repetitions were set up in this experiment, and each repetition contains 20 twigs.

2.3 Identification of antenally-active compounds in host volatiles

Volatile extracts derived from C. camphora twigs were analyzed by coupled gas chromatography-electroantennogram detection (GC-EAD) using a 6890N chromatography-flame ionization detector fitted with a 30 m capillary column HP-5 (30 m × 0.25 mm × 0.25 µm, Agilent Technologies Inc, USA, California). The parameters are as follows: 1) The temperature of the injector and detector was 250°C using splitless injection. 2) The carrier gas was high-purity nitrogen at a flow rate of 2 mL/min. 3) The oven was programmed from 40°C, then ramped 10°C per min to 80°C, 5°C per min to 100°C, 1°C per min to 105°C, 5°C per min to 120°C, and finally 10°C per min to 250°C (3 min hold time). 4) The GC column effluent was divided into two deactivated capillary columns by a Y-connector in equal length with nitrogen added as a make-up gas (flow rate: 8 mL/min) by another Y-connector. The GC effluent was delivered into the airflow (charcoal-purified and humidified air, flow rate: 300 mL/min) tube (15 mm ID) of EAD and then passed over the antenna preparation. The antennae of virgin female and male adults were cut from the base with scissors and forceps. And then the end of the antenna was quickly cut off about 0.5 mm with anatomical scissors. The two ends of the antenna were mounted on the two electrodes of EAG with a capillary glass tube (the tube was filled with normal saline, the silver wire was immersed in it and connected with the electrode, and the bubbles were removed in the capillary before the test to avoid interference). After connection, we align the antenna with the center of the air outlet and 1 cm away from the air outlet and waited until the baseline of the antenna potential was stable. An analog-to-digital conversion board (IDAC-232, Syntech) was used to input the signals of electroantennographic (EAG) and FID into a computer, and the Syntech GC-EAD software was used to display and analyze signals. Antennae from ten females and ten males were respectively used for GC-EAD analysis, and the trial for each antenna was repeated three times.

2.4 Chemical analyses of host volatiles

Volatile extracts derived from C. camphora were identified using coupled gas chromatography-mass spectrometry (GC-MS) (Thermo Electron, USA). The related parameters of gas chromatography were the same as described in the GC-EAD experiment. Mass spectra were taken in EI mode (70 eV). The temperatures of both the ion source and transfer line were 250°C. The scanning range was 30 to 350 u. All compounds in the volatile extracts were preliminarily identified via the matching of mass spectra and database spectra and then confirmed by the retention time and mass spectrum matching of key compounds and those of authentic standards. A set of linear alkanes from C₈-C₂₈ was used for GC-MS analysis under the same conditions to calculate Kovat’s index. The information on pure compounds used in this experiment is listed in Table 1.

Table 1

Pure compounds used for chemical identification, laboratory, and field assays.
Maker	Compound	CAS no.	Purity (%)	Source
A	Ocimene	3779-61-1	90	Shanghai Yuanye Bio-Technology Co., Ltd
B	D(+)-Camphor	464-49-3	96	Shanghai Aladdin Biochemical Technology Co., Ltd
C	Mineral oil	8042-47-5	96	Shanghai Aladdin Biochemical Technology Co., Ltd
D	Isopropanol	67-63-0	99	Shanghai Aladdin Biochemical Technology Co., Ltd

2.5 Electroantennographic (EAG) assays

Different doses (0.01, 0.1, 1, 10, 100 µg) of two main compounds (i.e., D(+)-camphor and ocimene) and mixed components [D(+)-camphor : ocimene = 1 : 1, 10 µg] were prepared with mineral oil.

Electrophysiological activities of female and male adult antennae in response to D(+)-camphor and ocimene at different doses or mixed components [D(+)-camphor : ocimene = 1 : 1] were examined using the EAG technique. EAG responses of P. tsushimanus adult antennae flagellomeres (seventh to ninth) to all treatments were tested. Before each test, 10 µL of each solution was dropped uniformly onto a piece of filter paper strip (2 cm length × 0.5 cm width), which was inserted into a Pasteur pipette (15 cm length) and used as odor stimuli. Mineral oil was prepared as a solvent control treatment.

