Chemical Composition, Larvicidal and Ovicidal Activities, and Enzyme Inhibition Capacity of Thymus serpyllum Essential Oils Against Spodoptera litura (Fabricius)

Abstract

Due to their effectiveness at low doses and relative safety for non-target species, plant essential oils (EOs) are considered ideal alternatives to conventional pesticides for pest control. In this study, the chemical composition of Thymus serpyllum (T. serpyllum) EO was construed by Gas Chromatography-Mass Spectrometry (GC-MS), and its larvicidal and ovicidal activity against omnivorous pests Spodoptera litura (S. litura) was assessed. The effects of T. serpyllum EO on the activities of antioxidant detoxification enzymes were also measured. GC–MS analysis revealed that the main constituents of T. serpyllum EO were thymol (42.1%), p-cymene (22.4%), and γ-terpinene (18.6%). In the larvicidal toxicity experiment, the T. serpyllum EO demonstrated LC50 values of 0.606 and 0.664 mg/mL against the second- and third-instar larvae of S. litura, respectively, after 48 h exposure. Moreover, an EC₅₀ value of 0.905 mg/mL was measured against S. litura eggs. In S. litura, T. serpyllum EO treatment reduced the enzymatic activity of ESTs and GST and, conversely, increased the enzymatic activity of AChE. Overall, this study demonstrated that T. serpyllum EO has the potential to be implemented as a novel eco-friendly insecticide against S. litura.

1. Introduction

Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae) is a typical destructive crop pest worldwide [1]. Over 300 plant species have been identified as its hosts, including cotton, maize, vegetables, rice, groundnut, and soybean, strongly impacting the agricultural industry [2,3,4]. S. litura (Spodoptera litura Fabricius, 1775) (Lepidoptera: Noctuidae) exhibits a high reproductive and developmental ability, resulting in five to six overlapping generations annually. S. litura larvae are characterized by their ability to feed on leaves, buds, fruits, and flowers. If not treated in time, it might cause severe crop losses or even destruction [5,6]. Globally, synthetic insecticides are often used to control S. litura. However, due to their extensive application and due to long-term interactions between pesticides and insects, S. litura has developed resistance to many conventional pesticides, including organochlorides, organophosphates, cyantraniliprole, pyrethroids, abamectin, avermectins, and indoxacarb [1,7,8,9]. To date, severe insecticide resistance has been observed in S. litura in numerous countries, including China, Puerto Rico, Mexico, India, Pakistan, and Thailand [1,3].

In recent years, considering the non-selectivity and persistence of chemical pesticides, plant essential oils (EOs) and their derivatives garnered a growing interest due to their effectiveness at low doses [10] and relative safety to non-target organisms [11]. They can be completely degraded in the environment, leaving no residues, and are regarded as a valuable resource for the development and formulation of environmentally friendly pesticides with low toxicity. Recently, extracts or EOs from various plants such as Crithmum maritimum L. [12], Couroupita guianensis (Aubl.) [13], Piper betle L. [14], Zanthoxylum armatum DC. [15], Inula racemosa (Asteraceae) [16], Zanthoxylum alatum Roxb. [17], Vernonia anthelmintica L. [18], Wedelia prostrata (Hook. et Arn.) Hemsl. [19], Acorus calamus L. [20], Alpinia galanga (Linn.) Willd, and Ocimum basilicum L. [21] have been assessed on S. litura to examine their toxicity and antifeedant potential. Essential oils and their major constituents can serve as safe alternatives for pest control. Mahajan et al. [20] reported that β-caryophyllene exhibited a satisfactory inhibitory activity on the growth and development of S. litura. Yooboon et al. [21] discovered that piperine and β-asarone showed acute toxicity against S. litura, with their combination demonstrating greater acute toxicity than the individual compounds. Additionally, thymol [22], pogostone [23], and pulegone [3] were found to be effective against S. litura.

Thyme L. plants belong to the Lamiaceae family and are perennial herbs or low shrubs. More than 250 species of this genus have been recorded worldwide, with wide distribution in Northern Africa, temperate regions of Europe, and Asia [24]. Essential oils have been extensively adopted and utilized by the pharmaceuticals sector and the food sector for their potential biological properties and activities [25]. Previous studies have shown that Thymus serpyllum (L.) (T. serpyllum) EO has valuable biological activities, including antibacterial [26], antimicrobial [27], and antifungal activities [28]. Regarding its insecticidal activity, T. serpyllum EO exhibited notable efficacy in the control of Frankliniella occidentalis (Pergande) [29], Acanthoscelides obtectus (Say) (A. obtectus) [30], Musca domestica L. (M. domestica) [24], Varroa destructor (V. destructor) (Anderson and Trueman) [31] and Reticulitermes dabieshanensis (R. dabieshanensis) according to Wang et Li [32]. The composition of T. serpyllum EO has been inadequately studied [24,32], and its toxicity against S. litura remains unknown.

