Electrophysiological Mechanism and Identification of Effective Compounds of Ginger (Zingiber officinale Roscoe) Shoot Volatiles Against Aphis gossypii Glover (Hemiptera: Aphididae)

Abstract

Aphis gossypii Glover (Homoptera: Aphidinae), a major pest of Chinese pepper (Zanthoxylum bungeanum Maxim), causes significant agricultural damage. Ginger (Zingiber officinale Roscoe) has shown potential as a source for developing botanical pesticides due to its strong bacteriostatic and insecticidal properties; however, the underlying mechanisms remain poorly understood. This study evaluated the repellent activity of ginger shoot extract (GSE) across four solvent phases—petroleum ether, trichloromethane, ethyl acetate, and methanol—against A. gossypii. The results demonstrated that GSE exhibited significant repellent effects, with the methanol phase showing the most pronounced activity. Twelve fractions were chromatographically separated from the methanol phase, and electroantennography (EAG) analysis revealed that fraction 4 induced strong EAG responses in both winged and wingless aphids. Further identification of active compounds in fraction 4 by gas chromatography–mass spectrometry (GC–MS) indicated the presence of terpenes, aromatics, alkanes, esters, and phenols as major constituents. Subsequent EAG analysis identified several key compounds—octahydro-pentalene (C1), (Z)-cyclooctene (C2), dimethylstyrene (C3), tetramethyl-heptadecane (C5), tetrahydro-naphthalene (C6), and heptacosane (C9)—as responsible for eliciting EAG responses in both aphid forms. Additionally, results from Y-tube olfactometer assays showed that (Z)-cyclooctene and heptacosane were significantly attractive, while octahydro-pentalene acted as a strong repellent to both winged and wingless aphids. These findings offer valuable insights for the development of synthetic attractants and repellents for A. gossypii and provide a theoretical foundation for utilizing ginger in the creation of botanical pesticides targeting this pest.

1. Introduction

The Aphid (Aphis gossypii Glover; Homoptera: Aphidinae) is a major global pest affecting over 100 crop species, including maize (Zea mays), sorghum (Sorghum bicolor), and Chinese pepper. As a widespread agricultural pest, A. gossypii directly damages crop leaves, reducing photosynthesis and ultimately lowering yields. Indirectly, it also poses a threat through virus transmission and contamination from aphid honeydew [1]. In China, control of A. gossypii largely depends on chemical insecticides. However, this reliance has led to significant resistance to numerous insecticide active ingredients, along with potential risks to human and environmental health [2]. This highlights the urgent need for effective, eco-friendly, biodegradable, and residue-free alternatives [3]. Botanical pesticides, derived from plants, have emerged as viable solutions to regulate pest populations and diseases. These products, owing to their natural origin, are considered promising alternatives to chemical pesticides. In recent years, numerous plant-based pesticides have been explored. These can generally be classified into insecticides that control pest populations, repellents that deter pests from feeding on host plants, and attractants used as baits to eliminate pests [4]. For instance, peppermint oil serves as an organic insecticide against A. gossypii [5]. Key compounds in Chinese pepper oil, such as carvacrol and diterpenes in Lamiaceae species, contribute significantly to A. gossypii mortality [6,7]. Additionally, allelochemicals, such as tannic acid and gossypol, increase A. gossypii mortality [8]. Moreover, compounds such as gastrodene, cucurbitacin B, epigallocatechin gallate, and benzoate derivatives exhibit strong repellent activity against A. gossypii [9,10,11]. These findings collectively highlight the potential of botanical pesticides as effective, sustainable alternatives to synthetic chemical agents, demonstrating their diverse modes of action—whether insecticidal, repellent, or attractant—while maintaining environmental compatibility.

Ginger (Zingiber officinale Roscoe) is a high-value medicinal spice crop primarily cultivated in the central, southeastern, and southwestern regions of China. Research has demonstrated that ginger possesses potent bactericidal, insecticidal, and insect-repellent properties [12,13,14,15,16]. For instance, ginger extract exhibits notable contact toxicity against Diaphania hyalinata larvae, insecticidal activity against grain storage pests, and repellent effects against both adult whiteflies (Bemisia tabaci) and maize weevils (Sitophilus zeamais) [13,14,15,16,17,18,19,20]. It has been demonstrated that ginger shoot extract (GSE) inhibits the growth of sorghum aphids by impairing digestive enzymes and affecting antioxidant and detoxification enzymes, positioning GSE as a promising resource for botanical pesticide development. Recent studies have isolated 194 volatile oils, 85 gingerols, and 28 diarylheptanes from ginger [21]. Active compounds, such as 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, zingerone, and diarylheptanes, such as bisabolene and curcumene, have been extensively used in pharmacological applications [22], although their effects on A. gossypii remain unexplored.