A sharp blade was used to sever the flagellomere part of the antenna completely from the base of the antenna, and then the tip of the antenna was cut off. The base and end of each antenna were inserted into a glass pipette containing Kaiserin saline solution (Kaissling et al. 1980). Electric continuity between antennae and the recording equipment using DC mode with AC/DC un-6 amplifier connected to a PC equipped with EAG 2.0 program (Syntech Laboratories, Hilversum, the Netherlands). Charcoal-filtered, humidified air (500 mL/min) continued to blow onto the antenna (seventh to ninth segments) passing through a stainless-steel delivery pipe (1 cm inner diameter), and the outlet was about 1 cm away from the antennae.

After the antenna connection was verified and the baseline was stable, the EAG test can be carried out. A given volatile compound was applied to a strip of filter paper strip placed in a new Pasteur pipette and connected to the stimulus source tube, and recording was started after gently pressing the pedal. The flow rate of each stimulation was 60 mL/s, the stimulation lasted for 0.2 s, and the interval between the two stimulation events was more than 60 s to ensure that the antennae recovered completely from the previous stimulation (Germinara et al. 2019). To keep the activity of the antennae, the time to treat the antennae was reduced as much as possible. At the beginning and end of each group of experiments, 10 µL of mineral oil (control treatment) were tested respectively. In the dose-response experiment, we used the lowest concentration first and then increased to progressively higher concentrations. Each treatment was tested on female antennae (N = 20) and male antennae (N = 20). Each antenna was tested 3 times.

2.6 Olfactometer assays

Olfactory responses of male and female adults to two main compounds (i.e., D(+)-camphor and ocimene) and mixed components [D(+)-camphor : ocimene = 1 : 1] at different doses were investigated in Y-tube glass olfactometer assays. The dimensions of the Y-tube glass olfactometer were as follows: main arm − 40 cm, two choice arms − 20 cm at 60° to each other, and the internal diameter of all arms − 4 cm. The olfactometer was placed in an environment with uniform light under a 500 W halogen lamp, and consistent background color at the bottom and Sidewalls. Tefon® tubing was used to connect two 250 ml glass flasks to the ends of two choice arms. The glass flask contains a piece of filter paper strip (5 cm length × 0.5 cm width) loaded with odor stimuli or solvent control treatment. A stream of air (500 mL/min/arm) filtered by carbon and humidified continuously flowed into each choice arm through the flasks.

Each compound dose (1, 10, 100 µg) and mixed components dose [D(+)-camphor : ocimene = 1 : 1, 10 µg] were prepared as odor stimuli. Mineral oil was prepared as solvent control treatment. Before each test, 10 µL of each solution was applied evenly to a piece of filter paper strip (5 cm length × 0.5 cm width), which was inserted into the flask. Healthy, virgin male and female adults in the same physiological state were selected for this experiment, which was placed individually in the plastic container (60 × 30 × 20 cm) at 26 ± 1°C and 60 ± 5% relative humidity for 24 h without food supply before the test. A single virgin male or female adult was placed into the opening at the starting end of the main arm and recorded as choosing if it crawled and reached the end of a choice arm within 20 min. Adults that did not crawl and reach the end of either choice arm within 20 min were recorded as having no response. Each adult was only used once for each test treatment. Before each assay, the components of the Y-tube olfactometer were cleaned with acetone, and then rinsed thoroughly in distilled water, then dried at room temperature. The treatment and control arms of the Y-tube olfactometer were reversed after every test. All tests were performed at the forest entomology laboratory of Nanjing Forestry University from 9 am to 5 pm under the same environmental conditions, such as 26 ± 1°C and 60 ± 5% relative humidity. Each treatment tested female adults (N = 36) and male adults (N = 36). Three repetitions were used for each treatment, respectively.

2.7 Field assays

Field assays were conducted at the plantations cultivated with 15-year-old C. camphora at Maogang (30°56′6.15″ N, 121°12′32.76″ E), Songjiang District, Shanghai, where an outbreak of P. tsushimanus infestation was first discovered in 2014 (Huang et al. 2014). Three 20 m×20 m sample plots were established and ten infested trees in each plot were randomly selected. All larval tunnels were dissected on the surface of the trunk of each tree, which were located by the presence of frass. The density of larvae below 2 m in the trunk of C. camphora plantations was 23.70 ± 10.33 larvae per tree (Table S1). The distance between trees was 3.0 m, mean height and the diameter at breast height of C. camphora trees were 5.0 ± 1.2 m and 21.1 ± 6.3 cm (Chen et al. 2021). In addition, a few other insects have occasionally been found to infest C. camphora plantations such as Orthaga olivacea Warren, Thalassodes quadraria Guenée, and Diaphania perspectalis Walker (Chen et al. 2021). Lures were prepared by heat-sealing polyethylene tubing (~ 7 cm×4.9 cm, wall thickness 0.05 mm; Uline, Pleasant Prairie, WI, USA) containing 1 mL solution [D(+)-camphor, ocimene, or mixed components diluted in isopropanol] or solvent control treatment (isopropanol).

