Insecticidal and Insectistatic Activity Assessment of Lantana camara (L.) (Verbenaceae) Essential Oil and endo-Borneol Against Tenebrio molitor (L.) (Coleoptera: Tenebrionidae)

Abstract

Tenebrio molitor is a common stored grains pest. The conventional way for its management involves the use of synthetic fumigants. Despite their effectiveness, these can cause environmental damage. The use of essential oils has emerged as an alternative for its management. Therefore, the aim of this study was to assess Lantana camara essential oil (EO) and endo-borneol biological activities against T. molitor. Insecticidal activity and weight gain were evaluated through the impregnated paper method against larvae and adults, while repellency was conducted with a Y-tube olfactometer; L. camara EO showed higher mortality for T. molitor adults (LC₅₀ = 7.2 μL EO L⁻¹ air) than for larvae (LC₅₀ = 13.7 μL EO L⁻¹ air) after 30 d. Furthermore, L. camara EO was found to be repellent for T. molitor adults (RC₅₀ = 0.08 μL EO cm⁻²). Regarding the EO composition, endo-borneol was identified by GC-MS as a major compound with 14.24% abundance. Larvae exhibited higher susceptibility (LC₅₀ = 7.8 μL L⁻¹ air) to endo-borneol than adults (LC₅₀ = 46 μL L⁻¹ air) after 72 h. Notably, endo-borneol demonstrated significantly higher repellent activity (RC₅₀ = 0.03 μL cm⁻²) than L. camara EO (RC₅₀ = 0.08 μL EO cm⁻²). These findings suggest that endo-borneol has potential as a natural source alternative for T. molitor management.

1. Introduction

The main food sources for humans are cereals, with corn, wheat, and rice as the most-cultivated around the world, with annual production at 850, 788.6, and 539.4 million tons, respectively [1]. However, the relevance of these cereals is not limited to their nutritional value. In addition to serving as food, grains can be used in animal feed, fuel generation, and other industrial applications [2].

Various factors deteriorate the quality of stored grains. Among the main abiotic factors are humidity, light, and temperature, which modify their texture and lead to biotic problems, such as diseases caused by fungi and bacteria, as well as rodent and insect pests, the latter being responsible for up to 35% of annual losses. The damage caused by insects is mainly due to consuming the cereal, in addition to affecting its quality because of alterations in the storage environment owing to their presence [3,4].

Generally, insects that attack stored cereals are of the orders Coleoptera (in their larval and adult stages) and Lepidoptera (in their larval stage). Some cosmopolitan species that infest stored cereals are Sitotroga cerealella (Oliv.) (Lepidoptera: Gelechiidae); Cryptolestes pusillus (Schon.) (Coleoptera: Laemophloeidae); Prostephanus truncatus (Horn.) (Coleoptera: Bostrichidae); and Tribolium castaneum (Herbst.), Tribolium confusum (Duval.), and Tenebrio molitor (Coleoptera: Tenebrionidae) [5].

Tenebrio molitor (L.) (Coleoptera: Tenebrionidae), commonly named “yellow mealworm”, infests stored cereals and their sub-products. It is a dark-brown holometabolous beetle with nocturnal habits. This species is cosmopolitan and mainly found in regions with temperate climates [6,7].

The main negative effects of T. molitor infestation include losses of up to 15% of stored gains and flour. Furthermore, its feces and body parts contaminate the stored commodities. Additionally, it indirectly spreads saprophytic microorganisms, causing a decrease in the product’s quality [8,9].

Synthetic chemical fumigants supplied in storage structures are the most common way to control T. molitor. The most commonly used products are methyl bromide, aluminum phosphide, and magnesium phosphide [10]. In addition, sulfuryl fluoride and carbon disulfide are synthetic alternatives to these conventional fumigants [11]. Despite their effectiveness, these chemicals are characterized by high environmental persistence, widespread dispersal through air currents, low water solubility, and bioaccumulation. They infiltrate and persist in trophic chains while also causing resistance development in the target insect [12].

Given these problems, environmentally friendly alternatives have been proposed, including the use of plants and their extracts that contain active compounds that can be employed as botanical insecticides against T. molitor. Essential oils (EOs) from plants have proven to be viable alternatives to toxic products. These plant-derived substances are usually effective, cause less damage to human health and the environment, are biodegradable, and are safer than their synthetic counterparts [13].

Within the Verbenaceae family, several plant species whose EOs show insecticidal activity against stored grain insect pests have been reported. In this regard, Arango-Gutiérrez and Vásquez-Villegas [14] found that Verbena officinalis (L.) EO demonstrated insecticidal activity against larvae and adults of Sitophilus granarius (L.) (Coleoptera: Curculionidae). They performed a phytochemical analysis identifying the following functional groups: phenols, tannins, steroids, flavonoids, anthraquinones, sesquiterpene lactones, and cardiotonic glycosides. They attributed the biological activity of this plant to the mixture of these secondary metabolites.

