Contact toxicity and repellent activity of essential oils from Alpinia zerumbet cv. ‘Variegata’ against stored product insects

ABSTRACT

Alpinia zerumbet cv. ’Variegata’, as a special spice, has attracted social attention for its potential to replace synthetic chemicals as insecticides and repellents in food protection. In this work, the essential oils (EOs) from different organs of Alpinia zerumbet cv. ’Variegata’ (stem and leaf, flower, and rhizome) were extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). The contact toxicities and repellent activities of the extracted EOs, along with their common components, against three insect species (Tribolium castaneum, Lasioderma serricorne, and Liposcelis bostrychophila) were evaluated. GC-MS analysis identified 25 components, and eucalyptol and camphor were the common primary components in the three EOs. The result showed that EOs and the main components (eucalyptol and camphor) exhibited varying degrees of contact toxicity and repellent activity against three insect species. Camphor exhibited more obvious contact toxicity than eucalyptol against both insect species (L. serricorne and L. bostrychophila), but the toxic effect was opposite for T. castaneum. The gray relation analysis explained that REOA had the strongest repellency against L. serricorne, with a coefficient of 0.52. The results in this study suggest that EOs of A. zerumbet cv. ’Variegata’ have the potential to be developed into botanical insecticides and repellents to control stored-product insects.

KEYWORDS:

• Alpinia zerumbet cv. ‘Variegata’
• essential oils (EOs)
• Tribolium castaneum
• Lasioderma serricorne
• Liposcelis bostrychophila

Introduction

In recent years, interest in essential oils and their use in food protection has gradually increased due to the growing range of storage insect hazards as well as the increasing dangers of synthetic chemicals.^[1] In this work, Tribolium castaneum (Coleoptera: Tenebrionidae), Lasioderma serricorne (Coleoptera: Anobiidae), and Liposcelis bostrychophila (Psocoptera: Liposcelididae) are selected as the target insects. Tribolium castaneum is mainly parasitic on wheat, corn, rice, and other grains. It can secrete foul-smelling liquid, produce fishy tastes, and may also secrete carcinogenic substances.^[2] Lasioderma serricorne is an omnivorous insect, mainly stored in tobacco leaves and tea but also found in grains and legumes. It is extremely destructive and eventually causes parasitic foods to produce mold.^[3] Liposcelis bostrychophila lives in a variety of habitats, often growing in books and grains. Outbreaks may occur in storage warehouses and food processing sites.^[4]

The control methods for stored-product insects generally include physical, chemical, and biological control.^[5] Due to the fact that chemical control methods are simple and efficient, synthetic insecticides are commonly used to control stored-product insects, such as DEET and PH₃. With the widespread use of synthetic insecticides, insect resistance and environmental pollution are gradually increasing, and human health is endangered.^[6] Furthermore, the use of plant materials such as essential oils (EOs) for insect management is a conventional method that is less toxic, degradable, safe against non-target insects, and less damaging to the environment. This series of reasons makes the development of new insecticides necessary.^[7] Essential oils act on insects through contact, ingestion, and fumigation, causing repelling, inhibiting feeding, reducing spawning, and even increasing mortality.^[8] Studies have shown that the toxicity of EOs may be due to their properties, which can disrupt or alter the normal functioning of the insect nervous system.^[9]