2.7.1 Experiment 1– Effect of different kinds of traps on trapping efficiency

Three kinds of traps were selected in this experiment, including a cross baffle trapping device (Fig. 4-A), a self-made trapping device (Fig. 4-B), and a trunk-glued trapping device (Fig. 4-C). Trapping device B was refitted from a 5 L mineral water bottle. One-third of the bottle was cut off and placed against the trunk. Several openings larger than the insect body were made on the top and side of the bottle. The inner side of the bottle was coated with colorless and tasteless glue. The tasteless glue was replenished weekly. The bottles were fixed in position with thin iron wire and against the trunk of C. camphora. Lures were respectively hung in the center of trapping devices A and B. The lure was fixed in the middle of the trapping device C, and the back of the lure was propped up with branches to avoid being stuck by glue and affecting the release of volatiles. All trapping devices (including lures) were randomly placed on the trunk of C. camphora 1.5 m above the ground and ≥ 20 m apart. Collection cups of trapping devices A and B contained a solution of antifreeze and water (1 : 2) to kill and preserve trapped adults. This experiment was conducted from 1 June to 1 July 2020. Trapped female and male adults were collected and lures were replaced weekly. Each trapping device and control group used 6 repetitions in the experiment and each treatment had 24 replicates. Each trapping device (A, B, C) was baited with: (1) 5 mg D(+)-Camphor in 1 mL isopropanol (N = 18) or (2) 1 mL isopropanol (N = 18).

2.7.2 Experiment 2– Effect of the height of the trapping device on trapping efficiency

This experiment was also conducted from 1 June to 1 July 2020 with D(+)-camphor (5 mg/mL) combined trapping device B. Trapping device B was placed against the trunk of C. camphora at three heights: high (2 m above the ground), middle (1.5 m above the ground), and low (0.3 m above the ground) (Fig. 5). Each height included: (1) 5 mg D(+)-Camphor in 1 mL isopropanol (N = 18) and (2) 1 mL isopropanol (N = 18). The related methods were the same as in experiment 1.

2.7.3 Experiment 3– Effect of dose of host volatile components on trapping efficiency

This experiment was run from 1 June to 1 July 2020 with trapping device B. The volume of each lure, the method of setting up trapping device B, and the use of the control group were the same as in Experiment 1. Lures were replaced, trapped adults were collected, and tasteless glue was replenished weekly. All treatments included: (1) 1 mg D(+)-camphor in 1 mL isopropanol (N = 6), (2) 5 mg D(+)-camphor in 1 mL isopropanol (N = 6), (3) 10 mg D(+)-camphor in 1 mL isopropanol (N = 6), (4) 1 mg ocimene in 1 mL isopropanol (N = 6), (5) 5 mg ocimene in 1 mL isopropanol (N = 6), (6) 10 mg ocimene in 1 mL isopropanol (N = 6), and (7) 1 mL isopropanol (N = 6).

2.7.4 Experiment 4– Effect of combination of host volatile components on trapping efficiency

According to the screening results of the above trapping conditions, the lures of D(+)-camphor (5 mg), ocimene (5 mg), and mixed components (D(+)-camphor : ocimene = 1:1, 5 mg) were used in trapping device B. Traps were placed against the trunk of C. camphora at middle height (1.5 m above the ground) to test of trapping effect in C. camphora plantations from 1 July to 30 September 2020. Tasteless glue (applied to the inside of trap B) was replenished, lures were replaced, and trapped adults were collected weekly. Treatments included: (1) 5 mg D(+)-camphor in 1 mL isopropanol (N = 6), (2) 5 mg ocimene in 1 mL isopropanol (N = 6), (3) 5 mg D(+)-camphor and 5 mg ocimene in 1 mL isopropanol (N = 6), and (4) 1 mL isopropanol (N = 6). The related methods were the same as in experiment 1.

