Chemical composition and acaricidal properties of essential oils from Cinnamomum mollifolium, Eucalyptus globulus, and Pimenta dioica against Hyalomma rufipes (Acari: Ixodidae)

Abstract

Background

Hyalomma rufipes (H. rufipes) ticks are widespread in Africa, Europe, and Asia. They transmit crimean-congo hemorrhagic fever virus, rickettsia, and midichloria, posing a significant risk to public health and livestock development. Developing acaricides based on plant essential oils (EOs) have risen, yet matters regarding their mechanism and feasible methods for tick tests remain unanswered. The aim of this study was to identify the chemical constituents of three EOs and evaluate their acaricidal activity against larval and adult ticks of H. rufipes, as well as to establish an artificial membrane feeding technique to explore the feasibility of Cinnamomum mollifolium EO for tick control in vitro.

Results

In this study, gas chromatography-mass spectrometry (GC-MS) was used to analyze the constituents of C. mollifolium, Eucalyptus globulus, and Pimenta dioica EOs, as well as to investigate their potential application in H. rufipes control. The main compound in C. mollifolium EO is (E)-Cinnamaldehyde (93.15%). Eucalyptus globulus EO comprises eucalyptol (61.02%) and l-(+)-Ascorbic acid 2,6-dihexadecanoate (35.89%). Pimenta dioica EO predominantly includes eugenol (73.67%), 1,2-dimethoxy-4-(2-propenyl)-benzene (13.04%), and caryophyllene (7.24%). Larval packet tests revealed that the EOs of C. mollifolium, E. globulus, and P. dioica exhibited notable larvicidal activity, with lethal concentration (LC₅₀) values of 8.84, 51.16, and 34.58 mg/ml, respectively. Adult immersion tests indicated that the EOs of C. mollifolium, E. globulus, and P. dioica had significant acaricidal activity against adult ticks, with LC₅₀ values of 15.51, 43.96, and 29.83 mg/ml, respectively. Artificial membrane feeding technology enabled successfully in vitro rearing of adult male and female ticks with attachment rates of 83.4% and 75.0% respectively. The most effective EO (C. mollifolium) was applied on ticks after attachment to an artificial membrane, and showed acaricidal activity with high adult mortality at higher concentrations.

Conclusion

This study offers useful information on the chemical makeup and tick-killing ability of essential oils from C. mollifolium, E. globulus, and P. dioica against ticks. The acaricidal potency of C. mollifolium EO significantly surpasses that of both E. globulus and P. dioica EOs. The effective use of artificial membrane feeding technology also highlights its value for future tick control research and applications.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-05131-x.

Introduction

Ticks are capable of transmitting a wide range of viruses, bacteria, protozoa, and fungi that have a significant impact on human health and livestock development [1]. Hyalomma rufipes are two-host ticks that are widely distributed in Africa and the Inner Mongolia, Shanxi, Ningxia, and Xinjiang regions of China [2, 3]. Hyalomma rufipes tick-borne pathogens can cause crimean-congo haemorrhagic fever, Francisella, Rickettsia, and Midichloria [4, 5].

Currently, chemical acaricides and repellents are the main tools used to control ticks. In regions with high pest prevalence, farmers often use synthetic acaricides organophosphates, amidines, antiparasitic agents, phenyl pyrazolones, and growth inhibitors [6, 7]. These have caused serious environmental pollution, leading to tick resistance to these chemicals. These reasons have promoted the search for new plant-derived compound for use in tick control research [8]. Secondary metabolites in plants may have some botanical properties with acaricidal and tick repellent activity after extraction [9, 10]. The problem of insect resistance, and drug residues in the environment and food has been addressed using synthetic oils [11].