This study investigated the effects of GSE and its major active compounds on the electrophysiological and behavioral responses of A. gossypii. Electroantennography (EAG) is a powerful tool for assessing the olfactory responses of insects to odorants, with the EAG amplitude reflecting the bioelectrical potential generated by antennal odorant receptors (ORs) in response to stimuli [23]. Initially, repellent assays using four different GSE phases were conducted to identify the phase with the strongest repellent activity. The most effective extraction phase was then chromatographically separated, and EAG recordings of its major components were evaluated. Components that elicited significant EAG amplitudes were analyzed using gas chromatography–mass spectrometry (GC–MS) to identify the most likely active compounds. Finally, the attractancy and repellency of these identified active compounds were assessed using a Y-tube olfactometer in dual-choice assays. This study sheds light on the behavioral effects of ginger’s major active compounds on A. gossypii, offering an alternative approach to its biological control.

2. Materials and Methods

2.1. Aphid Collection and Rearing

Wingless A. gossypii aphids were collected from Zanthoxylum bungeanum plants at the experimental field of Yangtze University Agricultural Industrial Park and identified as A. gossypii by the Hubei Key Laboratory of Spices & Horticultural Plant Germplasm Innovation & Utilization (Yangtze University). These aphids were reared on faba bean (Vicia faba L.) plants in a controlled growth chamber maintained at 22 ± 2 °C, 65 ± 5% relative humidity, and a 12L:12D photoperiod for five generations, during which both wingless and winged aphids were obtained. Seven-day-old adults (both wingless and winged) were randomly selected for subsequent treatments.

2.2. Plant Materials

Ginger plants (Zingiber officinale cv. Fengtou) were harvested after 120 days of growth from the Agricultural Science and Technology Industrial Park of Yangtze University, Hubei Province, China (E: 112.026207, N: 30.361273). The leaves and shoots were washed with distilled water, shade dried, and ground into a fine powder, which was stored in sealed containers at room temperature for later use.

2.3. Preparation and Separation of GE

2.3.1. Preparation of GE

The ginger leaf powder was extracted using a Soxhlet extractor. Ten grams of the powder were soaked in 100 mL of water and thoroughly mixed. The mixture was heated at 80 °C for 6 h, and the extract was concentrated by evaporation at 80 °C. The resulting thick paste extract was collected and preserved at −80 °C.

2.3.2. Separation of GSE

To separate the GSE, liquid–liquid extraction was employed [24]. A 10% methanol solution of ginger extract (GE) was prepared at a concentration of 100 mg·mL⁻¹ for a total volume of 50 mL. This solution was placed into a liquid separation funnel and extracted sequentially with petroleum ether (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; CAS: 8032-32-4), trichloromethane (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China; CAS: 865-49-6), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; CAS: 141-78-6), and methanol–water (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; CAS: 67-56-1) in a 1:10 ratio. Each solvent was used for 3–5 extractions, with 50 mL of solvent per extraction. The combined extracts were concentrated into a thick paste using a rotary evaporator and preserved at −80 °C [25].

2.4. Aphis gossypii Glover Repellent Assay

Choice tests were conducted to investigate the repellent effects of ginger leaves, ginger extract, and the four extraction phases on A. gossypii (Figure 1), following the method of da Camara with minor modifications [26]. In each 15 cm Petri dish lined with moistened filter paper, a Vicia faba L. leaf of uniform age and size was sprayed with deionized water (control) or one of the following treatments: (1) ginger extract, (2) petroleum ether extraction, (3) trichloromethane extraction, (4) ethyl acetate extraction, and (5) methanol extraction using a hand sprayer. The GSE and four extracts were prepared as 10 mg·mL⁻¹ solution in 10% methanol.

Ginger leaves of uniform age, sprayed with deionized water as a control, were used. In each Petri dish, two leaves were placed, separated by wet cotton to avoid interference between them. The left/right position of the leaves was alternated between experimental replicates.