Meanwhile, Ringuelet et al. [15] evaluated the insecticidal activity of Lippia alba (Mill.) N. E. Br. (Verbenaceae) EO against adults of T. castaneum, finding insecticidal and repellent activities when the EO was applied through impregnated paper for fumigation. They attributed these effects to the major compounds limonene and carvone. Furthermore, Zandi et al. [16] determined the insecticidal activity of Lantana camara L. EO against larvae and adults of Callosobruchus maculatus (Fab.) (Coleoptera: Chrysomelidae), finding activity in both the larval and adult stages. Similarly, Aisha et al. [17] used the EO of L. camara to evaluate its effectiveness as a bioactive substance against three stored product insect pests: T. castaneum, Lasioderma serricorne (Fab.) (Coleoptera: Anobiidae), and Callosobruchus chinensis (L.) (Coleoptera: Chrysomelidae). The EO showed fumigant toxicity for T. castaneum, L. serricorne, and C. chinensis, while also showing effective repellent activity toward the tested insects. Regarding the use of borneol as a product with insecticidal and insectistatic activity, research has demonstrated that this compound has insecticidal and repellent effects against the housefly, Musca domestica (L.) (Diptera: Muscidae), and the mosquito Culex quinquefasciatus (Say.) (Diptera: Culicidae), in their adult and larval stages. Moreover, Bhat et al. [18] synthesized several isoborneol derivatives, including esters, ethers, and thioethers, from isoborneol under mild conditions.

Therefore, we aimed to evaluate the insecticidal and insectistatic activity of L. camara EO and endo-borneol against larvae and adults of T. molitor.

2. Materials and Methods

2.1. Collection of Plant Material

One kilogram of aerial parts (leaves, stems, and flowers) of L. camara during its flowering stage was collected on 26 February 2024, at 10:00 a.m. (UTC-6) at the Autonomous University of Querétaro gardens (20°35′26.7″ N, 100°24′42.1″ W), Querétaro, México. The soil at this site is originally vertisol, characterized by a clay texture; however, it has undergone anthropogenic modification [19]. The plant species was authenticated by Biol. José García Pérez and deposited at the Isidro Palacios Herbarium at the Autonomous University of San Luis Potosí with voucher specimen number 072128. Subsequently, the samples were transferred to the Laboratory of Natural Insecticidal Compounds of the Autonomous University of Querétaro (LNIC-AUQ) for EO extraction.

2.2. Extraction of the Essential Oil

The L. camara EO was obtained using a hydrodistillation method based on the work of González-Chávez et al. [20]. Fresh plant material (500 g) was cut into 2 cm pieces, placed in a 3 L round-bottom flask with 1.5 L of distilled water, and subjected to hydrodistillation. The resulting distillate was subjected to liquid–liquid extraction using diethyl ether (J.T. Baker, Mexico City, Mexico) to recover EO components from the hydrosol. The organic phase was separated using a separatory funnel and concentrated in a rotary evaporator (Büchi Rotavapor R-200) at 35 °C. The concentrate was dried with anhydrous sodium sulfate (J.T. Baker, Mexico) to remove residual moisture, and solvent residues were eliminated under vacuum. The addition of this salt is not included in the standardized method described by ISO 6571:2008 [21]. The EO obtained was stored in amber glass vials at 4 °C until use.

2.3. Identification of the Major Compound of the Essential Oil

The essential oil, 1.00 μL of the sample, was analyzed in duplicate. GC-MS analysis was performed using a GC 7890 system coupled to a mass spectrometer (MSD) 5975C (Agilent Technologies, Wilmington, DE, USA). Separation was achieved using an HP-5MS capillary column (0.25 mm internal diameter × 30 m, 0.25 μm film thickness) (J&W, Folsom, CA, USA). The injector was operated in split mode 1:30 at 250 °C with a helium flow rate of 1 mL min⁻¹. The oven temperature was programmed to 100 °C for 1 min and then increased by 6 °C min⁻¹ to 220 °C, 10 °C min⁻¹ to 290 °C, and finally by 40 °C min⁻¹ to 310 °C. The MSD was operated at 70 eV with the ion source set at 230 °C and the transfer line at 250 °C.

Compounds were identified by interpreting the fragmentation patterns of their mass spectra in an m/z range of 20-1000. Data acquisition was performed using the Chemstation software (Agilent Technologies, version E.02.02). Individual compounds were identified by comparing their mass spectra with those of reference compounds in the National Institute of Standards and Technology database (NIST11). The identity of endo-Borneol was confirmed by comparing its retention time and mass spectrum with an analytical standard (97% pure, Merck, Naucalpan de Juárez, Mexico). The identities of the compounds were confirmed using the linear retention index, calculated for each peak with reference to n-alkane standards (C6–C28) analyzed under the same conditions.

2.4. Tenebrio Molitor Rearing

The insects used for this study were obtained from an established colony at the LNIC-AUQ. Larvae were reared in medium density fiber boxes (50 cm large × 50 cm wide × 12 cm height) on a rearing substrate (

Table 1. Constituents of 500 g of Tenebrio molitor rearing substrate.

Constituent	Quantity
Sawdust	395 g
Wheat	75 g
Oat	25 g
Fresh carrot	5 g

) until the pupal stage. Subsequently, the pupae were transferred to plastic containers (25 cm × 15 cm × 7 cm) until adult emergence. Then, the beetles were placed in plastic boxes (25 cm × 20 cm × 10 cm) with rearing substrate for oviposition. After hatching, the neonate larvae were reared under the same initial conditions until they reached the desired larval instar or adult age for experimentation. Tenth instar larvae were randomly selected based on the morphological characteristics described by Park et al. [22] (mean body length = 1.34 ± 0.19 cm).

2.5. Insecticidal Activity by Fumigation on Larvae and Adults

Bioassays were conducted on the larval and adult stages of T. molitor using the fumigation method described by Mishra et al. [23]. Test solutions were prepared, using acetone as a volatile vehicle, at concentrations of 10, 16, 24, 30, and 60 µL L⁻¹ air of EO or endo-borneol, calculated based on the Petri dish volume (8.79 cm diameter × 1.64 cm height, internal measures; 0.1 L, volume). Acetone alone served as the negative control. Square pieces of Whatman filter paper (3 × 3 cm) were impregnated with 100 µL of each test solution and adhered to the centers of the glass Petri dish lids. The acetone was allowed to evaporate for one minute before introducing the insects.