Since ancient China, plants of Alpinia have been utilized in traditional Chinese medicine and spice.^[10–12] In addition, some Alpinia plants have been confirmed for their insecticidal and repellent activities against stored-product insects by previous investigations. The EO of Alpinia conchigera and Alpinia officinarum have been reported to possess fumigant, contact toxicity, and repellent activity against T. castaneum and L. serricorne, respectively.^[13,14] The rhizome extract of Alpinia galanga (L.) Willd, phenol 2,4-bis (1,1-dimethylethyl), showed great larvicidal potential as a promising candidate for the control of mosquito populations.^[15] Alpinia zerumbet cv. ’Variegata’, a kind of Alpinia, is a cultivar of Alpinia zerumbet (Pers.) Burtt et Smith, widely distributed in the south and southeast of China, and it is used as an ornamental plant year-round due to its beautiful flowers and pure fragrance, but the investigations of A. zerumbet cv. ’Variegata’ insecticidal activity against stored product insects are limited.^[16,17] Therefore, this work selected A. zerumbet cv. ’Variegata’ as the object to analyze the diversity of EO components and the biological activities. The chemical components of EOs in different organs (stem and leaf, flower, and rhizome) of A. zerumbet cv. ’Variegata’ were determined by GC-MS analysis. The contact toxicity and repellent activity of EOs and their main compounds (eucalyptol and camphor) from A. zerumbet cv. ’Variegata’ were evaluated against Tribolium castaneum, Lasioderma serricorne, and Liposcelis bostrychophila, and to evaluate the correlation between the relative content of the main compounds in EOs and their biological activity.

Materials and methods

Materials and essential oil extraction

The Alpinia zerumbet cv. ’Variegata’ was harvested from Pu’er (107º55’E, 26º35’ N), Yunnan Province, China, in April 2022. The voucher specimens (BNU-dushushan-2022-04-13) were deposited in the College of Resources Science and Technology, Faculty of Geographical Science, Beijing Normal University. The specific sample information was listed in Table 1. The EOs were extracted in a modified Clevenger-type apparatus and kept at 4°C in sealed containers. The supplier of n-hexane (97%) was Beijing Chemical Works in Beijing, China. Fluon was purchased from Beijing Sino-Rich Material Science in Beijing, China.

Table 1. The information of Alpinia zerumbet cv ’Variegata’.

Time	Site	Organs	Weight (g)	Volume (mL)	Density (g/mL)	Yield (%)
2022.04	Pu ‘er City, Yunnan Province, China	Stem and leaf	9460	5.7	1.04	0.06
		Flower	2190	5	0.99	0.23
		Rhizome	2560	1.6	1.02	0.04

Insects rearing

Tribolium castaneum and Lasioderma serricorne were fed on mixed feed (wheat flour with 10% dry yeast). Liposcelis bostrychophila was reared on wheat flour, dry yeast, and milk powder with a mass ratio of 10:1:1. The target insects were cultured in incubators with controlled atmospheric temperature (28 ± 2°C) and relative humidity (75 ± 2%). After 3–4 generations of culture and purification, two-week-old and unsexed adults were selected for bioassay.

Gas chromatography-mass spectrometry (GC-MS)

The chemical composition of essential oils was analyzed by gas chromatography-mass spectrometry (GC-MS). EOs were analyzed on an Agilent 6890N gas chromatograph coupled with an Agilent 5973N mass selective detector (70 eV). The quartz capillary column was a DB-5 MS. The chromatographic conditions were as follows: helium as carrier gas at 1 mL/min; the sample injection volume was 1 μL of 1% solution diluted in n-hexane, and the split ratio was 1:20 injection. The injector temperature was 250°C, and the initial column temperature was 60°C for 5 min, increased to 290°C at 7°C/min, and stayed for 5 min. The mass spectrum conditions were as follows: The scanning range is 50–550 m/z, and the scanning mode is full scan. Under the same operating conditions, the retention index of a homologous series of n-alkanes (C₅-C₃₆) was determined. The identification of components was determined by comparing mass spectra and RI values with the data stored in NIST 05 (Standard Reference Data, Gaithersburg, MD, USA), and the relative percentage of each component was determined by the GC-FID peak area% reports.

