Toxicity and Physiological Effects of Nine Lamiaceae Essential Oils and Their Major Compounds on Reticulitermes dabieshanensis

The volatile metabolites of Salvia sclarea, Rosmarinus officinalis, Thymus serpyllum, Mentha spicata, Melissa officinalis, Origanum majorana, Mentha piperita, Ocimum basilicum and Lavandula angustifolia were determined by gas chromatography–mass spectrometry. The vapor insecticidal properties of the analyzed essential oils and their compounds were screened using Reticulitermes dabieshanensis workers. The most effective oils were S. sclarea (major constituent linalyl acetate, 65.93%), R. officinalis (1,8-cineole, 45.56%), T. serpyllum (thymol, 33.59%), M. spicata (carvone, 58.68%), M. officinalis (citronellal, 36.99%), O. majorana (1,8-cineole, 62.29%), M. piperita (menthol, 46.04%), O. basilicum (eugenol, 71.08%) and L. angustifolia (linalool, 39.58%), which exhibited LC50 values ranging from 0.036 to 1.670 μL/L. The lowest LC50 values were recorded for eugenol (0.060 μL/L), followed by thymol (0.062 μL/L), carvone (0.074 μL/L), menthol (0.242 μL/L), linalool (0.250 μL/L), citronellal (0.330 μL/L), linalyl acetate (0.712 μL/L) and 1,8-cineole (1.478 μL/L). The increased activity of esterases (ESTs) and glutathione S-transferase (GST) were observed but only alongside the decreased activity of acetylcholinesterase (AChE) in eight main components. Our results indicate that S. sclarea, R. officinalis, T. serpyllum, M. spicata, M. officinalis, O. marjorana, M. piperita, O. basilicum and L. angustifolia essential oils (EOs) and their compounds, linalyl acetate, 1,8-cineole, thymol, carvone, citronellal, menthol, eugenol and linalool could be developed as control agents against termites.


Introduction
Termites are significant agricultural and forestry pests across the world and can seriously threaten the survival of plants and buildings [1]. According to statistics, there are more than 2800 recorded termite variants in the world, 185 of which are considered pests [2]. They cause global economic losses of more than USD 40 billion annually [3]. There is no doubt that chemical pesticides are some of the most effective and widely used methods for termite control [3]. However, the excessive use of pesticides has led to a series of problems, such as the development of insect resistance, ecological imbalance and harm to mammalian and human health [4].
Lamiaceae are annual or perennial herbs or shrubs, which include 10 subfamilies, 236 genera and more than 7000 species [5]. They are mainly distributed in Asia, Europe and Africa. There are more than 99 genera and more than 808 species in China, which are distributed throughout the country, with higher numbers found in the southwest and south. Lamiaceae plants are famous for their rich aromatic oils, many of which can be used for medicine. In particular, the genus Mentha possesses anti-inflammatory, antiemetic, antispasmodic, analgesic, anticancer, anti-obesity, antidiabetic, anti-bloating, and immunomodulatory actions [6].
However, there are almost no reports on the fumigant efficacy of Lamiaceae species EOs against Reticulitermes dabieshanensis. Thus, the objective of the present study was (1) to evaluate the fumigant activities of Salvia sclarea, Rosmarinus officinalis, Thymus serpyllum, Mentha spicata, Melissa officinalis, Origanum majorana, Mentha piperita, Ocimum basilicum and Lavandula angustifolia EOs; (2) to investigate eight kinds of EOs' constituents; and (3) to determine the activities of detoxification enzymes and acetylcholine esterase.