A total of 10 tenth instar larvae or 10 adult beetles (24–72 h post-emergence) were placed in each dish, along with 0.25 g of gelled artificial diet prepared according to Rodríguez-Cervantes et al. [24], as described in

Table 2. Formulation of gelified artificial food for Tenebrio molitor (50 g).

Ingredient	Quantity
Dried carrot powder	4.50 g
Wheat powder	3.75 g
Oat powder	3.75 g
Yeast	1.00 g
Methyl 4-hydroxybenzoate	0.30 g
Formaldehyde	0.20 g
Ascorbic acid	0.15 g
Neomycin sulfate	0.09 g
Potassium phosphate and B complex	0.30 g
Distilled water	50.00 mL

. Each treatment consisted of three replicate dishes. The dishes were sealed with Parafilm M and maintained in darkness at room temperature. Insect mortality was assessed every three days for a total observation period of 30 days. The insects were maintained in a bioclimatic chamber with a temperature of 27 ± 2 °C, a photoperiod of 10:14 h (light/dark), and a relative humidity of 40%.

2.6. Growth Inhibition by Fumigation on Larvae

Two methodologies were used to evaluate insecticidal activity. The first methodology assessed weight reduction in larvae exposed to different concentrations of EO and endo-borneol compared with normal development in control larvae. For this assay, the same fumigation methodology described by Mishra et al. [23] for insecticidal activity determination was employed, using only tenth instar larvae. The weights of 10 larvae contained in each Petri dish were recorded every 5 days over a 30-day period.

2.7. Repellent Activity on Adults

The second bioassay determined repellent activity on adults using a Y-tube vertical olfactometer (TYVO) modified from the construction model proposed by Clemente et al. [25]. The system consisted of a Y-shaped glass tube with an inner diameter of 2 cm, a 9 cm stem, and two 10 cm arms assembled at a 120° angle. Each arm was connected via rubber stoppers and automotive clamps to silicone tubing (0.4 mm internal diameter). One arm was directly connected to an automotive clamp, while the other was first connected to the side arm of a filtration flask containing filter paper impregnated with EO or endo-borneol. The flask was sealed, and silicone tubing was connected and attached to the clamp. The resulting tubing was connected via a side arm to a second filtration flask containing activated carbon for air purification. Finally, the system was connected to a water pump providing an air flow of 14.70 L min⁻¹ (Elite 802). The entire system was enclosed in a cardboard box lined with black cardboard to provide the required darkness.

Before and after each repellency test, the Y-tube was cleaned with acetone and air-dried for 15 min at room temperature. Gelled food (0.15 g) was placed in each arm. Filter paper (2 × 2 cm) was impregnated with 0.01, 0.1, 0.4, 1.0 µL of L. camara EO or endo-borneol in 1 µL of acetone to have concentrations of 0.0025, 0.025, 0.1 and 0.25 µL of L. camara EO or endo-borneol cm⁻² and it was placed inside the filtration flask. A negative control using 1 µL to impregnate the filter paper was also included. The acetone was allowed to evaporate for one minute before introducing the insects.

To begin each test, the air pump was activated and allowed to stabilize for 30 s before introducing an adult T. molitor specimen (24–72 h post-emergence, fasted for 72 h) through the base of the stem. The insect’s trajectory was observed for one minute, recording the pathway and time required to choose one of the two arms for feeding. Selection of the pathway without EO indicated repellent activity. The test was performed 10 times per concentration, using a new insect for each replication.

The repellency classification for each concentration of the tested EO and endo-borneol was established based on the obtained percentage of repellency, following the criteria by Visakh et al. [26]. The classification system is as follows: Class 0, 0–0.01%; Class I, 0.2–10%; Class II, 20.1–40%; Class III, 40.1–60%; Class IV, 60.1–80%; and Class V, 80.1–100% of repellency.

2.8. Ethical Guidelines

This work was submitted for review to the Bioethics Committee of the Faculty of Chemistry at the Autonomous University of Querétaro, which accepted the use of experimental animals under the conditions of the methodology outlined above. The resolution was “approved with regard to the bioethical aspects of the project” under official document number CBQ24/047.

2.9. Statistical Analysis

A completely random experimental design was employed. The data obtained from the evaluation of insecticidal and insectistatic activity were analyzed using non-parametric tests, namely, Ryan–Joiner and Levene, to determine the normality and homoscedasticity of the data with a confidence level of 95%. Subsequently, one-way analysis of variance was performed, followed by Tukey’s mean adjustment test with a significance level of 0.05 and a Probit analysis to calculate the LC₅₀. All results were processed with the statistical program Systat 9 [27].

3. Results

3.1. Yield of Lantana camara Essential Oil

A performance rate of 0.8% was achieved through the hydrodistillation of L. camara aerial parts.

3.2. Insecticidal and Insectistatic Activity of Lantana camara Essential Oil Against Tenebrio Molitor

Table 3. Insecticidal activity of Lantana camara essential oil against larvae of Tenebrio molitor.