Plant essential oils (EOs), also known as volatile or aromatic oils, contain the volatile chemical components found in plants [12]. These oils usually consist of the natural metabolic secretions of aromatic plants and have a variety of biological activities including anti-inflammatory, antibacterial, antifungal, antioxidant and insecticidal [13]. These active ingredients are able to act through a variety of mechanisms, such as interfering with the insect’s nerve conduction system, disrupting body wall structure, inducing oxidative stress, and inhibiting the activity of key enzymes [14, 15]. In the field of tick control, available studies have shown that essential oils of different cinnamon varieties exhibit differential activity against a wide range of tick species. It is known to play a toxicological role in insects, and the essential oil of Cinnamomum verum and its main component cinnamaldehyde ((E)-cinnamaldehyde) have shown significant acaricidal activity against ticks of Haemaphysalis longicornis [16]. Cinnamomum cassia has high repellent activity against insects [17]. However, the acaricidal effect of Cinnamomum mollifolium on the Hyalomma rufipes has not been studied. In addition, the essential oil of Pimenta racemosa is toxic to all larvae, nymphs, and adults of H. longicornis [18]. Eucalyptus globulus EO has also been shown to act as a mosquito deterrent, providing maximum protection against arthropod bites [19, 20].

In the present study, the chemical composition of essential oils of Cinnamomum mollifolium, Eucalyptus globulus, and Pimenta dioica was determined. The acaricidal activity of three essential oils against unfed adults of H. rufipes was tested using both the immersion assay and larval packet test. The essential oil derived from C. mollifolium exhibits demonstrably superior acaricidal activity compared to both E. globulus and P. dioica EOs. The acaricidal activity of C. mollifolium EO was tested on a basal basis against H. rufipes biting on artificial membranes, revealing that these essential oils have potential for application as environmentally friendly industrial acaricides [21].

Materials and methods

Tick rearing

The ticks H. rufipes (larvae and adults) were collected from the vegetation in Wuhai County, Inner Mongolia Autonomous Region, northern China. Laboratory-reared tick strains were confirmed to be the target species by comparison of morphological characteristics by stereo microscope [22]. The local farmers have consented to the utilization of this tick species for experimental research purposes. Non-reared ticks lived in an incubator [27 ± 1℃ 80 ± 5% relative humidity (RH), 6:18 h (light/dark; L:D) photoperiod]. The unfed larvae and adults were placed in a gauze bag sewn to the back of the rabbit’s ear and allowed to suck blood naturally; the rabbit was housed in a room at 27 ± 1℃, RH 80 ± 5%, supplemented with natural light. The New Zealand white rabbits, which are individually farmed, have been authorized for use in scientific research. The animal feeding experiments were all approved by the Animal Ethics Committee of Hebei Normal University under approval number 2023LLSC040.

Essential oils

The essential oils of C. mollifolium, E. globulus, and P. dioica used in this study were purchased from global organic farms’ essential oil ingredients direct supply mall.

Gas chromatography-mass spectrometry (GC/MS)

The composition of these three EOs was determined using a gas chromatograph-mass spectrometer (Agilent^® 7890 A/5975 C, Agilent Technologies, Santa Clara, USA). GC conditions: column (DB-5MS, 30 m×0.25 mm×0.25 μm), carrier gas HE, split ratio 100:1, inlet temperature 300 °C, injection volume 1 µl, flow rate 1 ml/min, ramping program: initial temperature 60 °C (3 min), ramping up to 300 °C at 30 °C/min (5 min). Mass spectrometry conditions: the ion source was an EI source with an electron energy of 70 eV, the temperature of the ion source was 230 ℃, the temperature of the quadrupole was 150℃, the scanning mode: full scan, solvent delay of 2.9 min, and the scanning range of 40–550 amu.

The EOs were subjected to compositional analysis by gas chromatography-mass spectrometry (GC-MS) to obtain the total ion flow diagrams, and the mass spectra of the peaks were searched in the NIST 08 standard spectral library using a computer, and the chromatographic peaks were quantitatively analyzed by the area normalization method, and the relative percentage contents of the individual components were calculated.

Larval packet test (LPT)

The LPT method for young ticks was followed by Stone and modified by Leite (Leite, 1998). The EOs with original solubility of 1 g/ml were diluted to working concentration with 2% anhydrous ethanol. C. mollifolium, E. globulus, and P. dioica were selected as the initial concentration of essential oils at 25 mg/ml, 150 mg/ml, and 100 mg/ml, respectively, and then subjected to multiplicative dilution, and treatments of essential oils with no effect were not recorded. 30 larvae ticks were placed in a 4 × 4 cm filter paper packet (Whatman No. 1) and dipped by pipetting 0.8 ml of the prepared essential oil solution onto the packet for 5 min before transferring it to a new packet (72.25 cm²), which was sealed on three sides with small steel clips. After 24 h of incubation in an incubator (27 ± 1℃, RH ≥ 80%), the mortality of larvae was detected and the LC₅₀ was calculated. Three replications of the experiment were carried out, and anhydrous ethanol was used as the control group.

