Insecticidal Activities Against Odontotermes formosanus and Plutella xylostella and Corresponding Constituents of Tung Meal from Vernicia fordii

Simple Summary Odontotermes formosanus (Shiraki) (O. formosanus) and Plutella xylostella (Linnaeus) (P. xylostella) are common industrial and agricultural pests with a wide distribution range, multiple host species, few natural predators, and rapid reproduction. The control of these pests relies heavily on synthetic chemical pesticides; however, environmental pollution and the rise of insect resistance induced by the excessive use of synthetic chemical pesticides pose serious challenges. The present study was designed to systematically evaluate the insecticidal activities of different extracts of tung meal against the above two pests and to reveal the chemical constituents of the main active parts. The result confirmed the remarkable insecticidal activity of tung meal and provided a theoretical basis for its development as a potential plant insecticide. Abstract The environmental pollution, pesticide resistance, and other associated problems caused by traditional chemical pesticides with limited modes of action make it urgent to seek alternative environmentally-friendly pesticides from natural products. Tung meal, the byproduct of the detoxified Vernicia fordii (Hemsl.) seed, has been commonly used as an agricultural fertilizer and as a pesticide. However, its active insecticidal extracts and ingredients remain elusive. In the present study, the contact toxicities of tung meal extracts against the agricultural and forest pests like O. formosanus and P. xylostella were examined. Our results showed that ethyl acetate and petroleum ether extracts showed the strongest toxicity against O. formosanus and P. xylostella, respectively. In order to further explore the chemical profiles of the ethyl acetate and petroleum ether extracts, UPLC-Q/TOF-MS and GC-MS analyses have been performed, and 20 and 29 compounds were identified from EA and PE extracts, respectively. The present study, for the first time, verified the noteworthy insecticidal activities on the aforementioned agricultural and forest pesticides and revealed the potential active parts and chemical composition, which are conducive to further exploiting the potential of tung meal as a natural plant-derived insecticide for biological control of agricultural and forest pests.


Introduction
Vernicia fordii (Hemsl.) Airy Shaw, a subtropical perennial industrial crop, is widely distributed in the east of Asia. It originated from China and is also known as tung oil The termite-resistance activity of tung meal against O. formosanus was determined by contact with filter paper containing samples with different concentrations. Briefly, appropriate amounts of different extracts (1 mL) were evenly dropped on the qualitative filter paper (9 cm in diameter) and spread in a 9 cm diameter glass petri dish. Twenty worker termites of about the same size were put into each plate under 25 ± 1 • C and 70 ± 5% RH in the dark. Deaths of worker termites were recorded and eliminated every 24 h, and the observation was continued until all termites died. The test samples were diluted with dimethyl sulfoxide (DMSO) into the initial concentration of 10 mg/mL, and then diluted with distilled water to different concentrations (10, 5, 2.5, 1.25, 0.625 mg/mL). Distilled water and DMSO solution with the same concentration were used as controls. Abbott formula (Abbott, 1925) was used for survival rate correction, and the adjusted survival rate of 6 extracts after 24, 48, and 72 h were calculated, respectively.

Insecticidal Activity on Agricultural Pest P. xylostella
The insecticidal activity of tung meal against P. xylostella was measured by feeding a diet containing extracts with different concentrations. Twenty-four P. xylostella in the second instar larvae were used as test samples; the larvae of P. xylostella were fed with different formulated feed (1 mg sample/1 g normal feed) after starvation treatment for 6 h, and the deaths were counted and recorded every 24 h. The formulated feed was prepared as follows: tung meal extracts (0.005 g) were firstly dissolved in the solvents of 100 µL DMSO and 150 µL H 2 O; then the above solutions were mixed and stirred with 10 g of normal feed; finally, the prepared formulated feed was air-dried and stored at 4 • C. Distilled water and DMSO solution with the same concentration were used as controls. The analytical methods conducted were as the same as described in Section 2.3.1.