This study aimed to elucidate the insecticidal efficacy and mechanism of action of T. serpyllum essential oil against S. litura. In the present research, we hypothesized that it has considerable activity against S. litura larvae and eggs and can inhibit its growth by modulating enzyme activities. We investigated the effects of T. serpyllum EO on S. litura second- and third-instar larvae. The objectives of our study were to (1) analyze the composition of T. serpyllum EO using GC-MS; (2) evaluate the larvicidal and ovicidal activity of T. serpyllum EO on S. litura; and (3) explore the impact of T. serpyllum EO on S. litura detoxification enzymes. The findings will facilitate the formulation of efficient and low-toxicity botanical insecticides for the management of S. litura.

2. Results

2.1. Chemical Composition of T. serpyllum EO

The major constituents of the T. serpyllum EO are presented in Table 1. The T. serpyllum EO comprised 13 primary chemical constituents, accounting for 99.2% of its composition. The most abundant constituent was thymol (42.1%). Furthermore, other constituents included p-Cymene (22.4%), γ-Terpinene (18.6%), Carvacrol (3.6%), β-Pinene (2.7%), Linalool (2.5%), α-Pinene (1.6%), α-Terpinene (1.5%), Camphene (1.3%), Limonene (1.2%), Camphor (0.8%), Terpin-4-ol (0.6%), and α-Terpineol (0.3%).

Table 1.

Components of Thymus serpillum by GC-MS.

No	Compounds a	RI b	RI c	(%)
1	α-Pinene	937	936	1.6
2	Camphene	954	954	1.3
3	β-Pinene	977	979	2.7
4	α-Terpinene	1018	1017	1.5
5	p-Cymene	1025	1025	22.4
6	Limonene	1030	1029	1.2
7	γ-Terpinene	1060	1060	18.6
8	Linalool	1099	1097	2.5
9	Camphor	1146	1134	0.8
10	Terpin-4-ol	1180	1177	0.6
11	α-Terpineol	1191	1186	0.3
12	Thymol	1292	1290	42.1
13	Carvacrol	1307	1299	3.6

^a Components in order of elution from an HP-5 MS column. ^b RI, retention index computed on the HP-5MS column relative to C8–C28 n-alkanes. ^c Relative retention indices taken from Adams.

2.2. Larval Toxicity

The toxicity of T. serpyllum EO to S. litura second- and third-instar larvae was evaluated using the leaf-dipping approach (Table 2 and Table 3). T. serpyllum EO toxicity varied in a dose-dependent manner. As shown in Table 2, the LC50 and LC90 reached 1.632 and 4.463 mg/mL in second-instar larvae after 12 h of exposure. After 24 h, the LC50 and LC90 reached 1.033 and 3.294 mg/mL, after 36 h, 0.780 and 2.317 mg/mL; after 48 h, 0.606 and 1.749 mg/mL; and after 60 h, 0.444 and 1.313 mg/mL. Following 72 h of exposure, LC50 and LC90 were 0.300 and 0.959 mg/mL, respectively. The larval toxicity of T. serpyllum EO against S. litura 2nd-instar larvae showed an increased trend with increased concentration. At the same time, the effective concentration showed a decreasing trend with more prolonged treatment and exposure time.

Table 2.

Second-instar larvael activity of the Thymus serpillum EO.

Time (h)	Concentration (mg/mL)	Mortality (%) ± SD	LC50 (mg/mL) (95%CL *)	LC90 (mg/mL) (95%CL)	χ2
12	0.25	3.3 ± 2.9 b	1.632 (1.281–2.378)	4.463 (2.886–11.051)	15.980
	0.5	5.0 ± 5.0 b
	1.0	18.3 ± 5.8 b
	2.0	66.7 ± 10.4 a
		F3,8 = 60.317, p < 0.0001
24	0.25	11.7 ± 2.9 c	1.033 (0.815–1.393)	3.294 (2.186–7.114)	15.789
	0.5	16.7 ± 2.9 c
	1.0	35.0 ± 5.0 b
	2.0	83.3 ± 10.4 a
		F3,8 = 85.481, p < 0.0001
36	0.25	15.0 ± 0.0 c	0.780 (0.667–0.919)	2.317 (1.794–3.384)	10.300
	0.5	23.3 ± 7.6 c
	1.0	53.3 ± 7.6 b
	2.0	93.3 ± 2.9 a
		F3,8 = 120.600, p < 0.0001
48	0.25	20.0 ± 5.0 c	0.606 (0.517–0.707)	1.749 (1.386–2.460)	10.254
	0.5	35.0 ± 8.7 c
	1.0	65.0 ± 5.0 b
	2.0	98.3 ± 2.9 a
		F3,8 = 108.062, p < 0.0001
60	0.25	31.7 ± 2.9 c	0.444 (0.369–0.520)	1.313 (1.049–1.835)	8.827
	0.5	46.7 ± 10.0 c
	1.0	80.0 ± 5.0 b
	2.0	100 ± 0.0 a
		F3,8 = 81.784, p < 0.0001
72	0.25	46.7 ± 2.9 b	0.300 (0.228–0.365)	0.959 (0.767–1.357)	9.744
	0.5	65.0 ± 10.0 b
	1.0	90.0 ± 10.0 a
	2.0	100 ± 0.0 a
		F3,8 = 33.640, p < 0.0001