Adult immersion test (AIT)

The activity of EOs against unfed adults was evaluated using AIT. Essential oils were diluted with anhydrous ethanol to a working concentration, and 10 unfed adult (male: female; 1:1) H. rufipes were taken with tweezers and placed in a 5 ml centrifuge tube. The initial concentrations of C. mollifolium, E. globulus and P. dioica selected as essential oils were 50 mg/ml, 150 mg/ml and 75 mg/ml, respectively, followed by multiplicative dilution. Essential oils solution was pipetted into the centrifuge tubes to submerge the ticks, and after 5 min of treatment, the solution was aspirated, and the ticks were removed and placed on fresh filter paper. Ticks were blotted dry, placed in new centrifuge tubes, and incubated in an incubator (27 ± 1℃, RH ≥ 80%). The mortality of adults was detected and the LC₅₀ was calculated after 24 h. Three replications of the experiment were carried out, and anhydrous ethanol was used as a control group.

Artificial membrane feeding test (AMFT)

The experiment was conducted using aseptic defibrinated bovine blood for feeding ticks, and glucose was added at a ratio of 4 g/l aseptic defibrinated bovine blood. The blood was dispensed into 50 ml centrifuge tubes under aseptic conditions and stored at 4 ℃, protected from light. Gentamicin solution was prepared at a concentration of 10 mg/ml and placed at 4 ℃. Then, 0.9% NaCl solution was used to prepare the ATP solution. A ratio of 5 µl of gentamicin solution and 100 µl of ATP solution per 10 ml of blood was used. Put 10 g of freshly collected cow hair into a beaker, add 50 ml of dichloromethane to the beaker, mix the cow hair with the dichloromethane, and let it stand for 30 min. Dispense the liquid into a 50 ml centrifuge tube (3000 rm, 20 min, 4 ℃), remove the supernatant, and then pour it into a new beaker. Repeat the above steps once. The beaker was placed in a fume hood overnight for evaporation and this greasy substance was scraped off with a glass rod and the solution of bovine hair extract was configured with dichloromethane at a concentration of 42 mg/ml. Cling film was laid flat on a glass plate and secured with clear tape to ensure that there were no air bubbles. Mirror wipe paper was adhered to the cling film. Mix 00–10 Ecoflex silica gel components A and B in a 1:1 ratio, then add n-hexane in a 10:2 ratio and mix well. The mixture was evenly applied to the mirror wipe paper with a spatula and allowed to dry at room temperature. After drying, the thickness was measured with a spiral micrometer, and artificial films with a thickness between 70 and 110 μm were selected.

An artificial membrane feeding system was constructed using a clear Plexiglas tube (3.2 cm outer diameter, 2.8 cm inner diameter, 4.5 cm height, 0.2 cm wall thickness) and a 6-well cell culture plate. The height of the tubes was adjusted with a 3 cm diameter rubber ring. Mold Star 30 silica gel components A and B were mixed in a 1:1 ratio and applied to bond the Plexiglas tubes to the artificial membrane. After bonding and drying, the cling film was removed and one end of the plexiglass tube was immersed in 75% anhydrous ethanol to check for leaks in the artificial membrane. A leak-free feeding system was selected and set aside. Firstly preheat the sterile defibrinated bovine blood to 37 °C. Under aseptic conditions, bovine blood with ATP and gentamicin was added to a 6-well plate with 5 ml per well for a total of 3 wells. 75 µl of bovine hair extract was added dropwise to the surface of the artificial membrane in a plexiglass tube and allowed to dry. The experiment was divided into 4 groups of 3 plexiglass tubes each with 60 mg of H. rufipes feces and 5 pairs of unfed adult ticks, and the mouths of the tubes were sealed with cotton plugs to limit the space for the ticks to move. The tubes were placed in pre-warmed bovine blood 6-well plates and the rubber ring was adjusted so that the artificial membrane was immersed in 3 µl of bovine blood to avoid air bubbles. The results were observed after 48 h of feeding. Solutions were prepared with Mycophenolate, Dimethyl Sulfoxide (DMSO), and Dulbecco’s Phosphate-Buffered Saline (DPBS) in the ratio of 10:2:3.7. DMSO is commonly used to solubilize membrane components, to help construct artificial membranes, to facilitate the passage of certain drugs or molecules across membrane structures, and for the study of membrane permeability. DPBS is used to maintain pH and osmotic pressure stability in artificial membrane experiments, ensuring that experimental conditions are close to physiological. It can also be used to clean and dilute experimental materials to ensure the accuracy of experimental results. The sterile bovine blood and 6-well plates were replaced every 12 h and the surface of the artificial membrane was rinsed with a solution of mycobacteria and 0.9% sterile NaCl to prevent mold growth.