Treatment (μL EO L−1 Air)	Mortality (%)
	10 d	20 d	30 d	30 d Cumulative Mortality
60	20 ± 7.4 a	66.7 ± 8.8 a	0 ± ND a	86.7 ± 6.3 a
30	23.3 ± 7.9 a	60 ± 9.1 a	0 ± ND a	83.3 ± 6.9 a
24	10 ± 5.6 a	63.3 ± 8.9 a	0 ± ND a	73.3 ± 8.2 ab
16	13.3 ± 6.3 a	43.3 ± 9.2 ab	3.3 ± 3.3 ND a	60 ± 9.1 ab
10	23.3 ± 7.9 a	20 ± 7.4 b	0 ± ND a	43.3 ± 9.2 bc
Control	0 ± ND a	16.7 ± 6.9 b	0 ± ND a	16.7 ± 6.9 c
LC50	13.7 (6.3–19.3) μL EO L−1 air
LC90	49.89 (40.6–67.9) μL EO L−1 air
Chi-square	10.92

Values correspond to the average of 30 measurements ± SEM. Different letters indicate significant differences (p < 0.05). LC₅₀ of the 30 d cumulative mortality is shown with its fiducial limits. ND stands for not determinable.

and

Table 4. Insectistatic activity of Lantana camara essential oil against larvae of Tenebrio molitor.

Treatment (μL EO L−1 Air)	Average Weight per Larva (mg)				Weight Gain (%)
	0 d	10 d	20 d	30 d
60	11.5 ± 0.15 a	13.0 ± 0.23 a	12.0 ± 0.12 ab	12.3 ± 0.23 ab	6.4
30	11.6 ± 0.18 a	11.0 ± 0.12 ab	12.6 ± 0.18 b	12.7 ± 0.30 b	10.2
24	11.5 ± 0.19 a	11.5 ± 0.12 ab	12.7 ± 0.21 ab	12.9 ± 0.38 ab	11.8
16	11.4 ± 0.18 a	10.3 ± 0.2 b	13.2 ± 0.23 ab	13.6 ± 0.23 ab	19.4
10	11.5 ± 0.12 a	11.2 ± 0.12 ab	14.9 ± 0.29 a	15.4 ± 0.47 a	34.7
Control	11.5 ± 0.22 a	12.9 ± 0.18 a	13.5 ± 0.42 a	16.9 ± 0.32 a	47.0

Values correspond to the average of the surviving individuals ± SEM. Different letters indicate significant differences (p < 0.05).

show the insecticidal and insectistatic activity of L. camara EO against T. molitor larvae. After 10 days of exposure to the EO, no mortality was observed in the control group, while the highest mortality was recorded at concentrations of 10 and 30 μL EO L⁻¹ air, with 23.3%. At 20 days, mortality in the control group was 16.7%, while the highest mortalities were observed at concentrations of 24, 30, and 60 μL EO L⁻¹ air, at 63.3%, 60%, and 67.7%, respectively. The insecticidal activity of the EO persisted for 20 days, resulting in significantly higher cumulative mortalities than the control across all treatments. The highest cumulative mortalities were observed at 30 and 60 μL EO L⁻¹ air, reaching 83.3% and 86.7%. LC₅₀ was 13.7 μL EO L⁻¹ air after 30 d.

Regarding insectistatic activity, on day 10, the larvae in the control group increased their weight to 12.9 mg, similar to the larvae treated with 60 μL EO L⁻¹ air. After 20 days, weight gain continued in the control group, reaching 13.5 mg. By contrast, larvae exposed to 24, 30, and 60 μL EO L⁻¹ air showed lower weight gains of 12.6, 12.6, and 12.0 mg, respectively.

By the end of the 30-day trial, the control larvae reached a weight of 16.9 mg, corresponding to a 62.4% increase. Meanwhile, larvae in the highest concentration treatment (60 μL EO L⁻¹ air) reached only 12.3 mg, representing an 8.5% increase, which is 54% lower than the weight gain observed in the control group.

Table 5. Insecticidal activity of Lantana camara essential oil against Tenebrio molitor adults.

Treatment (μL EO L−1 Air)	Mortality (%)
	10 d	20 d	30 d	30 d Cumulative Mortality
60	66.7 ± 0.66 a	33.3 ± 0.66 a	0 ± ND	100 ± 0 a
30	70.0 ± 1.53 a	26.7 ± 1.20 a	0 ± ND	96.7 ± 5.77 a
24	53.3 ± 0.33 a	36.7 ± 0.88 a	3.3 ± 0.33 a	93.3 ± 11.55 a
16	53.3 ± 0.33 a	26.7 ± 0.33 a	10.0 ± 0.58 b	90 ± 10.0 a
10	50.0 ± 0.58 a	16.7 ± 0.66 a	10.0 ± 0.58 b	76.7 ± 11.55 a
Control	3.3 ± 0.33 b	6.7 ± 0.66 a	0 ± ND	10.0 ± 10.0 b
LC50	7.2 (4.0–9.7) μL EO L−1 air
LC90	18.8 (16.0–23.0) μL EO L−1 air
Chi-square	10.87

Values correspond to the average of 30 measurements ± SEM. Different letters indicate significant differences (p < 0.05). CV stands for the Coefficient of Variation of the 30 d cumulative mortality. LC₅₀ of the 30 d cumulative mortality is shown with its fiducial limits. ND stands for not determinable.

presents the insecticidal activity of L. camara EO against T. molitor adults. After 10 days, a mortality rate of 3.33% was recorded in the control group. The highest mortalities were observed at EO concentrations of 60 and 30 μL EO L⁻¹ air, with 66.67% and 70% mortality, respectively. After 20 days of exposure, the control group showed a mortality of 6.7%, while the highest mortalities were observed at 24 and 60 μL EO L⁻¹ air, with 36.7% and 33.3%, respectively. The 60 μL EO L⁻¹ air treatment reached 100% cumulative mortality on day 20.

Finally, at 30 days, a dose-dependent increase in mortality was observed. All treatments showed significantly higher cumulative mortalities than the control, with the highest values observed at 60 and 30 μL EO L⁻¹ air, reaching cumulative mortalities of 96.7% and 100.0%, respectively. The LC₅₀ value for adults was 7.2 μL EO L⁻¹ air.