Chemical Composition Analysis 2.4.1. UPLC-Q/TOF-MS Analysis of EA Extracts
The chemical constituents of the EA extracts from tung meal were analyzed using an Agilent 1290 LC coupled with an Agilent 6530 accurate mass time-of-flight mass spectrometer (Agilent, Santa Clara, CA, USA) equipped with an Acquity UPLC-BEH-C18 column (2.1 × 50 mm, 1.7 µm; Waters, Milford, MA, USA). The Q/TOF-MS instrument was equipped with a dual ESI source in the negative ionization mode. The mobile phase consisted of mobile phase A (0.1% formic acid in ultrapure water) and mobile phase B (acetonitrile). The LC gradient elution was as follows: 5-27% B, 0-29 min; 27-32% B, 29-35 min; 32-95% B, 35-45 min; 95% B, 45-50 min. The injection volume was 10 µL, and the flow rate was 0.2 mL/min. The MS was performed under the following conditions: Mass Hunter workstation (Agilent) was used to obtain profile data with a mass range of m/z 100-1100 at a rate of 1 spectra/s. The temperature and the gas flow rate of drying gas were set at 350 • C and 8 L/min, respectively. The nebulizer pressure was set at 35 psi. The capillary voltage (Vcap) and fragment voltage were set at 3500 V and 175 V. The fixed collision energies were 10, 20, and 40 V, and reference masses of m/z 112.9855 and 1033.9881 were continuously introduced for accurate mass calibration. Compounds in the extracts of tung meal were tentatively identified by comparing the parent ion, retention time, and mass fragments with the correlated references and database.

GC-MS Analysis of PE Extracts
The identification of compounds in PE extracts was carried out in accord with the previous methods with slight modification [19,20]. Identification was performed by the gas chromatographic system (Agilent 7890A, Agilent, Santa Clara, CA., USA) combined with MS system (Agilent 7000C, Agilent, Santa Clara, CA., USA) equipped with an HP-5 capillary column. Gas chromatography is mainly used for the separation of volatiles. The carrier gas is helium with a flow rate of 1 mL/min. The GC column temperature program was initially set at 100 • C for 8 min and gradually increased to 220 • C at 5 • C/min, then at 2 • C/min and gradually increased to 275 • C, and finally at 5 • C/min and gradually increased to 310 • C and maintained for 10 min. The electron ionization (EI) system was used to obtain the mass spectrum with a mass range of 25-500 Da; the ionization energy is 70 eV in GC-MS detection. Identification of chemical composition was performed by using the NIST 11 Mass Spectral Library. The relative percentages of each compound were calculated by computerized integrator using total ion chromatography.

Statistical Analysis
All statistical analysis was performed employing SPSS Statistics version 24.0 software (IBM Corporation, Armonk, NY., USA), and experimental results were expressed as mean value (obtained by three replicates) ± standard deviation. Statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) combined with Duncan's test (p < 0.05).

Insecticidal Activity of Tung Meal on Forest Pest O. Formosanus
The insecticidal activity of tung meal against O. formosanus was represented by the survival rate after 72 h of treatment. It can be seen from Table S1 and Figure S1 that the insecticidal activity of tung meal varied with the types of extracts, exposure time, and concentration.
Within the concentration range of 2.5 to 10 mg/mL, different tung meal extracts showed diverse degrees of insecticidal activities against O. formosanus, among which the EA extracts were superior to the others. The detailed results are shown in Table S1. When the concentration was 10 mg/mL, the above 6 different tung meal extracts displayed comparable insecticidal activities against O. formosanus, with the equivalent survival rate of 0% after treatment for 72 h; when the concentration was cut to 5 mg/mL, the survival rates of the EA and PE extracts remained at 0%, while the DCM, CE, n-Bu, and H 2 O extracts were 1.25%, 3.75%, 3.75%, and 13.75%, respectively; when the concentration came to 2.5 mg/mL, the survival rate of the EA extracts also remained the lowest at 23.75%, and the other groups were 41.25% (DCM), 47.5% (CE), 56.25% (n-Bu), 25% (H 2 O), and 85% (PE). All extracts, except the PE extracts, were significantly lower (p < 0.05) than that of distilled water (97.50%) and the DMSO control group (97.50%) after 72 h of treatment with 2.5 mg/mL of samples. When the concentration was lower than 1.25 mg/mL, the survival rate of different extracts and fractions was more than 95%, indicating that the effective From these results, it can be concluded that tung meal has remarkable toxic activity against O. formosanus. Meanwhile, the EA extracts were the most promising as an insecticide, and the active components are mainly some compounds with moderate polarity. Interestingly, our present results were similar to the previous study of Sillma [21], in which they evaluated the insecticidal effects of different extracts from Jatropha curcas, another common plant of Euphorbiaceae, on two insect larvae of Bactrocera zonata and Bactrocera cucurbitae. They found that J. curcas exerted significant insecticidal effects on the two larvae, among which EA extracts were the most active part.