A plastic disc, surrounded by wet cotton, was positioned equidistant from the two leaves to serve as the host area for the aphids. The plastic disc and the fresh Vicia faba L. leaves were connected with a cover slip. Fifty wingless aphids were quickly placed at the center of the plastic disc, allowing the aphids to move through the cover slip. The Petri dishes were sealed with parafilm to prevent escape. After 1, 3, 6, 9, and 12 h, the number of aphids that settled on each leaf disc was counted. Three replicates, each with 50 aphids, were conducted per treatment. Repellent activity was calculated using the following formula:

Repellent activity (%) = [(C − T)/(C + T)] × 100, where C and T represent the number of aphids on the control and treatment leaves, respectively [27].

2.5. Column Chromatography and Thin Layer Chromatography

Column chromatography and thin layer chromatography (TLC) were performed following the steps outlined by Li [27]. For column chromatography, a 30 mm × 500 mm column with a sand core was chosen, and 40 times the weight of silica gel (100–200 mesh) relative to the separated extract was used. This was mixed with methanol and quickly injected into the column to eliminate air bubbles. A small amount of methanol was used to dissolve 5 g of the methanol extraction phase, which was then uniformly added along the inner wall of the column using a long drip pipe. A series of solvents with increasing polarity—comprising petroleum ether, trichloromethane, and ethyl acetate—were used as eluents. The elution flow rate was maintained at 3–4 drops per second, collecting 50 mL per fraction, yielding a total of 44 fractions.

For TLC, a 0.5 cm line was drawn from one end of the TLC plate as the starting point. A 0.5 mm capillary was used to apply the sample. The TLC plate was then developed in a saturated tank with a solvent mixture of petroleum ether and ethyl acetate (10:1 ratio). The solvent front was allowed to rise to 3 cm from the top, and the front was marked with a pencil. The plate was then sprayed with 10% ethanol sulfate and placed in an oven for 20–30 min to develop color. The solution containing similar components was combined, yielding 12 fractions, which were vacuum concentrated and used for EAG recordings.

2.6. Electrophysiological Recordings

For the EAG assay, the method described by Du was followed [28]. The antenna bracket of the EAG probe served as the electrode, and conductive adhesive was used as the connecting medium at both ends of the electrode. The antenna was carefully removed under a dissecting microscope, and the EAG probe was connected to both the reference and recording electrodes. The sample consisted of the 12 fractions obtained from the previous assay, with each solution prepared to a concentration of 10 mg·mL⁻¹ in 10% methanol. The control solution was 10% methanol. The sample was absorbed into a 0.5 mm capillary, which was placed in the pore of the blowing pipe. During the assay, the antenna was exposed to a solvent control at the beginning and end of each antennal preparation. Stimuli were delivered as 0.5 s air puffs into a continuously humidified air stream, controlled by an air stimulus controller (Syntech Instruments Inc., San Francisco, CA, USA; Catalog No.CS-55). EAG signals were recorded for 10 s, beginning 1 s before the stimulus pulse. A recovery period of at least 1 min was allowed between each puff to ensure antennal receptor recovery. The analog signal was detected via the EAG probe, captured and processed using a data acquisition controller (Syntech Instruments Inc., San Francisco, USA; Catalog No.IDAC-2), and analyzed with EAG Pro software (Syntech Instruments Inc., San Francisco, USA; version 2.3).

2.7. Gas Chromatography–Mass Spectrometry (GC–MS)

The GC-MS system consisted of a TRACE™ 1300 gas chromatograph equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm film thickness) and a real-time connected thermal conductivity detector (TCD). Following the steps of Du [28], the GC temperature was programmed to start at 60 °C, then increased to 236 °C at a rate of 3 °C/min and held for 20 min, before being increased to 270 °C and held for 8 min. The spitless mode was used, with the injection temperature set to 240 °C and the transfer line temperature at 260 °C. The mass spectrometer operated at 70 eV in electron impact mode. Chemical identification of the compounds was confirmed by comparing retention times and mass spectra with those of known standards.