Contact bioassay

For T. castaneum and L. serricorne, the contact method was described by Liu and Ho.^[18] For each concentration of tested samples, as well as the control, 0.5 µL of solution was dropped onto the dorsal thorax of the insect. Every 10 treated insects were transferred into a glass vial (8 mL). For L. bostrychophila, the method was similar to Zhao.^[19] The circular filter paper (diameter 5.5 cm) treated with 300 µL of solution was fixed on the bottom of a Petri dish. Release 10 insects into the center of the Petri dish. The solvents n-hexane and pyrethrins were used as negative and positive controls, respectively. The experiment was repeated five times in each group, and the treated insects were placed in insect incubators. Mortality was recorded 24 hours later, and LD₅₀ or LC₅₀ values were estimated by Probit analysis.

Repellent bioassay

The area preference method was used to evaluate the repellent activity of EOs and the major components against T. castaneum and L. serricorne.^[3] Cut filter paper (diameter 9 cm) into semi-circular pieces. Half of them were treated with 500 µL of sample (concentration of 0.13–78.63 nL/cm²) and the other half with the control of the same dose. After the solvent had evaporated, the two semi-circular filter papers were recombined and pasted on the bottom of the Petri dish. Place 20 adults in the middle of the Petri dish, which was covered with a lid. The same experimental method was used on L. bostrychophila. The following points should be noted: replacing with 5.5 cm diameter circular filter paper and the Petri dish, applying 150 µL sample (concentration of 0.10–63.17 nL/cm²) and control solution on each half of circular filter paper, respectively. The treated insects were re-placed in insect incubators.

Solvent n-hexane was used as negative control, and DEET was used as positive control. The experiment was repeated five times in each group. The number of insects in the control group (Nc) was recorded after 2 and 4 hours. The percent repellency (PR) is calculated by the following formula:

$PR\left(\mathrm{\%}\right)=\left[\frac{Nc-Nt}{Nc+Nt}\right]\times 100$

Nc and Nt were the numbers of insects in the negative control and treated groups, respectively. In SPSS V 27.0, PR values were converted to arcsine values and then subjected to Analysis of Variance (ANOVA) and the Tukey’s HSD test at p < 0.05. Using the scale proposed by Liu and Ho,^[18] the mean values were then classified into various groups (0 to V): Class 0, I, II, III, IV, and V, with the PR ＜ 0.1, PR = 0.1–20.0, PR = 20.1–40.0, PR = 40.1–60.0, PR = 60.1–80.0, and PR = 80.1–100.0, respectively.

Statistical analysis

The data were analyzed using the Statistical Package of Social Science (SPSS) version 27.0 for Windows 11 (IBM Corp., Armonk, NY, USA). The LD₅₀ (LC₅₀) and LD₉₀ (LC₉₀), their 95% fiducial limits, chi-square values, and associated parameters were calculated using Probit analysis. R (4.3.1) was used to calculate the relative correlation degree of EOs repellent activity against the three target insects.

Results

Chemical compositions of EOs

The yield of EOs from the different organs of Alpinia zerumbet cv. ’Variegata’ varied, ranging from 0.04% to 0.23% (v/w). FEOA had the highest yield (0.23%), which was six times higher than that of REOA (0.04%). The chemical compositions of the three EOs were reported in Table 2. The GC-MS results identified 16, 16, and 15 components, representing 93.56%, 94.27%, and 89.89% for SLEOA, FEOA, and REOA, respectively. Among the three EOs, eucalyptol (11.43–61.67%) and camphor (8.05–32.00%) were the main common components. It was worth noting that the relative content of m-Cymol in REOA was relatively high (14.80%), while the content in FEOA was very low (0.14%) and even missing in SLEOA. The chemical classes of the three EOs showed great similarity (Figure 1), dominated by oxygenated monoterpenes, and SLEOA, FEOA, and REOA accounted for 69.86%, 69.94%, and 46.13%, respectively. Although the oxygenated monoterpene content of REOA was the lowest, it was associated with an increase in monoterpene hydrocarbons (26.01%) compared with the other EOs.

Figure 1. The chemical classes of A. zerumbet cv. ’Variegata’ EOs.