Simulation of C. mollifolium EO contact toxicity test

The EOs with the best lethal effect were screened to explore contact toxicity experiments of C. mollifolium EO on blood-sucking adults on artificial membranes. There were 5 groups in the experiment, and each group consisted of 3 plexiglas tubes. Each tube contained 10 unfed adult ticks, one of which served as the control group, and the remaining four groups were dosed with 0.5 µl, 1 µl, 2 µl and 4 µl of cinnamon oil (1000 mg/ml). Two days after the unfed ticks attach to the artificial membrane, the drug was carefully applied to the ticks’ body surface using a pipette with a slow accelerating rate, to completely cover the ticks’ body surface. The results were recorded 30 min, 1 h, 5 h, and 24 h after treatment with cinnamon essential oil, and each group of experiments was repeated 3 times.

Statistical analysis

The lethal concentration of 50% (LC₅₀), 95% confidence interval (95% CI), and statistical analysis were calculated using Polo Plus 1.0 software (LeOra Software, CA, USA) for the ticks. Statistical analyses were calculated using Probit regression with SPSS 22.0 (IBM, USA) p-values less than 0.05 were considered significant. Significance of differences between groups: chi-square was tested by Levene’s method, followed by one-way ANOVA (one-way ANOVA), and multiple comparisons were performed by Tukey HSD test (α = 0.05). The SPSS 22.0 software was used to calculate the coefficient of determination (R²), and regression equations were obtained by regression analysis. The GraphPad Prism 8.02 software (GraphPad Software Inc., CA, USA) was used to generate the graphs.

Results

Chemical composition of the essential oils

The chemical composition of the EOs detected is analyzed and screened for important constituents (Figure S1). Three chemical components were identified in the essential oil of C. mollifolium. The most abundant chemical component was (E)-Cinnamaldehyde, which accounted for 93.15% of the total composition of C. mollifolium EO. This was followed by 9-Methoxybicyclo[6.1.0]nona-2,4,6-triene and Phenacylidene diacetate, which accounted for 4.24% and 0.08% of the total constituents of the essential oil, respectively (Table 1).

Table 1.

Chemical composition of C. mollifolium

Peak	RT (min)	Constituent	RIa	RIb	Area (%)
1	5.48	Phenacylidene diacetate	1699	1697	0.08
2	7.5	(E)-Cinnamaldehyde	1142	1145	93.15
3	8.08	9-Methoxybicyclo[6.1.0]nona-2,4,6-triene	1035	1065	4.24
Total					97.47

RT: retention time. Area (%): Percentage of the chemical compound. RIa: Retention index calculated by linear interpolation relative to retention times of a standard mixture of n-alkanes (C7–C40) using an HP-5MS column. RIb: Retention index from literature

Nine chemical components were identified in the essential oil of E. globulus (Figure S2), among which 3 chemical components with a relative content of more than 1% were identified. The most abundant chemical components were eucalyptol and l-(+)-Ascorbic acid 2,6-dihexadecanoate, which accounted for 61.02% and 35.89% of the total constituents of the essential oil, respectively (Table 2).

Table 2.