CL *: confidence limit which has been calculated with 95% confidence. The symbols “a”, “b” and “c” in the table denote levels of statistical significance, indicating meaningful differences between groups.

Table 3.

Third-instar Larval activity of the Thymus serpillum EO.

Time (h)	Concentration (mg/mL)	Mortality (%) ± SD	LC50 (mg/mL) (95%CL)	LC90 (mg/mL) (95%CL)	χ2
12	0.25	1.7 ± 2.9 c	1.725 (1.458–2.174)	4.401 (3.205–7.486)	13.964
	0.5	5.0 ± 0.0 c
	1.0	15.0 ± 5.0 b
	2.0	63.3 ± 2.9 a
		F3,8 = 235.933, p < 0.0001
	0.25	11.7 ± 2.9 c	1.159 (0.962–1.462)	4.218 (2.918–7.646)	9.307
	0.5	15.0 ± 5.0 c
	1.0	35.0 ± 5.0 b
	2.0	78.3 ± 2.9 a
		F3,8 = 169.333, p < 0.0001
36	0.25	13.3 ± 2.9 d	0.818 (0.696–0.972)	2.554 (1.945–3.843)	4.581
	0.5	23.3 ± 2.9 c
	1.0	55.0 ± 5.0 b
	2.0	88.3 ± 2.9 a
		F3,8 = 276.000, p < 0.0001
48	0.25	18.3 ± 2.9 d	0.664 (0.560–0.786)	2.166 (1.657–3.244)	5.220
	0.5	35.0 ± 5.0 c
	1.0	60.0 ± 5.0 b
	2.0	93.3 ± 2.9 a
		F3,8 = 191.667, p < 0.0001
60	0.25	31.7 ± 2.9 c	0.467 (0.389–0.547)	1.398 (1.112–1.967)	8.629
	0.5	45.0 ± 5.0 c
	1.0	76.7 ± 5.8 b
	2.0	100 ± 0.0 a
		F3,8 = 142.500, p < 0.0001
72	0.25	43.3 ± 10.4 c	0.317 (0.245–0.382)	0.994 (0.796–1.399)	6.136
	0.5	65.0 ± 5.0 b
	1.0	88.3 ± 5.8 a
	2.0	100 ± 0.0 a
		F3,8 = 45.667, p < 0.0001

The symbols “a”, “b” and “c” in the table denote levels of statistical significance, indicating meaningful differences between groups.

T. serpyllum EO demonstrated considerable efficacy against third-instar larvae of S. litura. As indicated in Table 3, the LC₅₀ and LC₉₀ values were 1.725 mg/mL and 4.401 mg/mL after 12 h of exposure. At a 0.25 mg/mL concentration, the mortality rate was 1.7 ± 2.9%, while it reached 63.3 ± 2.9% at 2.0 mg/mL. The toxicity of T. serpyllum EO to the third instar larvae increased with prolonged exposure. Mortality rates at each concentration rose progressively, with LC₅₀ and LC₉₀ values diminishing over time. After 72 h, they fell to 0.317 and 0.994 mg/mL, respectively. During this time point, the mortality rate reached 43.3 ± 10.4% at 0.25 mg/mL and increased to 100 ± 0.0% at 2.0 mg/mL. No mortality larvae mortality was observed in the control group treatment with acetone. One-way ANOVA analysis revealed a significant difference in mortality rates across the different EO concentrations (p < 0.0001). Tukey’s post hoc test also confirmed significant differences between the concentrations (p < 0.05). Logistic regression modelling indicated an EC50 value of 0.30 mg/mL at 72 h of exposure. These findings confirm that the toxicity of T. serpyllum EO to third-instar larvae of S. litura significantly increases with both exposure duration and concentration, while the effective concentration is reduced with increased exposure duration.