2.8 Statistical analysis

For laboratory assays, the negative polarity deflection (mV) of a stimulus was used to test the EAG response of each compound or mixed solution (Light et al. 1992). The EAG responses (mV) of each stimulus were such that the absolute EAG responses (mV) to each stimulus were subtracted by the mean response of the two nearest mineral oil controls (Raguso et al., 1998). The mean EAG responses (mV) of each compound or mixed solutions tested were analyzed by analysis of variance (ANOVA) and means were compared by Tukey’s HSD (Honestly Significant Difference) test (P = 0.05). The mean EAG responses (mV) of females and males to each stimulus were compared using the Student’s t-test (P = 0.05). Binomial tests (expected value = 0.5) were applied to compare the numbers of adults selected between two odor stimuli in Y-tube olfactometer bioassays (Xu et al. 2020). Non-responsive adults were excluded from the statistical analysis. In field bioassays, the Kruskal-Wallis H test followed by pairwise contrast tests were applied to statistically analyze differences in the mean number of trapped weevils across treatments. Bonferroni correction was used to adjust the multiple comparisons to control the experiment-wise error rate. Significant differences in sex ratio were analyzed by the Chi-square test. The significance level of various treatments was α = 0.05. All data were analyzed using SPSS 20.0 (IBM SPSS Statistics, Chicago, IL, USA) and plotted using Origin 2018 (OriginLab Inc., Northampton, UK).

3.1 Identification of antenally-active compounds in host volatiles

Two components (1 and 2) of headspace volatiles collected from C. camphora volatiles consistently stimulated EAD responses in the antennae of female (Fig. 1-A) and male (Fig. 1-B) adults. Two components were identified as ocimene [retention time (RT) 9.71 min, Cas No. 13877-91-3] and D(+)-camphor [RT 13.23 min, Cas No. 464-49-3] respectively by GC-MS library match and further confirmed by co-injection of authentic standard.

3.2 Electroantennographic (EAG) assay

The sensitivity of female and male P. tsushimanus adults to ascending doses of two single components and mixed components are shown in Fig. 2. For all doses tested of ocimene tested, there were significant differences among the mean EAG responses at different doses both in females (F = 1979.755, df = 4, P < 0.05) and males (F = 1648.967, df = 4, P < 0.05) (Fig. 2-A). The mean EAG responses of adult female (F = 1216.971, df = 4, P < 0.05) and male (F = 481.145, df = 4, P < 0.05) antennae to D(+)-camphor were also significantly different at all doses (Fig. 2-B). In the range of doses of ocimene tested, the mean EAG responses amplitude varied from 0.078 ± 0.001 mV to 0.455 ± 0.002 mV in females and from 0.058 ± 0.003 mV to 0.423 ± 0.001 mV in males. Amplitude of mean EAG responses of female adult antennae to all doses of D(+)-camphor is from 0.099 ± 0.003 to 0.636 ± 0.001. For male antennae, the mean EAG responses amplitude varied from 0.083 ± 0.006 to 0.526 ± 0.005. The EAG responses of female adult antennae to ocimene were significantly higher than that of male adults at 0.01 (t = 7.321, df = 4, P < 0.05), 1 (t = 6.061, df = 4, P < 0.05) or 100 µg (t = 18.755, df = 4, P < 0.05). The same difference was observed in the EAG responses of adults to D(+)-Camphor at 0.1 µg (t = 6.259, df = 4, P < 0.05), 10 µg (t = 6.375, df = 4, P < 0.05) or 100 µg (t = 22.157, df = 4, P < 0.05).

Strong EAG responses (> 0.488 mV) were elicited by mixed components in both females and males (Fig. 2-C). The mean EAG responses of females were significantly higher than that of males for mixed components (t = 4.311, df = 4, P < 0.05).

3.3 Olfactometer assays

Y-tube olfactometer bioassays showed that the numbers of both females and males selecting D(+)-camphor increased first and then decreased with the dose from 1 µg to 100 µg. D(+)-camphor showed significantly stronger attraction to females than controls at 100 µg dose (P = 0.035), but there was no significant difference for males (P = 0.189). D(+)-camphor at 10 µg dose showed significantly stronger attraction to both females (P < 0.001) and males (P = 0.001) than controls. Either females (P = 0.690) or males (P = 0.678) were not significantly attracted by D(+)-camphor at 1 µg dose (Fig. 3-A).