Chemical composition of E. globulus

Peak	RT (min)	Constituent	RIa	RIb	Area (%)
1	5.24	Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, (ñ)-	945	943	0.4
2	5.58	4,7,10,13,16,19-Docosahexaenoic acid, methyl ester, (all-Z)-	2525	2523	0.14
3	5.93	Cyclohexene, 1-methyl-4-(1-methylethylidene)-	1080	1083	1.42
4	6.06	Eucalyptol	1050	1050	61.02
5	6.22	3-Carene	1038	1040	0.32
6	6.84	(-)-Camphor	1141	1121	0.21
7	8.31	Gedunin	3325	3311	0.16
8	8.66	1 H-Indene,2,3,3a,4-tetrahydro-3,3a,6-trimethyl-1-(1-methylethyl)-	1378	1394	0.25
9	12.2	l-(+)-Ascorbic acid 2,6-dihexadecanoate	4631	4675	35.89
Total					99.81

RT: retention time. Area (%): Percentage of the chemical compound. RIa: Retention index calculated by linear interpolation relative to retention times of a standard mixture of n-alkanes (C7–C40) using an HP-5MS column. RIb: Retention index from literature

Nine chemical components were identified in the essential oil of P. racemosa (Figure S3), of which 5 chemical components with relative contents of more than 1% were identified. The most abundant chemical components were eugenol, 1,2-dimethoxy-4-(2-propenyl)-benzene, and caryophyllene, which accounted for 73.67, 13.04, and 7.24% of the total constituents of the essential oil, respectively (Table 3).

Table 3.

Chemical composition of P. racemosa

Peak	RT (min)	Constituent	RIa	RIb	Area (%)
1	5.84	à-Phellandrene	994	1002	0.86
2	6.05	Eucalyptol	1028	1025	1.59
3	7.01	L-4-terpineol	1155	1137	0.32
4	7.33	Chavicol	1250	1254	0.46
5	7.87	Eugenol	1364	1364	73.67
6	8.05	1,2-dimethoxy-4-(2-propenyl)-benzene	1314	1407	13.04
7	8.26	Caryophyllene	1500	1494	7.24
8	8.42	à-Caryophyllene	1440	1448	0.88
9	8.65	Cadina-1(10),4-diene	1462	1469	1.14
Total					99.2

RT: retention time. Area (%): Percentage of the chemical compound. RIa: Retention index calculated by linear interpolation relative to retention times of a standard mixture of n-alkanes (C7–C40) using an HP-5MS column. RIb: Retention index from literature

Effect of essential oils on larvae and adults

The acaricidal efficacy of three different concentrations of EOs on unfed larvae and adult H. rufipes was evaluated by the method of test (Table 4). The EOs with the most significant results in significant acaricidal activity unfed larvae H. rufipes was C. mollifolium EO. The LC₅₀ of the essential oils of C. mollifolium, P. racemosa, and E. globulus were 8.84, 34.58, and 51.16 mg/ml, respectively. As the concentration of essential oils decreased, the mortality of larval ticks was progressively reduced (Fig. 1). The most significant acaricidal activity against unfed adult H. rufipes was also C. mollifolium EO. The LC₅₀ values of C. mollifolium, P. racemosa, and E. globulus were 15.51 mg/ml, 29.83 mg/ml, and 43.96 mg/ml, respectively. At the same concentration, C. mollifolium was more acaricidal than P. racemosa and E. globulus essential oils against adult ticks (Fig. 2). Tick mortality increased progressively with increasing EO concentrations. At the same concentration, C. mollifolium EO had a stronger acaricidal activity on larvae than on adult ticks. The finding that C. mollifolium EO showed optimal acaricidal activity in all treatments points the way to a deeper investigation of its effects.

Table 4.

Contact toxicity of essential oils against H. rufipes

Stage	Treatment	N	DF	Regression eq. (Y=)	LC50 (CI 95%)	R 2
Unfed larvae	C. mollifolium	1200	3	17.043 + 2.727x	8.84a (6.80–11.28.80.28)	0.85
	E. globulus	1800	5	18.408 + 0.415x	51.16b (36.01–80.70)	0.83
	P. racemosa	1500	4	14.875 + 0.705x	34.58b (25.18–49.98)	0.86
Unfed adults	C. mollifolium	120	3	12.319 + 1.786x	15.51a (12.70–18.58.70.58)	0.82
	E. globulus	150	4	20.972 + 0.442x	43.96b (27.49–73.13)	0.84
	P. racemosa	120	3	8.116 + 1.073x	29.83b (21.77–41.39)	0.92

Note: LC₅₀ = lethal concentration (mg/mL) for 50% of individuals; CI = confidence interval; R² = correlation coefficient; The data followed with different letters are significantly different at P < 0.05

Fig. 1.