2.3. Ovicidal Activity

The ovicidal activity of T. serpyllum EO is shown in Figure 1. A dosage-dependent relationship was observed, with an EC₅₀ = 0.905 mg/mL. The hatching rate in the 0, 0.25, 0.5, 1, 2, 3, and 4 mg/mL T. serpyllum EO treatment groups were 100 ± 0%, 91.7 ± 3.6%, 75.0 ± 6.3%, 54.2 ± 9.5%, 37.5 ± 6.3%, 12.5 ± 6.3%, and 0 ± 0%, respectively.

Figure 1.

Ovicidal activity of the Thymus serpillum on eggs of Spodoptera litura. Values are mean ± SD of 3 replicates, and significant differences are indicated by different letters (a–e) (ANOVA, Tukey’s HSD, p < 0.05).

2.4. Activity Against S. litura Cellular Detoxification Enzymes

The potential role of T. serpyllum EO as an inhibitor of S. litura detoxifying enzymes involved in cellular detoxification was also investigated. CarE, GST, and AChE activities were measured, after 24 h of treatment with T. serpyllum (LC₅₀ = 1.033 mg/mL). In the “Control group”, 10 μL of distilled water was used instead of 10 μL of the solution, and the rest of the reagents were kept unchanged. Among these three enzymes, AChE was significantly inhibited after treatment with T. serpyllum EO (Figure 2D). Compared with the control group, the α-NA activity, β-NA activity, and GST activities of S. litura were significantly increased by T. serpyllum EO (Figure 2A–C).

Figure 2.

The effects of Thymus serpillum EO on the activities of detoxifying enzymes of Spodoptera litura. (A) Esterases (α-NA) activity for 24 h of LC₅₀ treatment; (B) esterases (β-NA) activity for 24 h of LC₅₀ treatment; (C) GSTs activity for 24 h of LC₅₀ treatment; (D) AChE activity for 24 h of LC₅₀ treatment. p < 0.05 were illustrated by the asterisk according to an independent sample t-test.

3. Discussion

The main constituent of T. serpyllum EO was thymol (42.1%), which is in accordance with the findings of Xie et al., Hýbl et al., and Yang et al. [24,31,32].

S. litura is a predominant polyphagousan pest [3] and has developed resistance to many chemical pesticides, such as benzoate, emamectin, carbamates, and pyrethroids [33]. Plant essential oils can serve as an alternative to biopesticides for pest control, targeting species of agricultural importance [17].

According to the study findings, the LC₅₀ of T. serpyllum EO against S. litura 2nd, and 3rd instar larvae after a 48 h exposure was 0.606 mg/mL and 0.664 mg/mL, respectively. These findings suggest that T. serpyllum EO exhibits substantial toxicity towards S. litura. GC-MS analysis identified the primary constituents of T. serpyllum EO, namely thymol, carvacrol, (S)-(+)-carvone, estragole, citral, linalool, (S)-(-)-limonene, and γ-terpinene, all known for their insecticidal properties against larvae of various pests [33]. A limonene analogue resulted in morphological and physiological alterations in Drosophila suzukii L3 larvae, contributing to elevated larval mortality. This aligns with the findings by Yang [34], which reported that T. serpyllum EO exhibited high toxicity to R. dabieshanensis. Additionally, Hýbl et al. highlighted the insecticidal properties of T. serpyllum EO on V. destructor (LC50 = 2.549 μL/L) [31]. Xie et al. demonstrated that this essential oil also has toxic effects on M. domestica (LC50 = 20.9 μL/L) [24]. Sertkaya (2021) documented an LC₅₀ of 1.12 μL/L for T. serpyllum EO against A. obtectus, along with a 90.0% repellent efficacy against Frankliniella occidentalis at a 0.5% concentration [35]. Similarly, Lantana camara EO exhibited potent insecticidal activity against Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus larvae [36]. These findings align with prior research conducted by Yang et al. [32]. Similarly, T. serpyllum EO exhibited significant toxicity towards R. dabieshanensis. In addition, Hýbl et al. [31] demonstrated the insecticidal effect of T. serpyllum EO against Varroa destructor (LC₅₀ = 2.549 μL/L). Xie et al. [24] showed T. serpyllum EO had toxic effects on M. domestica (LC₅₀ = 20.9 μL/L). Research conducted by Sertkaya [30] indicated that the LC₅₀ value of T. serpyllum EO against A. obtectus was 1.12 μL/L. Picard et al. [29] assessed the insecticidal ability of T. serpyllum EO, which showed a 90.0% repellent efficacy against F. occidentalis at 0.5%.