* SLEOA = EO of A. zerumbet cv. ’Variegata’ stem and leaf; FEOA = EO of A. zerumbet cv. ’Variegata’ flower; REOA = EO of A. zerumbet cv. ’Variegata’ rhizome.

Table 2. Chemical compositions of essential oils from different organs of Alpinia zerumbet cv. ’Variegata’.

No.	RI ^1	Compounds	Identification method ^2	Relative content (%)^3
				Stem and leaf	flower	rhizome
		Monoterpene hydrocarbons		3.84	3.43	26.10
1	980	D-Limonene	RI, MS	2.17	1.83	1.63
2	1003	ψ-Limonene	RI, MS	0.60	0.92	8.83
3	1023	p-Cymene	RI, MS	0.42	0.40	0.76
4	1030	m-Cymol	RI, MS	–	0.14	14.80
5	1083	4-Ethyl-o-xylene	RI, MS	0.27	–	–
6	1118	p-Menthatriene	RI, MS	0.39	–	–
7	1130	Cosmene	RI, MS	–	0.14	0.09
		Oxygenated monoterpenes		69.86	69.94	46.13
8	–	1-Propoxypropan-2-yl 2-methylbutanoate	MS	–	0.09	0.13
9	1031	Eucalyptol	RI, MS, CO	58.57	61.67	11.43
10	1088	Fenchone	RI, MS	–	–	0.17
11	1146	Camphor	RI, MS, CO	10.91	8.05	32.00
12	1181	Dill ether	RI, MS	0.13	–	–
13	1217	Fenchylacetate	RI, MS	–	–	2.41
14	1218	Benzylacetone	RI, MS	0.25	0.14	–
		Sesquiterpene hydrocarbons		3.41	4.14	0.29
15	1415	(Z)-α-Bergamotene	RI, MS	–	3.95	–
16	1437	trans-Bergamotene	RI, MS	3.21	–	–
17	1519	1,4,7,-Cycloundecatriene, 1,5,9,9-tetramethyl-, Z,Z,Z-	RI, MS	0.20	0.18	–
18	1545	Selina-3,7(11)-diene	RI, MS	–	–	0.29
		Oxygenated sesquiterpenes		0.21	0.23	0.00
19	1615	Humulene-1,2-epoxide	RI, MS	0.21	–	–
20	1632	γ-Eudesmol	RI, MS	–	0.23	–
		Other		15.54	15.63	17.37
21	1006	Benzene, 4-ethyl-1,2-dimethyl-	RI, MS	–	10.02	0.67
22	1319	Durenol	RI, MS	0.12	0.11	0.42
23	–	Benzoic acid, 2,4-dimethyl-, (2,4-dimethylphenyl) methyl ester	MS	12.33	–	–
24	1397	Methyl cinnamate	RI, MS	2.87	5.04	15.83
25	1860	Dicyclohexyl oxalate	RI, MS	0.22	0.45	0.45
		Total		92.86	93.35	89.89

¹RI: retention index as determined on DB-5 MS column using the homologous series (C₅-C₃₆) of n-hydrocarbons.

²Co: co-injection with standard compound.

³Relative area (peak area relative to the total peak area).

Contact toxicity of essential oils and major compounds

The three EOs of Alpinia zerumbet cv. ’Variegata’ and their main components had diverse toxic effects against the three target insects (Table 3). For T. castaneum, REOA showed the strongest contact toxicity, with LD₅₀ values of 12.28 μg/adult, followed by FEOA (LD₅₀ = 15.80 μg/adult). While camphor showed the weakest contact toxicity against T. castaneum, the LD₅₀ was 54.21 μg/adult, which was nearly 208 times higher than pyrethrins (LD₅₀ = 0.26 μg/adult). FEOA exhibited the strongest contact toxicity against L. serricorne (LD₅₀ = 8.28 μg/adult), followed by REOA (LD₅₀ = 14.45 μg/adult). As for L. bostrychophila, the contact toxicity of SLEOA (LD₅₀ = 18.42 μg/cm²) was the strongest, which was even better than that of pyrethrins (LD₅₀ = 18.72 μg/adult). The contact activity of the tested EOs against the target insects was great, and the mortality even reached 100% at the highest tested concentration (Table S1). The insects treated by EOs showed wing tremor, loss of coordination, and the black appearance of dead insects, which were typical neurotoxic reactions.^[20,21] Therefore, it can be inferred that EOs may cause abnormal performance or even the death of insects by acting on their nervous systems.