GC-MS Analysis
The chemical compositions of Lamiaceae EOs are shown in Table 1 Limonene

Fumigation Activity of Lamiaceae EOs and Its Major Constituents
According to Table 2  The fumigation activity of the major components was further determined, and the results are shown in Table 3. Among the eight components tested, those with the highest toxicity were eugenol (LC 50 = 0.060 µL/L), followed by thymol (LC 50
To explore the relationship between the constituents of plant EOs and termiticidal activity, eight main components were tested for insecticidal activity against R. dabieshanensis. In this study, linalyl acetate, 1,8-cineole, thymol, carvone, citronellal, menthol, eugenol and linalool displayed effective vapor activity against R. dabieshanensis, which are the major components of the nine selected EOs. In general, the insecticidal activity of the EOs may be attributed to their major component, as has also been reported in some previous studies [1,29,34,35]. Here, the O. basilicum EO showed the highest insecticidal activity in comparison with its major constituent, eugenol, against R. dabieshanensis. Similarly, Piri et al. [36] found that the Ajwain EO showed the highest insecticidal activity in comparison with its constituents against Tuta absoluta larvae. Shahriari et al. [37] reported that the Ajwain EO was more toxic to Ephestia kuehniella larvae than thymol. Results from this study suggested that the EOs exhibited termiticidal activity which can be attributed to their major active chemical constituents.
EOs comprise lipophilic and low-molecular-weight volatile compounds, with terpenoids and phenylpropanoids as the most common constituents. Our results demonstrate that the linalyl acetate, 1,8-cineole, thymol, carvone, citronellal, menthol, eugenol and linalool display effective vapor activity against R. dabieshanensis. Previously, monoterpenes were found to possess varying insecticidal activities on the various insect species [38][39][40]. From the results of the present study, it is expected that monoterpenes will be able to be used successfully as a control agent against R. dabieshanensis.
In addition, it is known in the literature that most of the EOs and their major components can exert their toxic efficacy on insects, notably through inhibition of P450 cytochromes (CYPs) [41], GABA receptors [42], octopamine synapses [43], tyramine receptors [44] and the inhibition of acetylcho-linesterase (AchE) [1]. Furthermore, these com-ponents from various plant kingdoms can also regulate the intracellular pathways of mitochondrial biogenesis, through the removal of damaged mitochondria (mitophagy) and the generation of new ones required to preserve the cellular and mitochondrial homeostasis [45].
To further explore the physiological effect of Lamiaceae EOs on R. dabieshanensis, the changes of two detoxification enzymes (esterase, glutathione transferase), one hydrolase (acetylcholinesterase) and the activity of acetylcholinesterase in vitro in R. dabieshanensis were measured. The results in Table 4 show that the activities of esterase and glutathione transferase increase and the activities of acetylcholinesterase decrease after the termites are treated with the main ingredients. With the increase in concentration, the inhibitory activity of acetylcholinesterase in vitro also increased. These studies indicate that the essential oil of Lamiaceae may lead to the death of R. dabieshanensis by inhibiting the activity of acetylcholinesterase.
Shahriari et al. [37], Piri et al. [36], Wang et al. [46] and Yang et al. [1] found that after treatment with an essential oil or its components, the activities of ESTs and GST of insects increased significantly, indicating that ESTs and GST may participate in the detoxification process of insects. Table 4 shows that the activities of ESTs and GST of termites after treatment are significantly increased. In addition, studies have shown that essential oils and their main components can produce toxic effects on insects by inhibiting acetylcholinesterase (AchE) [36,46]. For instance, carvone showed the effect of inhibiting acetylcholinesterase (70.20% at 0.05 M) in Tribolium castaneum [47], while dihydrocarvone showed strong acetylcholinesterase inhibitory activity (IC 50 = 1.60 mg/mL) in Blattella germanica [48].
Our results indicate that S. sclarea, R. officinalis, T. serpyllum, M. spicata, M. officinalis, O. majorana, M. piperita, O. basilicum and L. angustifolia EOs and their compounds could be developed as control agents against termites. For the practical use of these oils and their constituents as novel termite-control agents, the safety of the oils and their compounds in humans and nontarget organisms and their modes of action should be investigated further.

Termites
Three colonies of R. dabieshanensis were collected from Linglong Mountain Scenic Area, Lin'an District, Hangzhou City, Zhejiang Province (longitude 30.2251 • N, latitude 119.6843 • E), and reared with water and newspapers in a laboratory. The healthy and active termite workers of uniform size were selected for further experiments.