More recently, plant essential oils (EOs) have been implemented as a control agent against S. litura due to their effectiveness at low doses [10,11] and relative safety for non-target species [12]. Suresh et al. [10] reported that C. maritimum exhibited larvicidal activity against I-VI instar larvae of S. litura with LC₅₀ values ranging from 102.1 to 237.0 μL/L. The larvicidal activity of a C. guianensis flower extract against S. litura third instar larvae (with an LC₅₀ of 223 ppm) has been demonstrated by Ponsankar et al. [11]. The LC₅₀ value of Piper betle L. EO against third instar S. litura larvae was 0.48%, as reported by Vasantha-Srinivasan et al. [12]. Similarly, Kaleeswaran et al. [13] reported that n-hexane (pericarp) Z. armatum pericarp extracts (0.209%), followed by Ethyl acetate (0.450%) and Methanol (0.654%) extracts, respectively, displayed larvicidal activity against third instar S. litura at 72 h. Benelli et al. [17] demonstrated that W. prostrata exhibited significant toxicity toward fourth instar larvae of S. litura (LC₅₀ = 167.46 μL/mL). Strong toxicity of A. galanga (LD₅₀ = 13.26 μg/larva) and O. basilicum (17.71 μg/larva) EO against S. litura has been reported by Ruttanaphan et al. [19].

The primary constituent of essential oils was identified as the determinant of their biological activity [24,31,34,35,36]. Thymol is one of the major volatile components of T. serpyllum EO. Ruttanaphan and Bullangpoti [3] demonstrated that thymol had significant toxicity against third instar larvae of S. litura after 24 and 48 h (LC₅₀ = 5.610 μg/larva and 5.262 μg/larva). Koul et al. [22] reported a substantial insecticidal activity of thymol against S. litura, with LD₅₀ of 28.5 μg/larva. Furthermore, thymol was determined to be toxic to various pest species (Table 4), including R. dabieshanensis [32,37,38]. As shown in Table 4, thymol has a broad-spectrum insecticide activity.

Table 4.

Insecticidal activity of Thymus serpyllum EO and its major component thymol.

Oil/Compounds	Insect Species	LC50 or LD50	References
Thymus serpyllum EO	Reticulitermes dabieshanensis	0.092 μL/L	Yang et al. [32]
	Varroa destructor	2.549 μL/L	Hýbl et al. [31]
	Musca domestica	20.9 μL/L	Xie et al. [24]
	Acanthoscelides obtectus	1.12 μg/mL	Sertkaya [30]
	Frankliniella occidentalis	0.5%	Picard et al. [29]
thymol	Amblyomma sculptum;	0.0156 mg/cm2	da Silva Costa et al. [39]
	Rhipicephalus sanguineus	0.0041 mg/cm2
	Aedes aegypti	0.1 mg/mL 100% mortality	Nascimento et al. [40]
	Reticulitermes dabieshanensis	0.062 μL/L	Yang et al. [32]
	Tribolium castaneum;	24.65 μg/adult;	Xie et al. [41]
	Lasioderma serricorne;	9.9 μg/adult;
	Liposcelis bostrychophila	49.36 μg/adult
	Solenopsis invicta	0.98 μg/g, minimum repellent effective doses	Paudel et al. [42]
	Acromyrmex balzani	2.23 µg/mg	Dantas et al. [43]
	Chilo suppressalis	17.11 μg/larvae	Basij et al. [44]
	Plutella xylostella	2.45 mg/mL	Zhao et al. [45]
	Sitophylus oryzae	51.84%, repellency effects for 2%	Marsin and Muhamad [46]
	Tribolium confusum;	0.3%	Amari et al. [47]
	Supella longipalpa
	Sitophilus zeamais	196 μmol/cm2	Rodríguez et al. [48]
	Glyphodes pyloalis	32.18 μg/larva	Goharrostami et al. [49]
	Spodoptera exigua	9.54 μg/larva	Kumrungsee et al. [50]
	Spodoptera litura	5.610 μg/larva	Ruttanaphan and Bullangpoti [3]
	Acanthoscelides obtectus	Females, 98.4 mg/kg;	Lazarević et al. [51]
		Males, 66.0 mg/kg
	Plutella xylostella	27.94 mg/L	da Camara et al. [52]
	Spodoptera exigua	32.45 μg/larva	Pengsook et al. [53]
	Galleria mellonella	0.5 mg/adult	Sohail et al. [54]
	Podisus nigrispinu;	10.27 mg/g;	Lima et al. [55]
	Spodoptera frugiperda	4.91 mg/g
	Tuta absoluta	7.72 μL/mL	Piri et al. [56]
	Mythimna separate ;	6.67 μL/L;	Lu et al. [57]
	Myzus persicae ;	5.58 μL/L;
	Sitophilus zeamais ;	59.20 μL/L;
	Musca domestica ;	1.66 μL/L;
	Tetranychus cinnabarinus	2.14 μL/L
	Riptortus clavatus	70.0% repellent activity at 2.83 μg/cm2	Lee et al. [58]
	Culex pipiens	49 mg/L	Youssefi et al. [59]
	Leishmania infantum	7.22 μg/mL	Youssefi et al. [60]
	Musca domestica	13 mg/L	Scalerandi et al. [61]
	Sitophilus zeamais	84.06 μL/L	Oliveira et al. [62]
	Aedes albopictus	12.9 mg/L	Giatropoulos et al. [63]
	Hyalomma lusitanicum	100% at 5 mg/L	Navarro-Rocha et al. [64]
	Plutella xylostella	0.00018 ppm	Webster et al. [65]
	Aedes aegypti	35.71 ppm	de Mesquita et al. [66]
	Diaphania hyalinata	2.99 μg/mg	Melo et al. [67]
	Blatta lateralis	0.34 mg/nymph	Gaire et al. [68]
	Sitophilus zeamais	17.08 μg/mg	Oliveira et al. [69]
	Cryptotermes brevis	8.20 μg/mg	Santos et al. [70]
	Ixodes ricinus	100% at 1%	Tabari et al. [71]
	Aedes aegypti	11.1 μg/cm2	Ali et al. [72]
	Blattella germanica	100%, repellency effects for 10 μg/cm2	Lee et al. [73]
	Sitophilus oryzae ;	24.07 μg/cm2;	Kanda et al. [74]
	Tribolium castaneum;	11.21 μg/cm2
	Rhyzopertha dominica;	8.8 μg/cm2
	Aedes aegypti	0.013 mg/cm2	Rehman et al. [75]
	Aedes albopictus	100%, larvicidal activity, 0.1 mg/mL	Seo et al. [76]
	Stegomyia aegypti	68.05 μg/cm2	Huang et al. [77]
	Plutella xylostella	0.22 μg/larva	Kumrungsee et al. [78]
	Aedes aegypti	13.9 ppm	Tabanca et al. [79]
	Spodoptera litura	28.5 μg/larva	Koul et al. [22]
	Reticulitermes speratus	0.65 mg/Petri dish	Sekine and Shibutani [80]
	Crithidia fasciculata;	32.5 μg/mL;	Azeredo and Soares [81]
	Trypanosoma cruzi	62 μg/mL
	Aedes albopictus	9 μL/L	Park et al. [82]
	Tenebrio molitor	14.71 μL/L	Lima et al. [83]