Insecticidal Activity of Tung Meal on Agricultural Pest P. xylostella
The influence of different extracts obtained from V. fordii on the survival rate of P. xylostella is shown in Table 1. Among all the extracts, PE extracts showed the most significant toxic activity against P. xylostella with a survival rate of 58.33% after adding 1 mg of the active ingredients into 1 g of normal feed for 72 h, whereas the n-Bu extracts (60.42%) displayed the second-highest response. EA and CE extracts led to similar survival rates of 75.00%. The survival rate for the above four extracts was significantly lower than that of DMSO and water control (P < 0.05); the survival rates for the other two extracts were 79.17% (H 2 O) and 85.42% (DCM). In this study, PE extracts of tung meal presented the highest insecticidal effect on P. xylostella, indicating that the components with toxic activity to P. xylostella were mainly low-polarity compounds, and the PE extracts of tung meal were more suitable to be further developed as plant-derived insecticides against P. xylostella. Therefore, further analysis of the chemical components of PE extracts was later conducted to reveal the potentially responsible compounds that might play a role in the insecticidal activity of tung meal and to explore the potential of tung meal as a botanical insecticide.

Characterization of Chemical Components in EA Extracts by UPLC-Q/TOF-MS
In natural ecosystems, a wide variety of secondary compounds are involved in plant defenses against insects, such as insect repellents and toxins. Our study revealed the insecticidal activity of tung meal extracts against O. formosanus and P. xylostella. However, the complex chemical components in the tung meal extracts with very low content make them very difficult to be identified using the traditional methods often involving tedious isolation, purification, and NMR identification which greatly limit the exploration of their chemical components. UPLC-Q/TOF-MS has become an effective method for the identification of complex natural products combining its rapid and efficient separation ability of UPLC with the highly sensitive and accurate detection capacity of TOF-MS. In this study, UPLC-Q/TOF-MS was used to rapidly identify the chemical constituents in the  Table 1.
A total of 20 compounds were tentatively identified from the EA extracts of tung meal by comparing their retention times and MS fragments with the data in the references. Each compound was numbered according to the retention time of the base peak chromatography (BPC) (Figure 1). The retention times, molecular formula and weight, and MS fragments of all compounds are listed in Table 2.
secticidal activity of tung meal extracts against O. formosanus and P. xylostella. However, the complex chemical components in the tung meal extracts with very low content make them very difficult to be identified using the traditional methods often involving tedious isolation, purification, and NMR identification which greatly limit the exploration of their chemical components. UPLC-Q/TOF-MS has become an effective method for the identification of complex natural products combining its rapid and efficient separation ability of UPLC with the highly sensitive and accurate detection capacity of TOF-MS. In this study, UPLC-Q/TOF-MS was used to rapidly identify the chemical constituents in the EA extracts of tung meal which exhibited the best activity against O. formosanus as shown in Table 1.