Table 6. Repellent activity of Lantana camara essential oil against Tenebrio molitor adults.

Treatment (μL EO cm−2)	Repellency (%)	Repelling Class
0.25	70 ± 15.28 a	IV
0.10	60 ± 16.33 ab	III
0.025	60 ± 16.33 ab	III
0.0025	50 ± 16.67 b	III
Control	0 ± ND c	0
RC50	0.08 (−0.08–0.48) μL EO cm−2

Repellency values correspond to the average ± SEM of 10 data points. Different letters indicate significant differences (p < 0.05). RC₅₀ is shown with its fiducial limits. ND stands for not determinable.

presents the repellent activity of L. camara essential oil (EO) against T. molitor adults. When T. molitor adults were exposed to L. camara EO in a “TYVO” apparatus, no repellency was observed in the control group. At a concentration of 0.0025 μL EO cm⁻², repellency reached 50%. For concentrations between 0.025 and 0.10 μL EO cm⁻², repellency remained constant at 60%. The highest repellency, 70%, was achieved at the 0.25 μL EO cm⁻² concentration. All EO treatments showed significant differences in repellency compared with the control.

3.3. Identification of the Major Compounds in Lantana camara Essential Oil

Only 48 compounds were identified and quantified corresponding to 84.58% of the total of the compounds present in L. camara EO. The major compounds identified after GC-MS analysis were endo-borneol, spathulenol and β-phellandrene (

Table 7. GC-MS analysis of Lantana camara essential oil. The three main identified compounds are highlighted in gray.

No	Retention Time	Similarity (%)	Compound	Abundance (%)	Retention Index
1	4.793	97	Camphene	0.51	945
2	5.313	91	α-Sabinene	4.46	968
3	5.411	96	(1S)-(−)-β-Pinene	6.38	982
4	5.660	94	β-Myrcene	2.64	997
5	6.040	90	α-Tricyclene	0.42	1005
6	6.376	96	(+)-2-Carene	0.09	1016
7	6.593	97	p-Cymene	1.31	1023
8	6.744	94	β-Phellandrene	8.12	1028
9	6.918	96	1R-α-Pinene	0.33	1034
10	7.232	98	β-Ocymene	2.52	1044
11	7.579	97	γ-Terpinene	0.27	1056
12	7.883	97	p-Menth-8-en-1-ol	0.52	1066
13	8.522	96	α-Terpinolene	0.13	1087
14	8.913	86	(−)-β-Linalool	1.51	1100
15	9.444	83	2-Methyl-6-methylene-1,7-octadien-3-one	0.16	1115
16	9.704	96	(1R,4S)-4-Isopropyl-1-methylcyclohex-2-enol	0.22	1122
17	9.823	81	3-ethylidene-1-methyl-cyclopentene	0.24	1125
18	10.344	86	Isopinocarveol	0.36	1139
19	10.485	98	Camphor	4.29	1143
20	11.417	97	endo-Borneol	14.24	1168
21	11.775	95	(−)-4-Terpineol	2.24	1178
22	12.295	96	α-Terpineol	0.68	1192
23	12.501	97	(−)-Myrtenol	0.55	1198
24	12.968	98	(S)-(−)-Verbenone	0.30	1205
25	13.922	94	Anisole	0.30	1217
26	15.960	99	(−)-Bornyl acetate	3.54	1242
27	19.592	89	Copaene	0.20	1287
28	20.524	94	Cyperene	0.23	1299
29	21.348	98	Caryophyllene	2.46	1409
30	22.693	98	Humulene	0.18	1426
31	23.810	98	(−)-Germacrene D	1.99	1440
32	24.005	99	α-Selinene	0.38	1443
33	24.406	91	cubedol	2.07	1448
34	25.479	99	δ-Cadinene	0.68	1462
35	27.138	91	Nerolidol	0.40	1483
36	27.691	93	Spathulenol	12.05	1490
37	27.799	83	Caryophyllene oxide	2.37	1491
38	28.287	86	β-Gurjunene (calarene)	0.28	1498
39	28.623	84	(3E,3aR,7aR)-3-ethylidene-3a-methyl-2,4,5,6,7,7a-hexahydro-1H-indene	0.19	1603
40	28.775	80	Isoaromadendrene epoxide	0.41	1607
41	29.231	91	β-Calarene	0.72	1618
42	29.567	95	2-Isopropyl-5-methyl-9-methylene-bicyclo-1-decene(4.4.0)	1.15	1626
43	29.697	95	γ-Muurolene	0.23	1629
44	29.794	99	β-Eudesmol (β-selinenol)	1.01	1632
45	20.892	99	α-Cadinol	0.77	1417
46	30.694	94	Alloaromadendrene	0.23	1653
47	33.307	90	β-Selinene	0.08	1826
48	36.386	84	1,5-dimethyl-7-oxabicyclo[4.1.0]heptane	0.17	2110

) with 14.24%, 12.05% and 8.12% abundance, respectively, also in Figure 1, and showed that endo-borneol appears at 11.417 min and endo-borneol standard at 11.321 min as shown in Figure 2.

3.4. Insecticidal and Insectistatic Activity of Endo-Borneol Against Tenebrio molitor

Table 8. Larvicidal activity of endo-borneol against Tenebrio molitor.