Previous studies have documented the toxicity of terpenoid complexes from essential oils to S. litura [3,18]. Mahajan et al. [18] determined the efficacy of β-caryophyllene, which resulted in a 13.33% adult emergence in S. litura at 3125 ppm. Similarly, β-asarone was toxic to S. litura larvae with LD₅₀ values of 6.24 μg/larva [37]. Rotenone can be highly effective against third instar larvae of S. litura (LC₅₀ = 5043 mg/L) [38]. According to Huang et al. [23], pogostone had significant larvicidal activity against S. litura, including oral toxicity (LC₅₀ = 986.88 mg/L) and contact toxicity (LC₅₀ = 1041.42 mg/L). Ruttanaphan and Bullangpoti [3] reported that pulegone exhibits high larvicidal activity against third instar larvae of S. litura after 24 h (LC₅₀ = 8.348 μg/larva). Ruttanaphan et al. [21] discovered a remarkable insecticidal activity of linalool against S. litura, with an LD₅₀ of 32.271 μg/larva and 49.742 μg/larva, respectively.

ESTs and GSTs are detoxification enzymes that play key roles in detoxifying botanical pesticides, and their activities are typically induced and up-regulated by exogenous compounds [84]. As shown in Figure 2, the ESTs and GSTs activities of S. litura were significantly elevated after exposure to an LC₅₀ concentration of T. serpyllum EO. Yang et al. [32] reported that eight main components of EOs increased the activity of ESTs and GST of R. dabieshanensis; however, the activity of AChE was decreased. The EO from C. citratus, C. khasans, C. nardus and the main compound including citral, geraniol, and citronellal inhibited α-NA esterase activity and enhanced β-NA esterase activities [32]. The activities of ESTs and GST of R. dabieshanensis by treatment with M. citrate were significantly increased compared to the control [33]. This finding corresponded to studies by Yang et al. [32,84,85,86], which demonstrated the efficacy of T. serpyllum EO in enhancing the activity of detoxifying enzymes of treated insects.