GC-MS Analysis
The chemical analyses of EOs were determined by GC-MS. A gas chromatograph (Agilent 6890A, Santa Clara, CA, USA) was used with an HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness). The flow rate of helium carrier gas was set at 1.0 mL/min, the split ratio was set at 1:50 and a sample volume of 1.0 µL was injected. The injector and detector temperatures were set at 250 • C. The mass range was scanned from 15 to 500 m/z. The compound composition was identified by comparing its retention index with the NIST11.LIB database and the Adams [49] library.

Fumigant Toxicity
In order to conduct fumigations [36], filter paper strips (1.5 × 6 cm) were stuck to the lids of 1 L glass jars (10 cm diameter × 12.5 cm), and 0.04-3.0 µL of nine EOs, their major components or acetone as a control was added. Twenty healthy workers were put into a glass bottle, the bottle cap was quickly closed and a moist filter paper was placed on the bottom of the bottle as food. The experiment was repeated three times with three colonies, and the glass jars were kept at 25 ± 1 • C and 75 ± 5% RH. After 24 h, the number of dead termites was recorded.

Enzyme Assays
The effects of major constituents on the esterase enzymes, glutathione S-transferase and acetylcholine esterase against the worker adults of R. dabieshanensis were determined at the LC 30 concentrations. Enzyme extracts were prepared from five termite workers, homogenized in 1 mL 0.1 M phosphate buffer (pH 7.0) and centrifuged at 4 • C and 12,000× g for 15 min; then, the supernatants were placed in a 1.5 mL microcentrifuge tube and stored at −80 • C for later use.

Esterase (EST)
EST activity was determined utilizing the method of Yang et al. [1]. A total of 20 µL of 10 mM α-naphthyl acetate (α-NA) and β-naphthyl acetate (β-NA) was added separately, and, after that, 10 µL enzyme solution and 50 µL of 1 mM fast blue RR Salt were added. After mixing for 5 min at 27 • C, the OD value was measured at 450 nm with a 96-well microplate reader.

Glutathione S-Transferase (GST)
The GST activity was determined according to the method of Yang et al. [1]. The reaction solution contained 20 µL of 20 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 10 µL of enzyme solution. After incubation at 27 • C for 5 min, the OD value was measured at 340 nm using a 96-well microplate reader.

Acetylcholinesterase (AChE)
Acetylcholinesterase activity was determined using the method of Yang et al. [1]. The reaction solution was incubated at 25 • C for 5 min and contained 80 µL 0.1 M phosphate buffer (pH 7.0), 50 µL 10 mM acetylcholine iodide and 50 µL 10 mM of 5,5-dithiobis-2nitrobenzoic acid (DTNB), which was then added to 20 µL of enzyme solution. The OD value was measured at 405 nm using a 96-well microplate reader.

Acetylcholinesterase Inhibition
In an AChE inhibition test, five termites were ground using a porcelain mortar in 0.1 M Tris-HCl buffer (pH 7.8) (0.02 M NaCl and 0.5% Triton X-100). Then, the ground termites were centrifuged at 15,000× g for 15 min at 4 • C. The reaction solution contained 20 µL of the tested compound, 40 µL of enzyme solution, 50 µL of 10 mM acetylthiocholine iodide, 10 µL 4 mM DTNB and 100 µL of protein extraction buffer. After incubation at 27 • C for 30 min, the OD value was measured at 412 nm using a 96-well microplate reader.

Data Analysis
Toxicity data were subjected to probit analysis in order to estimate the LC 50 values of nine EOs, their major constituents and 50% inhibition AChE activity (IC 50 ). The data of the mortality and inhibition rates were analyzed by one-way ANOVA and Duncan's multiple comparison method, with a significance level of p < 0.05.
Funding: This work was supported by the Natural Science Foundation of Zhejiang Province (LZ20C040001).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.