Mortality of three essential oils against unfed larvae H. rufipes (Tukey’s honest multiple comparison test, *P < 0.05, **P < 0.01 ***P < 0.001)

Fig. 2.

Mortality of three essential oils against unfed adults H. rufipes (Tukey’s honest multiple comparison test, *P < 0.05, **P < 0.01 ***P < 0.001)

Artificial membrane feeding test

Ticks successfully attached into artificial membranes to suck blood, with attachment rates as high as 70% (Fig. 3). The results of the four groups showed that male ticks in the second and third groups had the highest attachment rate, which was 86.7% in both groups. Male ticks in groups 2 and 4 had the highest attachment rate with 80.0%. Female ticks in group 2 had the highest attachment rate of 80.0%. Female ticks in groups 1, 3, and 4 had the lowest attachment rate, with a bite rate of 73.3%.

Fig. 3.

A: Number of attached ticks; B: Tick attached rate (%)

Simulation of C. mollifolium EO contact toxicity test

After recording the attachment rate of blood sucking adult ticks on artificial membranes, the acaricidal activity against ticks was observed and recorded after treating the ticks with different doses of C. mollifolium EO (Table 5).

Table 5.

The mortality rate of H. rufipes attached to the artificial membrane treated with C. mollifolium EO

Dose (µl)	Number of ticks	Attached ticks	Attatchment rate (%)	Mortality (%) ± SEM (Time)
				0.5 h	1 h	5 h	24 h
0	30	23	76.7 ± 3.3	0.0 ± 0.0a	0.0 ± 0.0a	0.0 ± 0.0a	0.0 ± 0.0a
0.5	30	24	80.0 ± 5.8	4.2 ± 4.2ad	16.8 ± 4.2be	58.4 ± 2.1bf	74.7 ± 1.9bg
1	30	22	73.3 ± 3.3	8.9 ± 4.5abd	22.6 ± 4.3bcd	63.7 ± 4.2bce	81.5 ± 5.1bcf
2	30	20	66.7 ± 6.7	15.3 ± 1.4bd	29.2 ± 6.4bcd	65.3 ± 9.7bce	84.7 ± 9.7bce
4	30	21	70 ± 5.8	18.7 ± 3.2bd	33.1 ± 2.6ce	76.6 ± 3.5cf	95.2 ± 4.8cg

Note: Different letters represent significant differences between the experimental and control groups using one-way analysis of variance (ANOVA, P < 0.05)

In this experiment, there was a significant difference in the 0.5 h, 1 h, 5 h, and 24 h test results compared to the 0 µl effect for 0 mg, 0.5 mg, 2 mg and 4 mg. Significant difference was observed in the results of 1 h, 5 h, and 24 h tests at different concentrations at the same time, 0.5 µl as compared to 4 µl. It indicates that the mortality rate of C. mollifolium EO treated H. rufipes increased with the concentration. Significant differences were found among the four groups of 0.5 h, 1 h, 5 h, and 24 h for 0.5 µl, 1 µl, 2 µl and 4 µl of the same concentration at different times. This suggests that mortality of H. rufipes treated with C. mollifolium EO increased over time.

Discussion

Plant essential oils are derived from secondary metabolites of plants and have a wide range of biological activities that can affect insects in a variety of ways, including toxicity, fumigation, repellency, food repellency, inhibition of growth and development [23]. Tick control with natural products is a viable approach that reduces tick resistance and the problem of pesticide residues in the environment [24]. Therefore, assessing the toxicity EOs against ticks to optimize their use in tick control management is of critical environmental and public health importance.

In the present study, the chemical composition of EOs from C. mollifolium, E. globulus, and P. dioica was characterized. The main chemical composition of the C. mollifolium EO in this study was similar to that of C. zeylanicum and C. cassia [25]. (E)-Cinnamaldehyde (93.15%) was identified as the major component. Phenacylidene diacetate and 9-Methoxybicyclo[6.1.0]nona-2,4,6-triene have not been found in other cinnamon EOs [26]. The acaricidal activity of cinnamon was positively correlated with its high content of (E)-cinnamaldehyde [27]. (E)-Cinnamaldehyde is a natural compound with a wide range of biological activities as well as remarkable environmental friendliness. It has been shown to exhibit significant repellent and acaricidal activity against ticks [28], providing an efficient and environmentally friendly solution for tick control [29].