Furthermore, AChE plays a significant role in the mechanism of action of essential oils (EOs) or relevant constituents, resulting in insecticidal effects. This study indicated that essential oil exerted a substantial inhibitory effect on acetylcholinesterase in vivo in S. litura. This is in agreement with findings by Jin et al. [34], who demonstrated that three Cymbopogon EOs and their primary components effectively inhibited the AChE activity of R. ffaviceps in vitro and in vivo. Additionally, Wu et al. [31] found that Mentha spp. EOs and their major constituents exhibited significant inhibitory activity against AChE both in vivo and in vitro. Previous studies have indicated that EOs and their major constituents can bind to the active site of AChE to inhibit AChE activity [34]. In addition, it has been demonstrated that most EOs can also exert toxic effects on insects by inhibiting cytochrome P450 enzymes (CYPs) [87], GABA receptors [88], octopus amine synapses [89], and tyramine receptors [90]. Therefore, EOs and their main constituents would lead to insect mortality by causing dysregulation in the nervous, antioxidant, and enzyme-based metabolic systems.

4. Materials and Methods

4.1. Insect Rearing

The eggs of S. litura were purchased from the Henan Jiyuan Baiyun Industry Company (Jiyuan, China). After the hatching, the larvae were raised individually on Chinese cabbage leaves. The laboratory temperature was maintained at 26 ± 2 °C, with 12:12 h (light/dark) cycles and RH of 75 ± 5%. The bioassay was conducted using eggs and healthy and uniform-sized 2nd and 3rd instar larvae.

4.2. Essential Oil

T. serpyllum EO was purchased from the online shop of Shanghai Zixin (Shanghai, China).

4.3. GC-MS Analysis

EOs of T. serpyllum were analyzed by an Agilent 7890B/5977A (Santa Clara, CA, USA) coupled with an HP-5 MS capillary column (30 m × 0.25 mm ID × 0.25 μm film thickness) (Santa Clara, CA, USA) and electron impact ionization (70 eV). Oven temperature increased at a rate of 10 °C min⁻¹ from 50 °C (1 min) to 250 °C, subsequently kept at 250 °C for 2 min. Helium (99.999%) was applied as a carrier gas. Through GC-MS analysis of the essential oils, the peak areas of the major constituents were recorded in this study, and the relative percentage of each component was calculated by peak area normalization ($P_i$):

$$P_i={\displaystyle \frac{A_i}{A_{total}}}\times 100\mathrm{\%}$$

where i is the individual compound;$A_i$ is the peak area per compound; and $A_{total}$: is the sum of all compound peak areas corresponding to the total peak area.

The peaks of EOs of T. serpyllum were annotated by comparing the retention indices (RI) and mass spectra (NIST 11.0) and Wiley 275 library data with those of the literature.

4.4. Larvicidal Activity

Larvicidal bioassays were carried out using the modified leaf dipping method as described by Benelli et al. [17]. The T. serpyllum EO was evaluated in a series of concentration gradients (0, 0.25, 0.5, 1.0, and 2.0 mg/mL) diluted in the solvent Polysorbate 80 (P8010, Sigma-Aldrich, St. Louis, MO, USA). Then, Chinese cabbage leaves of uniform shape and size (3 cm × 3 cm) were immersed in the series of concentration gradients separately. After 10 s, the leaf discs were removed and dried, then placed in moisturized filter paper on plates. After that, each of the 20 larvae was placed onto Chinese cabbage leaves treated with different EO concentrations, respectively. The number of dead larvae was counted every 12 h and observed for 72 h. All experiments were performed in triplicate.

4.5. Ovicidal Bioassays

A total of 420 eggs were split into 7 groups (n = 60 eggs in each group). Each group was separately immersed in different concentrations (0, 0.25, 0.5, 1.0, and 2.0 mg/mL in Polysorbate 80) of T. serpyllum EO. Then, the number of eggs hatched in each group was counted. All experiments were performed in triplicate. Hatching rates were assessed at 120 h after the initiation of the treatment.

4.6. Enzyme Activities

4.6.1. Homogenate Preparation

The 2nd instar larvae were placed into the LC₅₀ concentrations of T. serpyllum EO to evaluate the impact on detoxification enzymes, specifically esterases and acetylcholinesterase. Each group (20 larvae) was homogenized, and centrifuged at 12,000× g for 15 min at 4 °C. Then, the supernatants were stored at −80 °C for subsequent use. Each assay was repeated a minimum of three times.