A total of 20 compounds were tentatively identified from the EA extracts of tung meal by comparing their retention times and MS fragments with the data in the references. Each compound was numbered according to the retention time of the base peak chromatography (BPC) (Figure 1). The retention times, molecular formula and weight, and MS fragments of all compounds are listed in Table 2.    Figure 1. b The relative content (%) was obtained by integrating the chromatogram peaks in Figure 1. Lignans are common components of Euphorbiaceae plants. As illustrated in Table 2, fourteen peaks (peaks 1, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19) were tentatively identified and classified as lignans. Compounds Figure 2 shows the MS spectrum and the corresponding proposed fragment pathways. Hence, peaks 8 and 11 were tentatively assigned as americanol A and isoamericanol A, which is in good agreement with the previous report [23]. Lignans are common components of Euphorbiaceae plants. As illustrated in Table 2, fourteen peaks (peaks 1, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19) were tentatively identified and classified as lignans. Compounds Figure 2 shows the MS spectrum and the corresponding proposed fragment pathways. Hence, peaks 8 and 11 were tentatively assigned as americanol A and isoamericanol A, which is in good agreement with the previous report [23]. The MS data were in agreement with those previously reported, and compounds 1 and 5 were identified as (+)-7-epi-sesamindicatechol and (±)-3,3′-bisdemethylpinoresinol, respectively [22,23].
Compound 12 displayed a deprotonated molecular ion [M-H] − of m/z 327 at tR 21.39 min, which was 2 Da (2 hydrogens) less than americanol A and isoamericanol A. The fragment at m/z 297 was attributed to the neutral loss of CH2O. Typical fragment ions of compound 12 were obtained at m/z 165, m/z 163, m/z 147, and m/z 135, which were similar to americanol A and isoamericanol A. Compound 12 was identified as isoamericanin A, where one hydroxymethyl group in isoamericanol A was oxidized to an aldehyde group, in comparison with their spectral features. This compound had also been found in the seed testa of Vernicia fordii [23].
Compound 12 displayed a deprotonated molecular ion [M-H] − of m/z 327 at t R 21.39 min, which was 2 Da (2 hydrogens) less than americanol A and isoamericanol A. The fragment at m/z 297 was attributed to the neutral loss of CH 2 O. Typical fragment ions of compound 12 were obtained at m/z 165, m/z 163, m/z 147, and m/z 135, which were similar to americanol A and isoamericanol A. Compound 12 was identified as isoamericanin A, where one hydroxymethyl group in isoamericanol A was oxidized to an aldehyde group, in comparison with their spectral features. This compound had also been found in the seed testa of Vernicia fordii [23].
There are six neolignan isomers including peaks 6, 10, 13, 14, 15, and 16, which were detected at t R  The fragment ion of m/z 165 was similar to americanol A and isoamericanol A. By comparing the fragmentation pathways with those of the references, compounds 6, 10, 13, 14, 15, and 16 could be tentatively identified as isoamericanol B1, isoamericanol B2, isoamericanol C1, isoamericanol C2, princepin, and isoprincepin [24], which was in accord with the findings of Chen et al [23]. Those compounds were also named sesquineolignans because they were structurally formed by the oxidative polymerization of three phenylpropyl. In addition, all of these chemical components had also been found in the seeds of Joannesia princeps Vellozo, another plant of the Euphorbiaceae [25].
Three dilignant isomers, compounds 17, 18, and 19, were detected at t R 31.28, 32.31, and 33.40 min with the same deprotonated molecular ion [M-H] − of m/z 657 and MS spectral data. The characteristic fragment ions of m/z 493 and m/z 163 resulted from the cleavage of 1,4-dioxane. Another major characteristic fragment ion of m/z 165 was also observed. Furthermore, fragment ions of m/z 493 and other fragment ions were similar to those of (iso) princepin. Those three compounds were tentatively identified as isodiverniciasin A, diverniciasin C, and diverniciasin B, which have been detected in the seed testa of V. fordii [23]. The distinguished fragment ion of m/z 119 indicated the loss of H 2 O, and another major fragment ion m/z 108 was obtained by the loss of CO. Considering that the above fragments were the same as in the previous report [28], compound 3 was tentatively identified as 2,4-dihydroxybenzaldehyde, which had been detected in the seed testa of V. fordii [23].