2.8. Electrophysiological and Behavioral Responses of A. gossypii to Potential Volatiles

The EAG assays were conducted as described previously. In this case, 10 mg·mL⁻¹ GSE was used as the positive control, with 10% methanol serving as the control (CK). Nine compounds were prepared at a concentration of 10 mg·mL⁻¹ with 10% methanol and tested as treatments. For the Y-tube olfactometer detection, the method followed Liu [29]. Six compounds, identified through the EAG assays, were selected for the behavioral response test. These six compounds were prepared with 10% methanol to a final concentration of 10 mg·mL⁻¹, with methanol used as a control to assess the aphid’s selectivity for different compounds. Prior to the assay, aphids were starved in the dark for 24 h. During the assay, the vacuum pump was turned on, and the aphids were introduced at the end of the Y-tube. After 30 min, the number of aphids in each arm was counted. The number of aphids in the two odor source tubes, as well as those remaining in the base tube, were used to calculate the reaction rate, selectivity coefficient, and choice:

Reaction rate = (sum of insects in each trapping tube/total number of insects) × 100%;

Choice = (number of insects in treatment or control trapping bottles/total number of insects) × 100%;

Selective coefficient = [(number of reactive insects in treatment trapping tube − number of reactive insects in control trapping tube)/total number of reactive insects] × 100%.

2.9. Statistical Analysis

Data were processed using Excel software (Microsoft, Redmond, WA, USA; version 2021). One-way ANOVA was used to analyze the repellent activity of the four extraction phases against A. gossypii, as well as the electrophysiological and behavioral responses under different compound treatments. Tukey’s HSD multiple comparison (α = 0.05) was applied to analyze the data. The percentage data were arcsine-square root transformed before ANOVA. The data were presented as mean ± standard error (SE) and analyzed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Graphs were produced using Sigma Plot 14.0 (Inpixon Corp., Palo Alto, CA, USA).

3. Results and Discussion

3.1. Repellency of GSE and 4 Extraction Phases Against A. gossypii

This study assessed the repellent efficacy of GSE and its four extraction phases against A. gossypii. As presented in

Table 1. Repellent activity of ginger extract and its four extraction phases against A. gossypii over a 12 h period.

	Repellent Activity (%)
	1 h	3 h	6 h	9 h	12 h
Ginger Extract a	42.22 ± 5.17 a	50.95 ± 2.86 a	64.82 ± 4.89 a	50.95 ± 2.86 a	51.28 ± 0.74 a
Petroleum ether a	35.28 ± 0.13 a	20.88 ± 0.31 c	8.83 ± 0.26 d	6.05 ± 1.08 d	5.28 ± 1.06 d
Trichloromethane a	18.68 ± 2.47 b	20.40 ± 0.28 c	26.71 ± 0.37 c	38.52 ± 0.30 b	35.74 ± 0.21 b
Ethyl acetate a	16.81 ± 0.98 b	20.23 ± 1.31 c	21.22 ± 0.45 c	13.69 ± 0.50 c	10.36 ± 1.84 c
Methanol a	32.86 ± 0.29 a	39.84 ± 0.09 b	47.37 ± 0.21 b	56.99 ± 2.50 a	55.19 ± 0.94 a
F	7.51	37.96	41.72	72.42	208.31
df	4, 10	4, 10	4, 10	4, 10	4, 10
p	0.005	<0.0001	<0.0001	<0.0001	<0.0001

^a The 10 mg·mL⁻¹ solution was prepared with 10% methanol for each treatment. Data are presented as mean ± SE. Prior to ANOVA, the data were arcsine transformed, and the table displays the transformed values. Values within a column followed by the same letter are not significantly different according to Tukey’s HSD Multiple Comparison test (p < 0.05).

, after 1 h of treatment, no significant differences in repellent activity were observed among GE, the petroleum ether phase, and the methanol phase, though all were more effective than the trichloromethane and ethyl acetate phases. After 3 h, GSE exhibited the highest repellent activity (50.95%), with the methanol phase showing significantly lower activity than the ginger extract but notably higher than the petroleum ether, trichloromethane, and ethyl acetate phases. After 6 h, ginger extract remained the most effective, followed by the methanol phase (47.37%), while the petroleum ether phase exhibited the least activity (8.83%). After 9 h, the repellent activities of the methanol phase and GSE were highest, at 56.99% and 50.95%, respectively, followed by trichloromethane (38.52%) and ethyl acetate (13.69%), with petroleum ether showing the lowest repellent activity (6.05%). After 12 h, the methanol phase and GSE had the highest repellent activities, at 55.19% and 60.53%, respectively, significantly surpassing those of trichloromethane (35.74%), ethyl acetate (10.36%), and petroleum ether (5.28%).