Table 3. Contact toxicity of essential oils from different organs of Alpinia zerumbet cv. ’Variegata’ against T. castaneum, L. serricorne and L. bostrychophila adults at 24 h.

Insects	Samples	LD50/LC50	LD90/LC90	Slope ± SE	Chi-Square (χ^2)
		(μg/adult; μg/cm^2) (95% CI) ^4	(μg/adult; μg/cm^2) (95% CI) ^4
TC	SLEOA	24.91(21.78–28.07)	47.31(42.11–55.13)	1.54 ± 0.19	13.93
	FEOA	15.80(1.24–19.80)	45.47(38.86–57.47)	1.06 ± 0.17	18.64
	REOA	12.28(3.78–17.44)	47.82(40.16–62.82)	0.94 ± 0.17	17.31
	Eucalyptol^1	18.83(17.13–2.69)	—	—	16.56
	Camphor^1	54.21(5.49–57.32)	—	—	17.13
	Pyrethrins^1	0.26(0.22–0.30)	—	—	13.11
LS	SLEOA	19.01(16.29–21.52)	36.19(32.23–42.43)	2.02 ± 0.27	7.87
	FEOA	8.28(6.00–1.25)	23.58(19.30–32.72)	2.05 ± 0.40	9.87
	REOA	14.45(7.80–18.89)	46.35(39.36–59.43)	0.20 ± 0.04	16.95
	Eucalyptol^1	15.58(12.88–18.02)	—	—	15.18
	Camphor^1	11.30(7.78–14.07)	—	—	16.13
	Pyrethrins^3	0.24 (0.16–0.35)	—	1.31 ± 0.20	17.36
LB	SLEOA	18.42(16.16–20.58)	32.28(28.96–37.50)	1.636± 0.22	10.24
	FEOA	55.27(46.13–59.14)	96.77(86.77–111.90)	3.64 ± 0.44	16.93
	REOA	44.76(38.64–5.39)	82.77(74.02–96.53)	4.21 ± 0.56	23.39
	Eucalyptol^2	344.00(331.00–358.00)	—	—	9.04
	Camphor^2	53.00(51.00–55.00)	—	—	11.20
	Pyrethrins^3	18.72 (17.60–19.92)	—	2.98 ± 0.40	10.56

¹Data from Zhang et al.^[49]

²Data from Liang et al.^[50]

³Data from Yang et al.^[51]

⁴Contact toxicity was considered significantly different when non-overlap of 95% CI.

*TC = T. castaneum; LS = L. serricorne; LB = L. bostrychophila.

*SLEOA = EO of A. zerumbet cv. ’Variegata’ stem and leaf; FEOA = EO of A. zerumbet cv. ’Variegata’ flower; REOA = EO of A. zerumbet cv. ’Variegata’ rhizome.

The bivariate correlation results between the relative content of the two components and contact toxicity against T. castaneum, L. serricorne, and L. bostrychophila were shown in Table 4. The results showed that eucalyptol positively correlated with contact toxicity against T. castaneum, but negatively correlated with that to L. serricorne and L. bostrychophila. The profile of camphor was opposite to that of eucalyptol.

Table 4. Pearson’s correlation coefficient of relative content of the two major compounds and insecticidal activity of EOs.