In the Y-tube olfactometer bioassays, the response rate of both females and males selecting Ocimene also increased first and then decreased with increasing doses. The tendency of attraction of both females (P = 0.006) and males (P = 0.008) to ocimene at 10 µg dose showed to be significantly stronger than controls. However, there was no significant difference in the response rate of females or males to 1 µg (P = 0.286, P = 0.230) and 100 µg (P = 0.076, P = 0.061) doses (Fig. 3-B).

Mixed components showed significantly stronger attraction to females (P = 0.002) and males (P = 0.001) than controls at 10 µg dose. The response rate of males to mixed components was greater than those of females (Fig. 3-C).

3.4 Field assays

3.4.1 Experiment 1– Effect of different kinds of traps on trapping efficiency

There was a significant difference in the number of adults captured by the three trapping devices baited with lures (D(+)-Camphor or isopropanol) (H = 21.138, df = 5, P = 0.001 < 0.05). The number of adults captured by trapping device B baited with D(+)-Camphor was significantly more than those of trapping device A and all isopropanol solvent control treatment (all P-values were less than 0.0001, respectively, P < 0.003, Bonferroni corrected significance level) (Fig. 4).

Trapping device B baited with D(+)-Camphor captured significantly more female adults than those of other treatments except the trapping device C (P = 0.001, in both comparisons, respectively, < 0.003, Bonferoni corrected), while there was no significant difference in the number of male adults among all treatments (Bonferoni corrected P > 0.003 in all pairwise contrasts).

Four females and three males were captured at trapping device B, and 1 female and 2 males were captured in trapping device C, but the differences were not statistically significant between the sexes of adults captured at trapping device B (χ² = 0.032, P = 0.858) or trapping device C (χ² = 0.139, P = 0.709) baited with D(+)-camphor respectively. However, no adults were captured at trapping device A.

3.4.2 Experiment 2– Effect of the height of the trapping device on trapping efficiency

The results showed that there was a significant difference in the number of adults captured at different heights of trapping device B baited with lures (D(+)-camphor or isopropanol) (H = 22.744, df = 5, P < 0.0001). Significantly more adults were captured at high height of trapping device B baited with 5 mg D(+)-camphor than those of all isopropanol solvent control treatments (P = 0.001 in both comparisons, respectively, < 0.003, Bonferroni corrected significance level) (Fig. 5).

The number of males captured by trapping device B baited with D(+)-camphor at high height was significantly more than those of other treatments except for middle height (P = 0.001 < 0.003, in all pairwise contrasts), while there was no significant difference in the number of female adults among all treatments (P > 0.003, Bonferroni corrected).

A total of 9 females and 6 males were captured in experiment 2. Specifically, there were 3 females and 4 males captured at high height, 4 females and 2 males were captured at middle height, and 2 females were captured at low height, but there was no sexual bias in adults captured at high height (χ² = 0.032, P = 0.858), middle height (χ² = 0.178, P = 0.673), or low height (χ² = 1.333, P = 0.248).

3.4.3 Experiment 3– Effect of dose of host volatile components on trapping efficiency

A significant difference was shown in the number of adults caught by different doses of all treatments (H = 34.458, df = 6, P < 0.0001). Trapping device B baited with 5 mg D(+)-camphor captured significantly more adults than other treatments except for 10 mg D(+)-camphor (P = 0.001, in all pairwise contrasts, < 0.0023, Bonferroni corrected significance level). No significant difference was detected between the number of adults caught by 10 mg and other treatments (P > 0.0023, in all pairwise contrasts) (Fig. 6).

There were 7 females and 6 males in total caught in this experiment, of which 5 females and 4 males were caught by 5 mg D(+)-camphor, and 2 females and 2 males were caught by 10 mg D(+)-camphor. There was no sexual bias in the number of adults caught by 5 mg D(+)-camphor (χ² = 0.020, P = 0.887) or 10 mg D(+)-camphor (χ² = 0, P = 1). However, no adults were captured at different doses of ocimene and solvent control treatment.