The principal compounds identified in E. globulus EO were eucalyptol (61.02%), l-(+)-Ascorbic acid 2,6-dihexadecanoate (35.89%), and cyclohexene, 1-methyl-4-(1-methylethylidene)- (1.42%). The eucalyptol content of Eucalyptus globulus was previously reported to be 63.1% in studies, and the percentage of eucalyptol as its main component fluctuates between 32.19% and 86.51%, which is widely recognized and cannot be disputed [30, 31]. Eucalyptol may be responsible for the acaricidal activity, which may act by inhibiting the detoxifying enzyme cytochrome P450 or by interfering with physiological functions [32]. Eucalyptol has been reported to inhibit acetylcholinesterase (AChE) in Rhipicephalus microplus larvae [33].

Compound analysis in P. racemosa EO revealed that the main components are eugenol (73.67%), 1,2-dimethoxy-4-(2-propenyl)-benzene (13.04%), caryophyllene (7.24%), eucalyptol (1.59%), and cadina-1(10),4-diene (1.14%). Eugenol substance can be present in very high concentrations in essential oils, sometimes more than 50% [34]. Eugenol is a natural phenolic compound with antibacterial, anti-inflammatory, and analgesic properties [35]. Eugenol also inhibits complex I activity of the mitochondrial respiratory chain in the oxidative phosphorylation pathway by binding to NADH dehydrogenase chain 2, leading to mite death [36]. Undiluted eugenol is lethal to poultry red mites (Dermanyssus gallinae) [37]. Differences in raw material sources, extraction plant organs, and extraction methods of EOs may affect their chemical composition and biological activities. Therefore, understanding the toxicological effects of individual components and combinations of essential oils (EOs) on insects is essential for the development of novel and environmentally friendly pest control strategies.

Plant essential oils are potentially toxic because of their complex composition, which can interfere with tick respiration, nerve, and energy metabolism [38–40]. At present, there are relatively few reports on the use of essential oils to control H. rufipes. The only known in vitro anti-tick effect is that of Tagetes minuta L. (Asteraceae) on H. rufipes [41]. Therefore, the present study investigated the effectiveness of acaricidal essential oils against unfed ticks in order to provide an experimental basis for developing environmentally friendly tick control agents.

The three plant EOs with the most significant acaricidal activity against unfed H. rufipes, both larvae and adult, were C. mollifolium EO with LC₅₀ of 8.84 mg/ml and 15.51 mg/ml. Low concentrations of (E)-cinnamaldehyde in Cinnamomum zeylanicum showed strong acaricidal activity against R. microplus [42]. Chinese cinnamon oil showed significant repellent activity against H. longicornis, Rhipicephalus haemaphysaloides, and Hyalomma asiaticum wakame, and significant poisoning activity against larvae and adults H. longicornis [16, 43, 44]. (E)-cinnamaldehyde, the main component of C. mollifolium, has direct experimental evidence of acaricidal activity. Cinnamon essential oil stimulates the detoxification enzymes glutathione S-transferase, acetylcholinesterase, carboxylesterase, and catalase in H. longicornis [26]. C. mollifolium similarly elicits a detoxification metabolic response in ticks.

Myrtaceae E. globulus and P. dioica EOs had weaker acaricidal activity than C. mollifolium EO. However, E. globulus and P. dioica EOs showed weaker acaricidal activity against adults than larvae, and higher contact concentrations were required for larvae. E. globulus EO has acaricidal activity against adult Rhipicephalus annulatus [45]. The significant toxicological effect of P. racemosa and a single component, eugenia caryophyllata, on H. longicornis was verified in a previous experiment [18]. 1,2-dimethoxy-4-(2-propenyl)-benzene and eugenol accounted for the highest percentage in this experiment. Eugenol has been used severely against R. annulatus [46], and their synthetic mixtures hold great promise for tick control [47].