Insecticidal toxicity ^1	Relative content (%) (N = number of valid sample)
	Eucalyptol	Camphor
LD50 to TC	0.676 (N = 3)	−0.635 (N = 3)
LD50 to LS	−0.141 (N = 3)	0.195 (N = 3)
LC50 to LB	−0.187 (N = 3)	0.113 (N = 3)

*TC = T. castaneum; LS = L. serricorne; LB = L. bostrychophila.

Repellent activity of essential oils and major compounds

The repellent activity of the three EOs and the major components against T. castaneum, L. serricorne, and L. bostrychophila were presented in Figure 2.

Figure 2. Percentage repellency of A. zerumbet cv. ’Variegata’ EOs against T. castaneum, L. serricorne, and L. bostrychophila.

a) and b) Percentage repellency of A. zerumbet cv. ’Variegata’ EOs against L. bostrychophila at 2 and 4 h post-exposure.

c) and d) Percentage repellency of A. zerumbet cv. ’Variegata’ EOs against L. serricorne at 2 and 4 h post-exposure.

e) and f) Percentage repellency of A. zerumbet cv. ’Variegata’ EOs against T. castaneum at 2 and 4 h post-exposure.

* SLEOA = EO of A. zerumbet cv. ’Variegata‘ stem and leaf; FEOA = EO of A. zerumbet cv. ’Variegata‘ flower; REOA = EO of A. zerumbet cv. ’Variegata’ rhizome.

Although EOs and the two major components showed different levels of repellent activities against target insects, the repellent effect (Class V) was best at the highest concentrations (78.63 nL/cm² and 63.17 nL/cm²), and gradually decreased with concentration (Table S2). For T. castaneum, the repellency of the major components was above 80.00% (Class V) after 2 h of exposure, at the highest concentration of 78.63 nL/cm². Among all tested samples, the repellent effect of REOA was even higher than that of DEET at 78.63 nL/cm² after 4 h of exposure. For L. serricorne, and L. bostrychophila, compared with other test samples, eucalyptol had the worst repellent effect, and the repellent rate was less than 60.00% (Class 0–III). After 2 hours and 4 hours of exposure, at the highest concentration, the three tested EOs had the most obvious repellent effect against L. bostrychophila, with repellent rates higher than 78.00%, and REOA had stable and effective repellent effects on all target insects, for which the repellent level was Class V.

The relative degree of correlation between the repellent activities of EOs and DEET against the three target insects was determined using gray correlation analysis, and the results were shown in Table 5. The repellent effect of FEOA on T. castaneum and L. bostrychophila was the weakest, and the relative correlation degree was 0.43 and 0.42, respectively. It was worth noting that the relative correlation degree of REOA to L. serricorne (0.52) was greater than 0.50, which was the most obvious repellent effect.

Table 5. Gray correlation of repellent activities of essential oils from different organs of Alpinia zerumbet cv. ’Variegata’ against T. castaneum, L. serricorne and L. bostrychophila.

Insect	Sample	Bestcoef	Worsecoef	Coef
TC	SLEOA	0.7010	0.8410	0.4546
	FEOA	0.6691	0.8777	0.4326
	REOA	0.7270	0.8109	0.4727
	DEET	0.9792	0.6077	0.6170
LS	SLEOA	0.7474	0.9147	0.4497
	FEOA	0.7980	0.8570	0.4822
	REOA	0.8497	0.7807	0.5212
	DEET	0.9248	0.7479	0.5529
LB	SLEOA	0.7447	0.8481	0.4675
	FEOA	0.6861	0.9316	0.4241
	REOA	0.7725	0.8299	0.4821
	DEET	0.9835	0.6597	0.5985

TC = T. castaneum; LS = L. serricorne; LB = L. bostrychophila.

SLEOA = EO of A. zerumbet cv. ’Variegata’ stem and leaf; FEOA = EO of A. zerumbet cv. ’Variegata’ flower; REOA = EO of A. zerumbet cv. ’Variegata’ rhizome.