Treatment (μL endo-Borneol L−1 Air)	Mortality (%)
	24 h	48 h	72 h	72 h Cumulative Mortality
60	96.7 ± 3.33	3.3 ± 3.33	0 ± ND	100 ± 0 a
30	90.0 ± 10.00	10.0 ± 10.0	0 ± ND	100 ± 0 a
24	66.7 ± 20.30	20.0 ± 10.0	13.3 ± 13.30	100 ± 0 a
16	46.7 ± 16.70	20.0 ± 10.0	20.0 ± 5.77	86.7 ± 23.1 a
10	3.3 ± 3.33	23.3 ± 3.33	46.7 ± 12	73.3 ± 23.1 a
Control	0 ± ND	6.7 ± 3.33	0 ± ND	6.7 ± 5.77 b
LC50	7.8 (5.6–9.7) μL endo-borneol L−1 air
LC90	15.3 (13.1–18.7) μL endo-borneol L−1 air
Chi-square	2.40

Values correspond to the average of 30 measurements ± SEM. Different letters indicate significant differences (p < 0.05). LC₅₀ of the 72 h cumulative mortality is shown with its fiducial limits. ND stands for not determinable.

shows the larvicidal activity of endo-borneol against T. molitor. After 24 h of exposure, no mortality was observed in the control group. However, the treatments with 60 and 30 μL endo-borneol L⁻¹ air exhibited insecticidal activity from the first contact, reaching mortality rates of 96.7% and 90%, respectively.

After 48 h, the control group showed a mortality rate of 6.7%, while 100% mortality was observed at the two highest concentrations (60 and 30 μL endo-borneol L⁻¹ air).

At 72 h, cumulative mortality ranged from 73.3% to 100%, with the 60, 30, and 20 μL endo-borneol L⁻¹ air concentrations being the most effective. The control group showed a cumulative mortality of only 6.67%, which was significantly lower than that of all treatments. LC₅₀ of endo-borneol against larvae was 7.8 μL endo-borneol L⁻¹ air after 72 h.

Table 9. Insecticidal activity of endo-borneol against Tenebrio molitor adults.

Treatment (μL endo-Borneol L−1 Air)	Mortality (%)
	24 h	48 h	72 h	72 h Cumulative Mortality (%)
60	40.0 ± 0 a	6.7 ± 5.77 a	16.7 ± 5.77 a	63.3 ± 3.33 a
30	16.67 ± 15.28 ab	6.7 ± 5.77 a	13.3 ± 15.28 a	36.7 ± 8.82 ab
24	13.33 ± 8.82 ab	3.3 ± 5.77 a	6.7 ± 5.77 a	23.3 ± 8.82 bc
16	13.33 ± 8.82 ab	3.3 ± 5.77 a	3.3 ± 5.77 a	20.0 ± 5.77 bc
10	13.33 ± 8.82 b	3.3 ± 5.77 a	3.3 ± 5.77 a	20.0 ± 5.77 bc
Control	0 ± NDb	3.3 ± 5.77 a	0 ± NDa	3.3 ± 3.33 c
LC50	46.0 (37.8–60.4) μL endo-borneol L−1 air
LC90	90.0 (71.5–128.0) μL endo-borneol L−1 air
Chi-square	2.37

Insecticidal activity values correspond to the average of 30 data points. LC₅₀ of the 72 h cumulative mortality is shown with its fiducial limits. Different letters indicate significant differences (p < 0.05). ND stands for not determinable.

shows the insecticidal activity of endo-borneol against T. molitor adults. After 24 h of exposure, no mortality was observed in the control group. The highest mortality at this time point was 40%, recorded at the 60 μL endo-borneol L⁻¹ air. At 48 h, a constant mortality rate of 6.7% was observed for the two highest concentrations, while the remaining treatments, including the control, showed a mortality rate of 3.3%.

By the end of 72 h, the highest cumulative mortality was 63.3% for the 60 μL endo-borneol L⁻¹ air concentration, followed by 36.7% for the 30 μL endo-borneol L⁻¹ air treatment. The control group accumulated only 3.33% mortality by the end of the experiment, which was significantly lower than that of all other treatments. The LC₅₀ for endo-borneol against adults was 46 μL endo-borneol L⁻¹ air after 72 h.

Table 10. Repellent activity of endo-borneol against Tenebrio molitor adults.

Treatment (μL endo-Borneol cm−2)	Repellency (%)	Repelling Class
0.25	100 ± 0 a	V
0.1	80 ± 13.33 ab	IV
0.025	60 ± 16.33 bc	III
0.0025	40 ± 16.33 c	II
Control	0 ± NDd	0
RC50	0.03 (0.01–0.08) μL endo-borneol cm−2

Repellent activity values correspond to the average ± SEM of 10 data points. Different letters indicate significant differences (p < 0.05). RC₅₀ is shown with its fiducial limits. ND stands for not determinable.

shows the repellent activity of endo-borneol against T. molitor adults. When they were exposed to endo-borneol in a “TYVO” device, no repellency was observed in the control (0%). By contrast, repellency values ranging from 40% to 80% were recorded for concentrations between 0.0025 and 0.1 μL endo-borneol cm⁻². The highest repellency, 100%, was achieved at 0.25 μL endo-borneol cm⁻². A clear dose–response relationship was observed, with higher concentrations resulting in higher repellency percentages. All treatments showed statistically significant differences compared with the control. The RC₅₀ of endo-borneol against adults was 0.03 μL endo-borneol cm⁻².