To understand the detrimental effects of EOs on non-target species, control experiments were conducted using artificial membrane-reared ticks [48]. This method is particularly suitable for pathogens that are difficult to study through conventional animal feeding systems, as it mimics the blood sucking behavior of ticks under natural conditions [49]. Artificial membrane rearing techniques can be used to assess the effects of compounds or microorganisms on ticks, as well as to study the physiological and ecological characteristics of ticks. Animal models are still necessary to assess tick-host interactions, and this is the first time that the artificial membrane technique has been used to successfully rear H. rufipes. Due to the complex rearing conditions when rearing hard ticks, natural biting conditions can be achieved using animal skin as a membrane [50]. In this study, membranes were prepared using materials such as silica gel, and ticks were successfully cultured to an engorgement state. The length of the mouthparts of different tick species varies, so the thickness of the artificial membrane must be adapted to improve the bite rate. Lewis et al. successfully reared adult ticks of Ornithodoros tartakovskyi using a modified artificial membrane rearing system, achieving a bite rate of 85% for males and 92% for females [51]. Adult Amblyomma americanum is reared with bite rates of 50% to 75% [52]. In the present study, we successfully reared unfed adult H. rufipes by improving the rearing conditions of the previous experimental method in conjunction with the above findings [53]. Biting rates of 83.4% for male ticks and 75.0% for female ticks were achieved in the experiment. Further studies can be conducted on this basis.

Based on the successful rearing of unfed adult H. rufipes on artificial membranes, the feasibility of the artificial membrane technology was further verified by incorporating experiments using EOs to contact ticks. Droplets of C. mollifolium EO, which has significant acaricidal activity, were added to the dorsum of deaf glaucoma tick biting on an artificial membrane to test the actual tick contact effect. The results were analyzed and it was concluded that the lethality of C. mollifolium EO against ticks was positively correlated with increasing the dose of essential oil. Tick mortality was 58.4% after 5 h of treatment with 0.5 µl of C. mollifolium EO. Tick mortality was 95.2% after 5 h of treatment with 4 µl of C. mollifolium EO. This suggests that C. mollifolium EO achieves some acaricidal effect on H. rufipes that bite on artificial membranes. This approach is feasible to rule out a complex immune relationship between the host animal and the tick and also reduces the use of host animals.

The limitations of this study are that the specific components of the essential oils with acaricidal activity and their targets of action have not been elucidated, and reproductive parameters have not been examined; these gaps will be filled in other follow-up work through sub-lethal dose exposure and transgenerational effects experiments. The potential risk of essential oils to non-target pests has not been assessed, and the differences in attachment rates between male and female ticks and their coping strategies have not been explained; the exact mechanism of action of the chemical components and the optimization of the ratios need to be further elucidated. Future research will be centered on component-activity correlation, environmental safety evaluation, and formulation engineering to promote the movement of plant essential oils from the laboratory to sustainable tick control practices in the field.

Conclusions

In this study, C. mollifolium, E. globulus, and P. dioica EOs were demonstrated to have significant acaricidal activity against H. rufipes. In vitro rearing of H. rufipes with the artificial membrane technique has been successful and can be used for subsequent drug testing on ticks. The 24 h mortality of C. mollifolium true contact ticks was modeled. The integration of artificial membrane rearing techniques with the screening of tick control drugs allows for better observation of tick physiology. This study provides a reference for screening tick-control drugs using artificial membrane reared ticks.

Author contributions

Xiaolong Yang: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Investigation. Songbo Zhang: Writing – original draft, Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation. Jingyao Gao: Writing – original draft, Validation, Supervision, Software, Methodology. Ruya Cao: Investigation. Weijia Xing: Methodology. Jiayi Zhang: Validation. Weikang Zhao: Software. Xinyu Zhang: Investigation. Zhihua Gao: Writing – review & editing, Validation, Funding acquisition, Supervision, Software, Project administration, Methodology, Investigation.

Funding

This work was supported by the Natural Science Foundation of Hebei Province (C2023205011), the Foundation of Hebei Normal University (L2023B56), and the National Natural Science Foundation of China (31472050).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

All experiments were performed in accordance with relevant guidelines and regulations. The animal feeding experiments were all approved by the Animal Ethics Committee of Hebei Normal University under approval number 2023LLSC040. The animals used in this study were obtained from a private source and the owners have given informed consent for their animals to be used in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.