Discussion

Chemical composition of the essential oils

The chemical compounds and contents of EOs from different organs of Alpinia zerumbet cv. ’Variegata’ showed great diversity. The chemical results in the present work had some similarities with Chen and Liu’s reports. Research analyzed the chemical components of A. zerumbet cv. ’Variegata’ EOs from the leaves (Fujian Province, China) and fruits (Guangdong Province, China), respectively, and the results showed that the main components of both included eucalyptol (43.56%, 35.73%), which was the same as the analysis results of the three EOs in this work.^[16,22] It was worth noting that there were some differences between the analysis results of this work and Shen’s research. Shen et al.^[23] analyzed the chemical composition of the leaves and flowers from A. zerumbet cv. ’Variegata’ EOs collected from Guangdong Province. The result exhibited that eucalyptol was the major component in leaf EO. While the main component of flower EO was (1 R)-(+)-α-pinene (22.09%), the components and relative contents of the majority released from flowers were significantly higher than those from leaves. Other studies focused on the EOs from different organs of A. zerumbet showed that the components of EOs were diverse, and some compounds were only present in specific organs.^[24,25] Although the composition and content of the previous results were different from this study, eucalyptol may be used as the main representative compound, and the irregular changes in EO components may have been influenced by organ dependence.^[26] The reasons for the variations may include environmental factors (e.g., climate, season, geographical conditions), genetics, and the photosynthesis and transpiration rates of the plants. In addition, different extraction methods also lead to changes in the composition and content of volatile substances.^[27,28]

Contact toxicity

With the decrease in concentration, the mortality of insects had a significant decreasing trend, which showed concentration-dependence. In addition to concentration, the contact toxicity of EOs was closely related to the insect species. Moreover, insects treated with different EOs showed varying sensitivity. According to the statistical data (the LD₅₀ and LC₅₀ values), SLEOA had the best insecticidal effect against L. bostrychophila, with the LC₅₀ value of 18.42 μg/cm². While FEOA and REOA showed the strongest insecticidal effect against L. serricorne and T. castaneum, with the LD₅₀ of 8.28 μg/adult, and 12.28 μg/adult, respectively. Previous studies also confirmed the above view. The EO of Alpinia zerumbet showed different contact toxicity levels against Tribolium castaneum, Ctenocephalides felis, and Rhodnius nasutus, with the LD₅₀ value of 6.59 µg/adult, the LC₅₀ value of 553.31 μg/cm², and 83.30% of mortality at the 125.00 µg/mL concentration, respectively.^[25,29,30] For the same insect species, such as L. serricorne, both the rhizomes EO from A. galanga and A. kwangsiensis exhibited contact toxicity, with LD₅₀ values of 12.20 µg/adult and 24.59 µg/adult, respectively.^[14,31] The insecticidal toxicity of EOs to target insects was simultaneously affected by multiple factors. For the same insect species, the chemical components of EO may interact with each other to enhance penetration through the cuticular layer and thus improve insecticidal activity.^[32] However, the same EO may affect the biological activity characteristics of insects through multiple modes of action, and even inhibit or change the activity of detoxification enzymes in insects, such as carboxylesterase and glutathione-S-transferases.^[33] In addition to the effects of EO components and insect enzyme activity, other factors, including insect sensitivity, growth inhibition of EO on insects, and even environmental factors, may change the biological activity of insects, so further research is needed.^[26,34]

The insecticidal effect of two monoterpene components (eucalyptol and camphor) was lower than that of the three EOs, which may interact with each other to improve their insecticidal activity.^[35] The EO of Salvia hispanica L. against the beet armyworm showed that the combination of its main components (alpha-thujone, (+)-camphor, eucalyptol, and alpha-caryophyllene) exhibited synergistic interaction.^[36] Valcarcel et al.^[37] also showed that there may be a synergistic effect between the main components of aromatic plant EOs, which needs further research to explain. Moreover, the two common major components showed different degrees of contact toxicity against T. castaneum, L. serricorne and L. bostrychophila, and the bivariate analysis found that there were no significant correlations between the relative content of the main compounds and insecticidal activity. The insecticidal activity of compounds may be affected by a variety of factors, such as temperature changes affecting the biological activity of the compounds. Studies showed that the insecticidal effects of thymol and carvacrol increased with temperature.^[38] It should be noted that the correlation can only infer that the insecticidal activity of EO was consistent with the content changes of the major compounds, but it cannot mean that there was a causal relationship between the component content and the insecticidal activity.^[39]