Fatty Acids
The According to the structures of these compounds shown in Figure 3, these components can be structurally characterized as lignans, fatty acids, flavonoids, disaccharides, and dihydroxybenzaldehyde. According to their basic carbon frames, those lignans can be divided into three categories, including monolignan (compounds 1, 5, 8, 11, and 12), sesquineolignan (compounds 6, 10, 13, 14, 15, and 16), and dineolignans (compounds 17, 18 and 19). By integrating the chromatogram, it was found that the contents of isoamericanol B1 and its related isomers (isoamericanol B2, isoamericanol C1, isoamericanol C2, isoprincepin, and isoprincepin) were the highest in the EA extracts of tung meal, accounting for 24.01% of the total, followed by (+)-7-epi-sesamin-dicatechol and its related isomers (±)-3,3 -bisdemethylpinoresinol, which also accounted for a high proportion of 13.58%; besides, americanol A and its related isomer isoamericanol A also accounted for a high proportion of 10.14%. , 12, x FOR PEER REVIEW 11 of 16 in the Human Metabolome Database (HMDB), and compound 9 was preliminarily identified as 5,7,4′-tri-O-methylcatechin. According to the structures of these compounds shown in Figure 3, these components can be structurally characterized as lignans, fatty acids, flavonoids, disaccharides, and dihydroxybenzaldehyde. According to their basic carbon frames, those lignans can be divided into three categories, including monolignan (compounds 1, 5, 8, 11, and 12), sesquineolignan (compounds 6, 10, 13, 14, 15, and 16), and dineolignans (compounds 17, 18 and 19). By integrating the chromatogram, it was found that the contents of isoamericanol B1 and its related isomers (isoamericanol B2, isoamericanol C1, isoamericanol C2, isoprincepin, and isoprincepin) were the highest in the EA extracts of tung meal, accounting for 24.01% of the total, followed by (+)-7-epi-sesamin-dicatechol and its related isomers (±)-3,3′-bisdemethylpinoresinol, which also accounted for a high proportion of 13.58%; besides, americanol A and its related isomer isoamericanol A also accounted for a high proportion of 10.14%.  Previous studies revealed that trehalose can remarkably reduce the mean day survival rate of sweet potato whitefly Bemisia tabaci (Gennadius) [29], and trehalose/mannose/maltose specific lectins exhibited promising insecticidal activities against stored product pests [30]. Azelaic acid was also detected at the active sites of a variety of plants with remarkable insecticidal activities [31]. The insecticidal activity of lignans has been rarely studied or not yet discovered. Some lignans, such as (±)-3,3 -bisdemethylpinoresinol, americanol A, americanin A, and isoprincepin, have been found in the fruit of Morinda citrifolia [32], and the fruit extracts of Morinda citrifolia have been reported to exert significant insecticidal activities against a variety of agricultural pests and have been used as an effective insecticide in the Philippines and Hawaii [33]. Princepin and isoprincepin are uncommon in natural products and their insecticidal activities have been poorly studied, but their structure-related compound haedoxan A has been reported to have prominent insecticidal activity [34,35]. Furthermore, J. curcas has been reported to have insecticidal activities on houseflies and insect pests of stored products [36,37]. In this study, we detected some major components in tung meal, such as isoamericanol A, americanol A, (+)-7-Epi-sesamindicatechol, (±)-3,3 -Bisdemethylpinoresinol, and isoprincepin, which are also the main components of J. curcas [38]. In conclusion, the insecticidal activity of tung meal could be attributed to the presence of those components; however, the insecticidal activities and their mechanisms based on these potential components need to be further verified.

Identification of Volatile Constituents in PE Extracts by GC-MS
The components in PE extracts were tentatively identified by comparing the MS fragments with the NIST database. Based on the literature, a compound whose similarity scores above 90% was taken into account [39]. The gas chromatograms of PE extracts and the corresponding compounds tentatively identified in the extracts, along with the retention times and percentages are shown in Figure 4 and Table 3, respectively. According to the retention times peaked in the GC-MS analysis, a total of twenty-nine compounds, accounting for 87.26%, were identified, which mainly consisted of unsaturated fatty acids and their ethyl ester, phytosterols, etc. Previous studies revealed that trehalose can remarkably reduce the mean day survival rate of sweet potato whitefly Bemisia tabaci (Gennadius) [29], and trehalose/mannose/maltose specific lectins exhibited promising insecticidal activities against stored product pests [30]. Azelaic acid was also detected at the active sites of a variety of plants with remarkable insecticidal activities [31]. The insecticidal activity of lignans has been rarely studied or not yet discovered. Some lignans, such as (±)-3,3′-bisdemethylpinoresinol, americanol A, americanin A, and isoprincepin, have been found in the fruit of Morinda citrifolia [32], and the fruit extracts of Morinda citrifolia have been reported to exert significant insecticidal activities against a variety of agricultural pests and have been used as an effective insecticide in the Philippines and Hawaii [33]. Princepin and isoprincepin are uncommon in natural products and their insecticidal activities have been poorly studied, but their structure-related compound haedoxan A has been reported to have prominent insecticidal activity [34,35]. Furthermore, J. curcas has been reported to have insecticidal activities on houseflies and insect pests of stored products [36,37]. In this study, we detected some major components in tung meal, such as isoamericanol A, americanol A, (+)-7-Epi-sesamin-dicatechol, (±)-3,3′-Bisdemethylpinoresinol, and isoprincepin, which are also the main components of J. curcas [38]. In conclusion, the insecticidal activity of tung meal could be attributed to the presence of those components; however, the insecticidal activities and their mechanisms based on these potential components need to be further verified.