3.4.4 Experiment 4– Effect of combination of host volatile components on trapping efficiency

The results of trapping for three months showed that a significant difference existed in the number of adults caught by major and minor host volatile components (Kruskal Wallis H test, H = 20.320, df = 3, P < 0.0001). Trapping device B baited with 5 mg D(+)-camphor caught significantly more females and the total of both sexes than those traps baited with the isopropanol solvent control or 5 mg ocimene (P = 0.001 in both comparisons, respectively, < 0.0083 Bonferroni corrected significance level). When combined with ocimene, fewer adults were caught by trapping device B baited with 5 mg mixed components [D(+)-camphor : ocimene = 1 : 1] than that of 5 mg D(+)-camphor alone, but the difference was not significant (P = 0.724 > 0.0083, Bonferroni corrected). Although the mixed component did not increase the number of captured adults, it was still significantly higher than those of ocimene (P = 0.003 < 0.0083, Bonferroni corrected) or the control group (P = 0.003 < 0.0083, Bonferroni corrected). No adults were captured by trapping device B baited with the single component ocimene, which was the same as the result of Experiment 3 (Fig. 7).

A total of 16 females and 10 males were caught in experiment 4. Trapping device B baited with 5 mg D(+)-camphor attracted 9 females and 5 males, and trapping device B baited with 5 mg mixed components [D(+)-camphor : ocimene = 1 : 1] attracted 7 females and 5 males. The number of females caught in both groups was more than that of males, but no significant difference between the sexes was detected [χ² = 0.152, P = 0.696, in 5 mg D(+)-camphor; χ² = 0.049, P = 0.825, in 5 mg mixed components [D(+)-camphor : ocimene = 1 : 1]].

The wood-boring monophagous pest P. tsushimanus damages the camphor tree C. camphora, its preferred host. Based on the above phenomena, the key olfactory signal components governing host preference behavior were identified in this study. The compounds D(+)-camphor and ocimene in the volatiles of the host plant C. camphora can elicit antennal electroantennographic response and orientation responses in the Y-tube, but the field activity test results show that only D(+)-camphor has the attractant activity, and there is no gender bias in the number of adults trapped. The attractant needs to be matched with a suitable trapping device to obtain a good trapping effect. The trapping device B (self-made trap device) is suitable for capturing adults. However, although trapping device C (trunk gluing trap device) can capture adults, the number is significantly less than that of the self-made trap device, which cannot be accurately counted because the adults struggle to fall. In addition, the trapping device A (cross baffle trap device) failed to trap adults.

4.1 Antennal electroantennographic response of adults elicited by host plant volatiles

With the rapid development of chemical analysis technology, a large number of key components involved in the chemical communication of wood-boring beetles have been identified (Mitsuyoshi et al. 2002; Xing et al. 2017; Xu et al. 2017; Xu et al. 2020). GC-EAD method is one of the optimal methods for screening active components, which has been applied to the identification of pheromone components in various insects, such as α-longipinene in Anoplophora glabripennis Motschulsky, (E)-2-cis-6,7-epoxynonenal in Aromia bungii Faldermann, and 4-Vinylanisole in locusts (Xu et al. 2017; Xu et al. 2020; Guo et al. 2020). In this study, we found that compounds D(+)-camphor and ocimene in the volatiles of the host plant C. camphora elicit a GC-EAD response in both female and male adults. At the same time, both male and female adults had greater EAG responses to the single or mixed components of D(+)-camphor and ocimene. The results of GC-EAD and EAG responses showed that D(+)-camphor and ocimene had antennal electrophysiological activities for both female and male adults. The components with electrophysiological activities may either be lure components or repellent components, which need to be comprehensively judged in combination with the results of laboratory and field behavior assays (Kong et al. 2001).

4.2 Bioactivity of host plant volatiles

After the preliminary identification of key active components of host plant volatiles, the bioactivity of these components was investigated and three kinds of field trapping devices were developed for this pest in this study, and the effects of different factors (e.g., lures, trap height) on the capturing efficiency were measured simultaneously.