4. Discussion

The L. camara essential oil obtained in this study had a yield of 0.8%. Valdez et al. [28] showed a yield of 0.022% when extracting L. camara EO through Clevenger-type apparatus and sodium sulfate to eliminate the traces of water, making our yield approximately 36 times higher. Similarly, Vacacela-Ajila et al. [29] reported a yield of 0.0069% using Clevenger-type apparatus, while the work of Aisha et al. [17] reported 0.24% from leaves with a Clevenger-type apparatus and sodium sulfate to remove the remaining water, the variations in yield observed among studies may be explained by environmental and biological differences across collection sites and years of the plant samples. Also, the higher yield observed in this study could be due to differences in the extraction methods used. Other variables that could induce this variation were the time and place of the collected samples. In this study, the plant material was collected in 2024 at the facilities of the Autonomous University of Querétaro, México; by contrast, Valdez et al. [28] collected samples in 2016 in Cuenca, Azuay Province, Ecuador; Vacacela-Ajila et al. [29] gathered their material in 2023 in Cantón Loja, Loja Province, Ecuador; while Aisha et al. [17] collected from Kerala Agricultural University, Thrissur, India in 2023.

The insecticidal activity of L. camara EO against other insect pests was previously evaluated. In this regard, Zandi-Sohani et al. [16] investigated the insecticidal activity of L. camara EO against larvae and adults of C. maculatus, observing significant activity in both life stages. The LC₅₀ values were 282.7 μL EO L⁻¹ air for females and 187.9 μL EO L⁻¹ air for males. Furthermore, Aisha et al. [17] reported that EO of this species exhibited fumigant toxicity LC₅₀ of 4.1 mg L⁻¹ air for L. serricorne, 6.2 mg L⁻¹ air for C. chinensis and 16.7 mg L⁻¹ air for T. castaneum after 24 h. These authors also reported contact toxicity with an LC₅₀ for L. serricorne and 6.2 mg cm⁻², 4.8 mg cm⁻² for C. chinensis and 8.9 mg cm⁻² for T. castaneum, at 24 h, while Natchiappan et al. [30] evaluated the EO of this species against adults of Sitophilus oryzae (L.) (Coleoptera: Curculionidae), Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae) and Carpophilus dimidiatus (Fab.) (Coleoptera: Nitidulidae), which showed LC₅₀ of 7.2 ppm, 13.2 ppm and 17.6 ppm, respectively. In addition, Liambila et al. [31] reported an LC₅₀ of 250 μL EO L⁻¹ after 72 h against second instar larvae of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). Wangrawa et al. [32] showed that this EO had an LC₅₀ of 49.2 ppm against larvae of Anopheles funestus (Giles) (Diptera: Culicidae) and 38.4 ppm against larvae of C. quinquefasciatus after 24 h. These previous findings align with the results found in the present study due to the significant insecticidal activity shown by L. camara EO against T. molitor, a stored grain pest. The alignment stands from the effectiveness of L. camara EO across different insect species. The fumigant results for adults show that they are more toxic than those reported by Zandi-Sohani et al. [16] for C. maculatus. Thus, L. camara EO possesses potent fumigant activity against different stored products and agricultural pests.

The repellent activity of L. camara EO has been studied against other stored product insects. For instance, Aisha et al. [17] observed a repellency of 64.4% against adults of C. chinensis at 0.5 mg cm⁻². Also, they assessed this EO against L. serricorne and T. castaneum where they exhibited 64.4% and 65.6% repellency at 5 mg cm⁻², respectively. While a direct comparison of an RC₅₀ value with a single percent repellency value is limited, our study reached a 50% repellent effect at a concentration that is much lower than that used by Aisha et al. On the other hand, Zandi-Sohani et al. [16] assessed the repellent activity of L. camara EO against C. maculatus adults where it exhibited a 100% repellency at 0.4 μL EO cm⁻². The RC₅₀ value found in this study is five times lower than their concentration, even considering that it achieves a 50% repellency. These findings support L. camara EO can be used for the management of T. molitor adults and other stored product pests.

In terms of the antifeedant activity, our results suggest that exposure of T. molitor larvae to L. camara EO caused a dose-dependent reduction in weight gain, with a 40.3% difference after 30 d between the highest tested concentration and the control. This finding is consistent with the marked antifeedant activity reported for L. camara EO and its organic extracts in different insect species, particularly lepidopterans. For instance, Chau et al. (2019) [33] assessed the antifeedant activity of this EO against second instar Spodoptera litura (Fab.) (Noctuidae) and Plutella xylostella (L.) (Plutellidae) larvae through no-choice assays. They found that 500 µL of EO exhibited an antifeedant index (AI) of 57.1% and 62.4%, respectively. On the other hand, Hùng et al. (2020) [34] evaluated the antifeedant activity of L. camara leaf EO against S. litura with leaf disks immersed in solutions of EO. At a concentration of 2.5%, the EO showed an AI of 63.0%. Furthermore, organic extracts have also demonstrated antifeedant activity in insects. In this context, Thanavendan and Kennedy (2016) [35] analyzed the antifeedant activity of different solvent extracts of L. camara against P. xylostella. In this study, the hexane extract had the strongest activity against fourth instar larvae with an AI of 94.6% at a concentration of 10%. Even against an isopteran, Yuan and Hu (2012) [36] assessed the antifeedant activity against Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) using no-choice paper test where the treatment of 0.212 mg cm⁻² exhibited a 78% reduction in feeding of this insect. Additionally, Melanie et al. (2019) [37] evaluated the ethanolic leaf extract of L. camara against Crocidolomia pavonana (Fab.) (Lepidoptera: Crambidae) third instar larvae. In their study, the ethanolic extract showed strong antifeedant activity against C. pavonana, with an AI of 60%. The correlation between the antifeedant activity in other insects and the growth inhibition in T. molitor, manifested as a lower food intake or altered metabolism, supports that the volatile compounds of L. camara EO have an impact on insect feeding behavior and physiology. The sublethal effects observed by this EO, such as growth inhibition and repellency, are classified as “insectistatic activities” due to their ability to interfere with growth and reproduction without leading to mortality, as described by Levinson (1975) [38]. This is the first study that describes the exposure effect of L. camara EO on T. molitor weight gain, confirming that its insectistatic action is not limited to previously studied orders but extends to this coleopteran species.