Ginger shows promise as a botanical insecticide. Recent field and laboratory studies revealed that co-cultivation of ginger with Chinese pepper disrupted A. gossypii behavior, and GSE inhibited the growth and development of sorghum aphids by affecting enzyme activities [29].

Previous research indicates that various plants exert repellent effects on aphids due to their diverse phytochemical compositions. For instance, extracts from Carica papaya L. and T. minuta demonstrated the strongest aphid suppression, likely due to insecticidal constituents, such as papain, alkaloids, terpenoids, and flavonoids [30].

Throughout the experiment, the repellent activity of the petroleum ether phase gradually declined from 35.28% at 1 h to 5.28% at 12 h. In contrast, the repellent activities of ginger extract, trichloromethane, ethyl acetate, and petroleum ether increased initially, peaked at 6 or 9 h, and then decreased. These results suggest that the residual effect of the petroleum ether phase was less persistent than the other extraction phases.

Moreover, during the entire treatment period, the repellent activity of the methanol, trichloromethane, and ethyl acetate phases was consistently stronger than that of the petroleum ether phase, with the methanol phase showing significantly greater repellent activity than both the trichloromethane and ethyl acetate phases. These results indicate that GSEs contain active compounds capable of modulating the behavior of A. gossypii. Additionally, this study identified the key components from the methanol extraction phase, which exhibited the strongest repellent effects on A. gossypii, and tested the antennal and behavioral responses to these major components.

3.2. EAG Responses Induced by Major Components of Methanol Extraction Phase in Wingless and Winged Aphids

Twelve chromatographic fractions were isolated from the methanol phase of ginger using column and thin-layer chromatography. These fractions were then subjected to EAG assays on both wingless and winged aphids.

As shown in Figure 2A, fractions 2, 3, and 4 elicited strong EAG responses in wingless aphids, with EAG amplitudes exceeding 300 μV. Specifically, fraction 4 produced the highest EAG amplitude among all fractions, followed by fractions 3, 2, 6, 7, and 9. In contrast, the EAG amplitudes of fractions 1, 5, 8, 10, 11, and 12 were all below 200 μV.

As shown in Figure 2B, fractions 4, 7, 8, and 9 induced strong EAG responses in winged aphids, with amplitudes greater than 400 μV, followed by fractions 6 and 12. Fractions 2, 3, 5, 10, and 11 elicited EAG amplitudes below 200 μV.

A comparison of the EAG responses in wingless and winged aphids revealed distinct patterns: fractions 2 and 3 elicited strong responses from wingless aphids (over 300 μV), but weak responses in winged aphids. Conversely, winged aphids responded strongly to fractions 7, 8, and 9 (over 400 μV), while wingless aphids showed minimal response. Notably, fraction 4 triggered substantial EAG responses in both wingless and winged aphids, indicating that this fraction contains active compounds capable of eliciting a robust response in both aphid types (Figure 2A,B).

The observed differences in EAG responses between winged and wingless aphids likely stem from their distinct ecological roles and physiological adaptations. Winged aphids, equipped for long-range dispersal, exhibit heightened sensitivity to volatile compounds, which aids in locating new host plants. In contrast, wingless aphids, which typically settle on a single host, show greater responsiveness to contact chemicals, facilitating their exploitation of local resources.

3.3. GC–MS Analysis of Fraction 4 Eliciting Strong EAG Responses of A. gossypii

Given that fraction 4 induced significant electrophysiological responses in both aphid types (Figure 2), a comprehensive GC-MS analysis was conducted to identify its chemical composition. The chromatographic profile revealed 27 tentatively identified compounds, which accounted for 98.60% of the total detected components (

Table 2. Identification of principal compounds in four fractions by GC-MS, along with the main properties and peak areas of their secondary metabolites.