4.6.2. Esterases (ESTs)

The EST assay was carried out based on the protocol of Piri et al. [56]. The EST was determined on two substrates, acetate-1-naphthyl ester (α-NA) (N1252, Sigma-Aldrich, St. Louis, MO, USA), and acetate-2-naphthyl ester (β-NA) (N1254, Sigma-Aldrich, St. Louis, MO, USA). The 20 µL substrate (α-NA or β-NA) (10 mM) and 50 µL Fast Blue RR Salt (1 mM) (F6250, Sigma-Aldrich, St. Louis, MO, USA) were mixed. Finally, 10 μL of the enzyme solution was supplemented into the mixture. In the control group, the 10 μL enzyme solution was replaced by 10 μL distilled water, and the rest of the reagents remained unchanged. After incubation at 27 °C for 5 min, the absorbance (OD value) was determined at 450 nm using a microplate reader (Synergy™ HTX, BioTek, Winooski, VT, USA). In addition, OD values were recorded every 1 min for 10 min. Each assay was repeated at least three times to ensure the reliability and reproducibility of the data. The EST activity was calculated using the following formula:

EST activity (U/mg) = (ΔA_test − ΔA_ck) × V_total ÷ V_test ÷ T

ΔA_test corresponds to the absorbance change value of the treatment group measured for 10 min. ΔA_ck corresponds to the change in absorbance of the control group measured for 10 min. V_total represents the total volume of the reaction system in each well. V_test represents the volume of the enzyme solution. T denotes the reaction time.

4.6.3. Glutathione S-Transferases (GSTs) Activity Assay

The enzymatic activity of GSTs was measured using the protocol employed by Piri et al. [56]. 20 μL CDNB (20 mM) and 510 μL enzyme solution were mixed. After incubation at 27 °C for 5 min, the absorbance was determined at 340 nm. Each assay was repeated a minimum of three times. The GST activity was calculated using the formula below:

GST activity (U/mg) = (Δ_OD340 × V_total) ÷ (ε × L)

Δ_OD340 corresponds to the GST-mediated absorbance change. V_total represents the total volume of the reaction system in each well. ε represents the molar extinction coefficient of GST (0.0096/(μmol·cm)), and L represents the optical range of the colourimetric cup (1 cm).

4.6.4. Acetylcholinesterase (AChE) Activity Assay

AChE (C4359, Sigma-Aldrich, St. Louis, MO, USA) activity was determined following the protocol of Ellman et al. [91]. 80 μL PBS solution (0.1 M, pH = 7.0), 50 μL ATCh (10 mM), and 50 μL 5,5-dithiobis-2-nitrobenzoic acid (DTNB) (D8130, Sigma-Aldrich, St. Louis, MO, USA) (10 mM) were mixed, respectively. After incubation at 27 °C for 5 min, 20 μL of enzyme solution was then added. Absorbance (OD) was measured at 405 nm using a microplate reader, and OD values were recorded at 1 min intervals for 30 min. Each assay was repeated at least three times to ensure the reliability and reproducibility of the data. The AChE activity was calculated using the following formula:

AChE activity (U/mg) = (Δ_OD405 × V_total) ÷ (ε × L)

Δ_OD405 corresponds to AChE-mediated absorbance change. V_total represents the total volume of the reaction system in each well. ε represents the molar extinction coefficient of AChE (0.0136/(μmol·cm)), and L represents the optical range of the colourimetric cup (1 cm).

4.7. Statistical Analysis

Larval mortality, hatching, and suppression rates were analyzed using nonparametric statistical methods. The Kruskal–Wallis test was implemented to evaluate overall differences among treatment groups (p < 0.05). Dunn’s post hoc test was used to identify specific groups with significant differences in pairwise comparisons. All statistical analyses were performed with SPSS v20.0 (SPSS Inc., Chicago, IL, USA). The LC₅₀ and EC₅₀ values were calculated by fitting dose–response curves with logistic regression models. Within-group differences were assessed using the independent samples Mann–Whitney U-test, denoting results with p < 0.05 with an asterisk (*).

5. Conclusions

In our study, thymol was the main constituent of T. serpyllum EO. Moreover, T. serpyllum EO was highly toxic to second- and third-instar larvae of S. litura. Moreover, it demonstrated significant inhibition of AChE activity, confirming that T. serpyllum EO can be developed and utilized control agent against S. litura. Before large-scale field application, the effects of T. serpyllum EO on non-target organisms must be determined. There is also a need to design slow-release formulations that can be used to extend the effectiveness of T. serpyllum EO. The findings disclosed in this study may facilitate the effective and environmentally friendly management of S. litura in the field.

Author Contributions

Conceptualization, J.Q., X.W. and L.W.; methodology, L.W.; validation, X.W. and J.Q.; formal analysis, S.J., S.W. and Z.X.; investigation, L.W., S.W. and Z.X.; data curation, L.W.; writing—original draft preparation, L.W. and S.J.; writing—review and editing, J.Q. and X.W.; supervision, J.Q. and X.W.; funding acquisition, J.Q., X.W. and L.W. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

Our study was backed up by Jilin Province Science and Technology Major Projects (20230304002YY); Yantai University Doctoral Science Research Foundation (SM15B01, SM19B70 and SM19B28); “double-hundredaction” of Yantai (2320004-SM20RC02).