The results of olfactometer assays showed that male and female adults had a strong response to 10 µg D(+)-camphor or ocimene, and the response rate of D(+)-camphor was higher than that of ocimene, respectively. Additionally, the results of field assays showed that D(+)-camphor also had attractive activity to both female and male adults. Previous surveys have shown that D(+)-camphor is considered a repellent for pests (Nenaah et al. 2011; Guo et al. 2016; Jiang et al. 2016; Singh et al. 2012). Interestingly, our results indicate that D(+)-camphor is used by P. tsushimanus adults as a key olfactory signal for host location. A similar result has been reported that the isothiocyanates released by cruciferous plants can strongly attract P. xylostella Linnaeus adults, but it is harmful to many other insects (Liu et al. 2020). Toxic isothiocyanates were used by P. xylostella for host location, which can effectively repel natural enemies or other food competitors. This survival strategy may be also used by P. tsushimanus to occupy this vacant niche and cause outbreaks. However, ocimene or isopropanol solvent control did not attract adults, and ocimene had no attractant activity by itself nor synergized attraction when combined with D(+)-camphor. This may be due to monophagous insects being more sensitive to the types and quality of host plant volatiles than polyphagous insects (Rajapakse et al. 2010), although ocimene exhibits certain attractive effects in indoor short-distance behavioral measurements. In addition, ocimene is one of the common and high content gas components released from the leaves and flower organs of various plants (Farré-Armengol et al. 2017), which may lead to the insensitivity of P. tsushimanus to ocimene in C. camphora forests. What’s more, ocimene is one of the important volatile components released by plants induced by insect feeding damage and it can induce plant defense reactions (Godard et al. 2008), which may be one of the important influencing factors. For example, ocimene is released by tea plants after being fed by insects such as Empoasca vitis Gothe, M. aurolineatus, or Ectropis oblique Prout (Sun et al. 2010; Cai et al. 2014). The mixed components containing ocimene have a strong repellent effect on M. aurolineatus (Han et al. 2017). The content of ocimene was high in the extract of Chloroxylon swietenia Apiaceae, which not only has a strong repellent effect but also poisons or inhibits food digestion on Spodoptera litura Fabricius (Kiran et al. 2006).

No adults were trapped by trapping device A (cross baffle trap device) with lures. We speculated that since P. tsushimanus adults always fly less and mainly move by crawling (Chen et al. 2021), they may be difficult to be captured after flying and hitting the baffles of trapping device A. The number of adults captured by trapping device C (trunk gluing trap device) with lures was less than that of trapping device B (self-made trap device). It is possible that the trapped adults can fall off after struggling, and consequently cannot be accurately counted. From these results, the most suitable trapping device for the attraction of P. tsushimanus adults in the field is the trapping device B (self-made). The number of adults captured by our self-made trap device with lures hung at high and medium heights was more than that of the low height, which may be related to the oviposition preference of P. tsushimanus adults in the high and medium height of the tree trunk. Therefore, they often come into contact with the high-height trapping device first. However, there is no significant difference in the number of adults captured by the trapping device B with lures hung at high and medium heights. Therefore, to facilitate ease of operation, the medium height of the trapping device should be a priority in the field assays. Dose-response tests showed that the number of adults captured by 5 mg was more than that of 10 mg D(+)-camphor, which was similar to the field assay result of A. glabripennis (Xu et al. 2020). These results indicated that the dose of lures in insect chemical communication needs to be moderate, not following the concept of the greater the better. Therefore, the suitable conditions for field monitoring and control of this pest are as follows: trapping device B (self-made), lure with 5 mg D(+)-camphor, hung at medium height of the trunk.

The volatiles from host plants in nature exist as mixed components, and consequently, multi-component mixtures are highly effective in luring the target insects (Binder et al. 1995; Xing et al. 2017). In this study, we preliminarily discovered a single component D(+)-camphor possessed a certain attractive activity, while other co-attractants or synergistic components remain to be identified in the future. In addition, field trapping is commonly affected by various environmental factors, such as temperature, humidity, and rainfall (Sainii et al. 1996; de Abreu et al. 1997; Hallett et al. 1999). Therefore, these factors affecting the trapping efficiency need to be further considered. Through the above-related research, the purpose is to improve or optimize the trapping effect of lures with D(+)-camphor as the key active component.