Compound	RT (min)	MF a	MW b	PA(%) c	Cas d
octahydro-pentalene	4.16	C8H14	110.197	58.98%	694-72-4
(Z)-cyclooctene	4.34	C8H14	110.197	6.19%	931-87-3
ethyl-methyl-cyclohexane	4.83	C9H18	126.239	0.26%	19489-10-2
methyl-decane	7.55	C11H24	156.308	2.05%	13151-35-4
dimethyl-nonane	7.82	C11H24	156.308	0.74%	17302-28-2
mesitylene	8.04	C9H12	120.192	0.17%	108-67-8
p-cymene	9.96	C10H14	134.218	0.25%	99-87-6
o-cymene	10.18	C10H14	134.218	0.27%	527-84-4
undecane	10.54	C11H24	156.308	1.32%	1120-21-4
dimethylstyrene	12.24	C10H12	132.202	0.22%	2234-20-0
ethyl-dimethyl-benzene	12.74	C10H14	134.218	1.55%	1758-88-9
cyclohexyldimethoxymethyl-silane	13.21	C9H20O2Si	188.339	0.68%	17865-32-6
cyano-L-phenylalanine	14.09	C10H10N2O2	190.199	0.47%	263396-42-5
dodecane	14.49	C12H26	170.335	7.05%	112-40-3
tetramethyl-heptadecane	17.11	C21H44	296.574	0.76%	18344-37-1
tetrahydro-naphthalene	17.34	C11H14	146.229	1.02%	1680-51-9
trimethyl-octane	17.48	C11H24	156.308	0.24%	62016-34-6
hexyl pentadecyl ester sulfurous acid	18.14	C21H44O3S	376.278	0.34%	NA
tetradecane	22.72	C14H30	198.388	2.30%	629-59-4
di-tert-butylphenol	27.74	C14H22O	206.324	0.39%	96-76-4
hexadecane	30.48	C16H34	226.441	0.83%	544-76-3
nonadecane	34.10	C19H40	268.529	7.72%	629-92-5
methyl-octadecane	35.95	C19H40	268.521	0.38%	1560-88-9
methyl-eicosane	42.90	C21H44	296.574	0.62%	1560-84-5
dibutyl phthalate	43.27	C16H22O4	278.344	1.43%	84-74-2
heptacosane	49.83	C27H56	380.733	0.61%	593-49-7
methylenebis (tert-butyl-methylphenol)	56.16	C23H32O2	340.499	1.76%	119-47-1
Total				98.60%

^a Molecular formula. ^b Molecular weight. ^c Peak area of each component at the detected retention time, calculated from GC–MS results. ^d CAS registry number: a unique digital identification number for the chemical substance.

). Structural characterization was achieved through matching with the NIST mass spectral library (Match Factor > 85%). The identified secondary metabolites were systematically cataloged by retention time and characterized by their molecular weights and relative abundances (peak area percentages).

Notably, octahydro-pentalene was identified as the predominant constituent, accounting for 58.98% of the peak area, followed by characteristic aliphatic hydrocarbons, such as nonadecane (7.72%) and dodecane (7.05%), as well as the cyclic alkene (Z)-cyclooctene (6.19%). While 26 compounds were successfully annotated with CAS registry numbers, one unidentified component (RT 12.34 min) remains, requiring further structural elucidation. This chemical profile diverges from previously reported ginger volatiles [31], likely due to variations in extraction methods, seasonal harvesting, and potential isomerization during sample preparation. The 27 compounds identified by GC–MS were cross-referenced with the ginger metabolomes established at the Spice Crops Research Institute, College of Horticulture and Gardening, Yangtze University [32], and nine principal volatiles of ginger were identified (

Table 3. Screening of principal volatiles in the methanol extraction phase of ginger by comparing the compounds in fraction 4 with the existing ginger metabolomes in the laboratory.

Code a	Compound b	Class I	RT	RSI c	SI c	Report
C1	octahydro-pentalene	Terpenes	4.16	920	906	Yes
C2	(Z)-cyclooctene	Terpenes	4.34	905	880	Yes
C3	dimethylstyrene	Terpenes	12.24	881	839	Yes
C4	ethyl-dimethyl-benzene	Aromatics	12.74	896	881	Yes
C5	tetramethyl-heptadecane	Alkanes	17.11	869	847	Yes
C6	tetrahydro-naphthalene	Terpenes	17.34	896	889	Yes
C7	di-tert-butylphenol	Phenols	27.74	878	870	Yes
C8	dibutyl phthalate	Esters	43.27	938	927	Yes
C9	heptacosane	Alkanes	49.83	899	885	Yes

^a Each component is coded as C1–C9. ^b Compounds in fraction 4 and ginger metabolomes showing high similarity after comparison. ^c The similarity index represents the degree of match between the GC–MS analysis results and the mass spectrum database from the GC–MS data system, as well as other published mass spectrum databases.