Identification of Volatile Constituents in PE Extracts by GC-MS
The components in PE extracts were tentatively identified by comparing the MS fragments with the NIST database. Based on the literature, a compound whose similarity scores above 90% was taken into account [39]. The gas chromatograms of PE extracts and the corresponding compounds tentatively identified in the extracts, along with the retention times and percentages are shown in Figure 4 and Table 3, respectively. According to the retention times peaked in the GC-MS analysis, a total of twenty-nine compounds, accounting for 87.26%, were identified, which mainly consisted of unsaturated fatty acids and their ethyl ester, phytosterols, etc.    Figure 4. b The relative content (%) was obtained by integrating the chromatogram peaks in Figure 4. c Molecular weight; d Similarity score obtained by matching NIST 11.
Among those compounds, the highest peak appeared at t R 33.00 min, accounting for 38.44% of the total content, which was estimated as ethyl α-linolenate or ethyl γ-linolenate according to the MS data. The third highest peak appeared in t R 33.82 min, accounting for 8.93% of the total content, and was tentatively identified as the isomer of the highest peak due to the same MS/MS fragments. The second highest peak appeared in t R 29.69 min, accounting for 14.36% of the total content, and was tentatively identified as ethyl linolate. In addition, ethyl oleate (6.34%), ethyl palmitate (3.82%), ethyl stearate (3.09%), stearic acid (2.38%), palmitic acid (2.09%), and β-sitosterol (1.58%) were also tentatively identified in PE extracts. These results were consistent with previous studies [40,41].
Fatty acids and their ethyl ester, and β-sitosterol may be the principal components exerting remarkable insecticidal activity against P. xyloides. For example, ethyl linolenate, ethyl oleate, ethyl palmitate, ethyl stearate, and ethyl linolate were found in Eupatorium odoratum extract, an insect repellent, by inhibiting oviposition of P. xyloides [42]. The volatile oils and crude extracts of Zingiber officinale have noteworthy insecticidal, repellent, and oviposition activities against many pests like Callosobruchus chinensis, Sitophilus zeamais, and P. xylostella [43]. In a similar study, five components with a content of more than 2% have also been detected in Z. officinale essential oil and oleoresins, including palmitic acid, ethyl palmitate, ethyl linolate, ethyl oleate, and stearic acid [44]. In addition, the ethanol extracts of Cynanchi auriculati showed superior insecticidal efficacy to common pesticides against P. xylostella, among which PE extracts was the highest, and the analysis of chemical composition showed that it was rich in β-sitosterol [45].

Conclusions
In the present study, different extracts from the deoiled tung meal of V. fordii were first revealed to exhibit diverse insecticidal activities against O. formosanus and P. xylostella, respectively. The corresponding active extracts and potential components, including lignans, fatty acids and their derivatives, and others, were further confirmed by UPLC-Q/TOF-MS and GC-MS, which provided strong support for alternative forest and agricultural pest control by tung meal. Our results suggest that tung meal extract or its derivatives may be used to control and manage the infestation of some pests like O. formosanus and P. xylostella, as well as to mitigate the damage caused by chemical pesticides currently in use.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/insects12050425/s1, Table S1: Insecticidal activity of different tung meal extracts on termite of O. formosanus, Figure S1: Insecticidal activity of different tung meal extracts on O. formosanus and P. xylostella. The different letters (a-c) after the mean value represent significant differences at p < 0.05 level according to Duncan Test.