4.3 Relationship among host plant, pest, and their parasitic natural enemy insect

After being attacked by herbivorous insects, plants will also release volatiles to recruit some natural enemies, described as an indirect defense (Veterans, 1992; Havill et al. 2010). For example, after being attacked by Spodoptera exigua Hübner, Zea mays Linn releases volatiles to attract the natural enemies Cotesia marginiventris Cresson of S. exigua (Tunings et al. 1992). Brassica oleracea releases volatiles after being damaged by Pieris rapae to attract parasitoid Cotesia glomerata of P. rapae (Blaakmeer et al. 1992). In the camphor tree plantations, a natural enemy parasitoid Pseudocyanopterus pagiophloeusis Samartsev & Li parasitizes the larvae of P. tsushimanus. The parasitoid can be found every year and is a new species described from Shanghai, China (Samartsev et al. 2021). However, this new parasitoid has never been lured using the trapping devices in the field assays. Based on the above results, it is speculated that P. tsushimanus can avoid eavesdropping on the information in interspecific chemical communication by the natural enemy parasitoid P. pagiophloeusis when using D(+)-camphor as an important olfactory signal in host recognition. Similarly, the defensive isothiocyanates of Cruciferae plants are used by specialized insect females Plutella xylostella Linnaeus as olfactory signal compounds to locate host plants for oviposition (Liu et al. 2020). Toxic compounds can be used by pests to repel other competitors or natural enemies, which are often selected as the key olfactory signal for locating hosts. It is a good survival and evolution strategy for pests to occupy vacant ecological niches (e.g., utilize toxic compounds for host location), which may be the main reason for pest invasion, population reproduction, and rapid expansion in host plants.

4.4 Conclusion and outlook

Previous results have shown that P. tsushimanus can adapt to and thrive in C. camphora plantations. Furthermore, C. camphora, a broad-spectrum insect-repelling tree species, was the unique host tree species of P. tsushimanus. This study has found that D(+)-camphor is the key olfactory signal of P. tsushimanus preference for C. camphora. D(+)-camphor is used by P. tsushimanus for the host location. Although the lures currently used need to be further optimized, for example, the use of synergistic components, component distribution ratio, etc., it is clearly shown that D(+)-camphor is an indispensable key component in the development and utilization of lures for traps. In addition to interspecific chemical communication, the reproduction of insect populations also includes the intraspecific chemical communication of courtship between female and male adults. Therefore, future studies should focus on the identification of pheromone components of intraspecific chemical communication between female and male adults to comprehensively explore the adaptive mechanism of P. tsushimanus to its host plant and continuously control this pest below the economic threshold.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

Cong Chen and Dejun Hao conceived and designed the experiments and wrote the manuscript. Tian Xu, Shouyin Li, Mingyu Xue, and Yadi Deng performed the experiments and analysed the data. Yadi Deng，Binqi Fan, and Chufeng Yang collected insects. All authors read and approved the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant number 32271884) and the Science and Technology Commission of Shanghai Municipality (Grant number 18391903200).

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No competing interests reported.

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The key phytochemical cue D(+)-camphor is a promising lure for traps monitoring the new monophagous camphor tree borer Pagiophloeus tsushimanus (Coleoptera: Curculionidae)

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Insects

2.2 Headspace aeration of host volatiles

2.3 Identification of antenally-active compounds in host volatiles

2.4 Chemical analyses of host volatiles

2.5 Electroantennographic (EAG) assays

2.6 Olfactometer assays

2.7 Field assays

2.7.1 Experiment 1– Effect of different kinds of traps on trapping efficiency

2.7.2 Experiment 2– Effect of the height of the trapping device on trapping efficiency

2.7.3 Experiment 3– Effect of dose of host volatile components on trapping efficiency

2.7.4 Experiment 4– Effect of combination of host volatile components on trapping efficiency

2.8 Statistical analysis

3 Results

3.1 Identification of antenally-active compounds in host volatiles

3.2 Electroantennographic (EAG) assay

3.3 Olfactometer assays

3.4 Field assays

3.4.1 Experiment 1– Effect of different kinds of traps on trapping efficiency

3.4.2 Experiment 2– Effect of the height of the trapping device on trapping efficiency

3.4.3 Experiment 3– Effect of dose of host volatile components on trapping efficiency

3.4.4 Experiment 4– Effect of combination of host volatile components on trapping efficiency

4 Discussion and Conclusions

4.1 Antennal electroantennographic response of adults elicited by host plant volatiles

4.2 Bioactivity of host plant volatiles

4.3 Relationship among host plant, pest, and their parasitic natural enemy insect

4.4 Conclusion and outlook

Declarations

References

Additional Declarations

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