). These included four terpenoids, one aromatic, two alkanes, one phenol, and one ester. The reverse similarity coefficient (RSI) and similarity coefficient (SI) of these nine compounds were all above 80. The RSI measures the similarity between the mass spectrum of an unknown compound and a reference spectrum by comparing the peaks absent in the unknown spectrum, while the SI reflects the overall similarity between the two spectra by comparing all peaks. Both indices are critical for evaluating the confidence in compound identification. Similarity indices (RSI and SI) exceeding the 800 threshold indicate over 95% confidence in compound identification when compared against authenticated reference spectra. The validated components, which include octahydro-pentalene (C1), (Z)-cyclooctene (C2), dimethylstyrene (C3), ethyl-dimethyl-benzene (C4), tetramethyl-heptadecane (C5), tetrahydro-naphthalene (C6), di-tert-butylphenol (C7), dibutyl phthalate (C8), and heptacosane (C9), are commercially available and can be classified into terpenes, aromatics, alkanes, phenols, and esters (Table 3). These compounds, previously reported in ginger studies [31], likely contribute to the elevated EAG amplitudes observed in fraction 4 for both wingless and winged aphids, which were further confirmed through EAG analysis.

3.4. Nine Principal Volatiles That Elicited the EAG Responses in Wingless and Winged Aphids

The EAG responses of both wingless and winged aphids to the nine principal compounds were analyzed (Figure 3). As shown in Figure 3A, C2 elicited a significant EAG response in wingless aphids, which, although slightly lower (not significant) than the GSE-induced response, was still substantial. The EAG responses elicited by C3, C5, C6, and C9 in wingless aphids were significantly higher than the CK but significantly lower than the GSE response. In contrast, C1, C4, C7, and C8 did not induce significant EAG responses compared to the CK.

As presented in Figure 3B, the EAG response of winged aphids to GSE (428.66 µV) was the highest among all treatments, followed by C2 (334.9 µV) and C3 (271.53 µV).

Compared to the CK, the EAG responses of winged aphids to C1, C2, C3, and C5 were significantly increased by 78.05%, 225.36%, 163.80%, and 69.83%, respectively, while the responses to C4, C6, C7, C8, and C9 showed no significant difference from the CK.

Thus, four terpenoids and two alkanes, namely C1 (octahydro-pentalene, Terpenes), C2 ((Z)-cyclooctene, Terpenes), C3 (dimethylstyrene, Terpenes), C5 (tetramethyl-heptadecane, Alkanes), C6 (tetrahydro-naphthalene, Terpenes), and C9 (heptacosane, Alkanes), which induced significant EAG responses in wingless and/or winged aphids, were selected for further Y–tube olfactometer dual-choice assays.

3.5. Behavioral Responses of Wingless and Winged Aphids to 6 Volatiles

In the Y-tube olfactometer dual-choice assays, both wingless and winged aphids exhibited significant responses to all volatiles, except for C5 (F = 13.34, df = 29, p < 0.05; F = 13.87, df = 29, p < 0.05). As shown in Figure 4A, heptacosane (C9) induced the highest reaction rate in wingless aphids, followed by (Z)-cyclooctene (C2), tetrahydronaphthalene (C6), and dimethylstyrene (C3). Octahydro-pentadiene (C1) and tetramethyl-heptadecane (C5) triggered the lowest reaction rates in wingless aphids. These results suggest that these six volatiles likely exert similar effects on the behavior of both wingless and winged aphids.

As shown in Figure 5A, no significant difference was observed between the effect of C5 (F = 0.51, df = 9, p = 0.494) and the CK. C1 (F = 19.68, df = 9, p = 0.002) and C6 (F = 7.97, df = 9, p = 0.022) exhibited significant repellent effects on wingless aphids. C2 (F = 725.50, df = 9, p < 0.01), C3 (F = 51.97, df = 9, p < 0.01), and C9 (F = 128.41, df = 9, p < 0.01) significantly attracted wingless aphids, with choice rates of 68.80%, 65.42%, and 28.94%, respectively.