Repellent activity

The EOs from three different organs of A. zerumbet cv. ’Variegata’ showed good repellent activity against the three target insects, and the repellent effect of EOs was time- and concentration-dependent. The present work exhibited that the repellent classes of the three EOs to the three target insects were class 0-Ⅴ, which showed that the sensitivity of insects to different EOs was varied. At the highest concentration, SLEOA had the best repellent effect on L. bostrychophila, while REOA showed the best repellent effect against T. castaneum and L. serricorne. In previous reports, the percentage repellency of Alpinia conchigera was 94.50% against T. castaneum, and the EO of Etlingera yunnanensis rhizomes against L. bostrychophila was 82.00%.^[40] For the difference, one key factor that should probably be considered is the metabolic detoxification effect of insects.^[41] Plant secondary metabolites and insecticides can be metabolized by the detoxification enzyme system of insects, which reduces the actual effect of toxic substances and even changes the tolerance and resistance of insects.^[42] However, it was not possible to predict the changes in the toxic effects of the compounds produced by metabolism. Therefore, the changes of metabolic components still need to be further studied.

Studies had shown that most of the monoterpenes had repellent effects against insects, such as limonene, thymol, p-Cymol, eugenol, alpha-terpineol, and carvone.^[43–47] In this study, compared with camphor, the contact toxicity and repellent activity of eucalyptol were significantly lower against L. serricorne and L. bostrychophila. The principle of repellence was mainly to provide a vapor barrier to prevent contact with insects, and the exposure time of the vapor barrier determined the duration of the repellent effect.^[48] Differences in the volatility and molecular structure of the two main compounds (eucalyptol and camphor) may affect the effective time of the repellent, resulting in different repellent effects. Compared with the repellent activity of the two main compounds, the repellent effect and stability of EOs were better, which was consistent with the contact toxicity of EOs. The effective repellent activity of EOs may be related to the chemical compounds and the synergistic effect between the compounds. Whether the synergistic effect between the two compounds or the effect of other components leads to the improvement of repellent activity, still needs further research. Overall, although the toxicity and repellent activity of EO were weaker than those of positive controls, they can be further studied as commercial insecticides due to their environmental friendliness, availability, and great resource potential.

Conclusion

In this study, the insecticidal and repellent activities of different organs (stem and leaf, flower, and rhizome) EOs from Alpinia zerumbet cv. ’Variegata’ were evaluated against Tribolium castaneum, Lasioderma serricorne, and Liposcelis bostrychophila. The extracted EOs showed insecticidal and repellent activities and were more effective than the two main components (eucalyptol and camphor) in controlling the three insect species. The work suggested that the EOs have the potential to be developed as an environmentally friendly insecticide and repellent for food protection, and the insecticidal mechanism still needs to be further explored.

Author contribution

Conceptualisation, J.-Z.W.; Methodology, Y.-T.Q.; Resources, S.-S.D. and Y.-K.Y.; Supervision, C.F. and S.-S.D.; Validation, J.-Z.W.; Visualisation, J.-Z.W.; Writing – original draft, J.-Z.W.; Writing – review and editing, J.-W.Z. All authors have read and approved the final manuscript.

Availability of data and materials

All data in this article will be available upon request.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10942912.2023.2286896

Additional information

Funding

This work was supported by the Second Tibetan Plateau Scientific Expedition and Research Program (2019QZKK0608).