Regarding the chemical characterization of L. camara EO, there are various studies that provide information about the chemical composition of this species. For instance, Zandi-Sohani et al. [16] identified 14 compounds in this oil and four as major compounds such as α-humulene (23.3%), cis-caryophyllene (16.2%), germacrene D (13.2%) and bicyclogermacrene (12.5%). Furthermore, Aisha et al. [17] reported 24 compounds, where caryophyllene was the most abundant (69.9%), followed by isoledene (12%). Also, Natchiappan et al. [30] showed 36 compounds, being α-selinene (6.7%) and aromadendrene (6.3%) the most predominant. On the other hand, Wangrawa et al. [32] identified 26 compounds, with caryophyllene oxide (14.8%), (+/-) germacrene D (7.3%) and byciclogermacrene (6.6%) being the most abundant. Chau et al. [33] reported 15 compounds and the most abundant were β-caryophyllene (29.7%) and α-humulene (10.2%), while Liambila et al. [31] analyzed L. camara EO from six different climatic zones of Kenya that showed 18 to 28 compounds, with major compounds including β-caryophyllene (5.11–11.65%), sabinene (2.84–12.54%), bicyclogermacrene (6.29–10.05%) and spathulenol (4.22–7.54%). Valdez et al. [28] showed 66 compounds, with germacrene D (19.29%), β-caryophyllene (14.55%), α-humulene (9.51%) found to be predominant. In this research 48 compounds were identified, and the major ones were endo-borneol (14.24%) and spathulenol (12.05%). While the chemical profiles differ across studies, similarities were observed too. For instance, endo-borneol, an isomer of borneol, was present in our study as a major constituent, and borneol was present in the research of Valdez et al. [28] with 0.04%, Romeu et al. [39] with 0.2% and Chowdhury et al. [40] with 1.13%. Our second major compound was spathulenol, and it was also present in the EO analyzed by other authors [30,33,34].

The differences in the composition of the L. camara EO could be due to variations in the collection year and location of the plant material, the chemical and physical properties of the soils where plants had growth. These differences in geographical location and time could have exposed the plants to different biotic and abiotic factors, causing a distinct EO yield, composition, abundance of the compounds. These factors can either increase or decrease essential oil production and include pest or herbivore attacks, changes in annual precipitation, winter cold, average daily temperature, evapotranspiration, edaphic factors (related to soil), and the plant’s age and genetics [41,42].

This is the first study that describes the biological activity of endo-borneol, a borneol isomer, against a stored product pest. However, there are studies that have shown that borneol and its isomers have insecticidal activity against stored product pests. For instance, Rozman et al. [43] assessed the fumigant activity of a mixture of this terpene, in a ratio of 87% borneol and 12% isoborneol, against S. oryzae, R. dominica, and T. castaneum. They observed that this terpene mixture caused a 100% mortality at 0.13 µL L⁻¹ with S. oryzae adults at 24 h of exposure, while the same mixture had a 100% mortality at 1.38 µL L⁻¹ against R. dominica at 24 h of exposure. In contrast, these terpenes only reached a 30% of mortality against T. castaneum at 168 h with a concentration of 138.88 µL L⁻¹. The fumigant activity of endo-borneol in the present study was lower than that observed by Rozman et al., with borneol and isoborneol mixture against S. oryzae and R. dominica. On the other hand, T. castaneum showed a lower susceptibility, even as shown in the present study, as it lasted 96 h more to cause half of the mortality than for T. molitor. In another study, Yildirim et al. [44] evaluated borneol fumigant activity against Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) without measuring the volume of the Petri dish (9 cm diameter) that exhibited a mortality of 100% with 30 µg per Petri dish after 72 h of exposure. Furthermore, Xiao-Meng et al. [45] assessed bornyl acetate, a borneol ester, against L. serricorne and found an LD₅₀ value of 9.42 µg adult⁻¹ after 24 h of contact toxicity. Thus, borneol monoterpenic structure in bornyl acetate may be involved in the insecticidal activity of this compound against this stored product pest. Additionally, this compound had repellent behavior towards L. serricorne as it caused 68% of repellency using 0.07 µL cm⁻² after 4 h, which is a lower repellency than observed in the present study as it causes a 50% of repellency with less than half of the used concentration (0.03 µL cm⁻²). This indicates that T. molitor is more susceptible to the repellent effect of endo-borneol at lower concentrations.

Overall, endo-borneol displayed a significantly superior performance to L. camara EO by itself. The susceptibility of larvae to endo-borneol suggests a faster insecticidal response as the results were achieved after 3 d of exposure instead of 30 d, as in the case of the EO. This supports the idea that the insecticidal activity of the EO is mainly due to its concentration of endo-borneol. Also, the pure compound showed higher repellent activity against T. molitor adults compared to the whole L. camara EO. This demonstrate that endo-borneol is the main active compound responsible for the insecticidal and insectistatic activities of L. camara EO against T. molitor.

5. Conclusions

This study concludes that Lantana camara EO and its major compound, endo-borneol, showed larvicidal and adulticidal activities. Also, these showed insectistatic activity due to their repellency against adults and growth inhibitory activity, decreasing the weight larvae against Tenebrio molitor. According to these results, endo-borneol can be used as an alternative in pest management programs due to its low environmental impact and being not harmful for non-target organisms. These characteristics extend the possible application of this EO as an ecofriendly insecticide.