As presented in Figure 5B, no significant differences were observed between the effects of C3 (F = 0.17, df = 9, p = 0.688), C5 (F = 0.06, df = 9, p = 0.807), and C6 (F = 0.75, df = 9, p = 0.413) compared to their respective controls for winged aphids. C1 (F = 19.34, df = 9, p = 0.002) significantly repelled winged aphids, with a choice rate of 57.30%. C2 (F = 122.69, df = 9, p < 0.01) and C9 (F = 272.97, df = 9, p < 0.01) significantly attracted winged aphids, with attraction rates of 66.03% and 61.62%, respectively.

These results indicate that C2 and C9 are strongly attractive to both wingless and winged aphids, whereas C1 is highly repellent to both aphid types. Furthermore, the response to these six volatiles was generally stronger in wingless aphids than in winged aphids.

In the Y-tube olfactometer assays, the selective ecoefficiency of each volatile was calculated based on the responses of both wingless and winged aphids to all volatiles, revealing significant differences in ecoefficiency (Figure 6) (F = 10.43, df = 29, p < 0.05; F = 10.89, df = 29, p < 0.05). As shown in Figure 6A, for wingless aphids, the selective coefficients of C1 and C6 were the lowest, at −0.14 and −0.12, respectively. In contrast, C2 and C9 had the highest selective coefficients, at 0.38 and 0.31, respectively, followed by C3 and C5.

As presented in Figure 6B, for winged aphids, the selective coefficient of C1 was the lowest (−0.15), with no significant difference between C1 and C3, or C5, or C6. C2 exhibited the highest selective coefficient of 0.32, followed by C9 at 0.20.

These results suggest that C2 and C9 are significantly attractive to both winged and wingless aphids. Although C1 and C6 exhibited repellent effects on wingless aphids, the responses were not significant compared to the control (Figure 5), and C6 had a lesser effect on winged aphids than on wingless aphids.

Previous studies have reported that alkane compounds, such as heptane, on the surface of Solena amplexicaulis flowers, significantly attracted Aulacophora foveicollis [33], and heptane in extracts of Bauhinia scandens showed insecticidal activity against Plutella xylostella [34]. Among the tested compounds, alkanes are considered important for aphid control, while terpenes also show potential for aphid management. Although octahydro-pentadiene (C1) and (Z)-cyclooctene (C2) are rarely used for pest control, their potential as repellents or attractants for A. gossypii requires further investigation.

4. Conclusions

This study elucidated the electrophysiological and behavioral responses of A. gossypii to GSE and its bioactive constituents. The results demonstrated that GSE exhibits significant repellent activity against both winged and wingless aphids, with the methanol extraction phase showing the highest efficacy. Subsequent chromatographic separation of the methanol phase yielded 12 fractions, among which fraction 4 induced the strongest EAG responses in both aphid morphs. GC-MS analysis of fraction 4 identified key compounds, including terpenoids, alkanes, aromatics, esters, and phenols, with octahydro-pentalene (C1), (Z)-cyclooctene (C2), dimethylstyrene (C3), tetramethyl-heptadecane (C5), tetrahydro-naphthalene (C6), and heptacosane (C9) being the most prominent. Behavioral assays using the Y-tube olfactometer further revealed that (Z)-cyclooctene (C2) and heptacosane (C9) acted as potent attractants, while octahydro-pentalene (C1) exhibited significant repellency toward both aphid forms.

These results underscore the dual role of ginger-derived volatiles in modulating A. gossypii behavior, suggesting their potential integration into sustainable pest management strategies. Specifically, the repellent properties of octahydro-pentalene could be leveraged to deter aphid colonization, whereas attractants such as (Z)-cyclooctene could serve as lures in trap-based control systems. The observed differential EAG and behavioral responses between winged and wingless aphids highlight the importance of considering morph-specific sensitivities when designing targeted interventions.

This study lays a foundational framework for developing ginger-based botanical pesticides, emphasizing the synergistic potential of terpenoids and alkanes in disrupting aphid-host interactions. Future research should focus on field validation of these compounds, optimization of formulation stability, and an assessment of their ecological impacts to ensure practical applicability. Ultimately, our findings contribute to the understanding of plant-insect chemical ecology and open new avenues for eco-friendly A. gossypii management.