Introduction

Mosquitoes are considered as important medical insect pests worldwide, and they, as vectors, transmit many diseases including malaria, filariasis, yellow fever and dengue fever1,2,3. Malaria has been reported as the most common mosquito-borne disease and present a great threat to human life mainly spread by Anopheles mosquitoes4. Every year, approximately 250 million people have been reported being infected with malaria, which has caused over one million deaths worldwide5. Anopheles sinensis is the No.1 vector of malaria in China6. The control of this vector is mainly managed with massive applications of various insecticides. However, the over uses of these toxic chemicals have resulted in strong insecticide resistance7,8.

Currently for mosquito control in China, pyrethroids are primary chemical insecticides being heavily used. N,N-Diethyl-3-methylbenzamide (DEET) is the most commonly repellent used for the prevention against An. sinensis. However, extensive use of pyrethroids has caused increasing insecticide resistance, and also created adverse effects on human health and their surroundings9,10,11. The toxicology of DEET has been more closely investigated and has found issues in safety for human use, including use on children, pregnant women, and lactating women12,13,14. There is an urgent need to develop alternative human and environmentally friendly mosquito control strategies. In recent years, natural products have gradually become the primary sources of novel mosquito repellents and larvicides due to low-toxic, quick biodegradation rates, and less side effects on hosts and non-target organisms15,16,17. Plant essential oils, extracted from Nepeta cataria, Severinia monophylla, Syzygium aromaticum and Cymbopogon citratus have been reported to be effective in repelling mosquito adults18,19,20. Besides, some of them have also exhibited larvicidal activity, including those from Laurencia dendroidea, Cunninghamia konishii and Azadirachta indica21,22,23.

The Chinese mugwort plant, Artemisia argyi, is a traditional medicine plant widely distributed in southern provinces with a long cultivation history. Extracts of this plant have been reported to possess immunomodulatory, neuroprotective insecticidal properties with effectiveness against bacteria, dampness, hemostasis, cancer, and inflammation24,25,26. Mugwort plants have been widely used to repel mosquitoes by burning the harvested dried leaves for thousands of years at countryside in China27. The essential oil of those plant leaves exhibits strong contact fumigant toxicity against Lasioderam serricorne adults28. The oils of four subspecies of mugwort plants including Ar. feddei, Ar. gmelinii, Ar. manshurica, and Ar. olgensis deterred mosquito biting of adult Ae. aegypti29. The essential oil of Ar. vulgaris provided 100% mortality against Ae. aegypti larvae after 8-h exposure at a concentration of 500 ppm.

In the study, we extracted the essential oils from plants of Ar. argyi collected from seven provinces/municipal areas using a steam distillation technique, and investigated their larvicidal, fumigant and repellent activity against An. sinensis. We also analyzed their chemical compositions using gas chromatography mass spectrometer (GC–MS), and evaluated the toxicity of four common predominant compounds of these oils against An. sinensis. The results from this study may provide some new insights for further research in use of the mugwort essential oil as an alternative and targeted control and preventative measure against Anopheles mosquitoes.

Results

Insecticidal and repellent activity of distilled Ar. argyi essential oils

The essential oil of Hubei province (HB) showed the highest larvicidal effect with a median lethal concentration (LC50) value at 40 μg/mL, followed by oils from mugwort plants collected in Chongqing (CQ) (LC50 = 49 μg/mL), Sichuan (SC) (LC50 = 54 μg/mL), Hainan (HN) (LC50 = 56 μg/mL), Gansu (GS) (LC50 = 55 μg/mL) and Shandong (SD) (LC50 = 59 μg/mL) and Yunnan (YN) (LC50 = 62 μg/mL) (Table 1). The larvicidal activity of all seven essential oils are significantly effective against An. sinensis in comparison of control in which no dead larvae were observed.

Table 1 Larvicidal activity of essential oils distilled from seven geographical regions of mugwort leaves in China against the 4th instar larvae of An. sinensis.
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All seven oils showed significant repellent activities against female An. sinensis adults compared to the negative control (Fig. 1). Similar levels of repellency as DEET were demonstrated from 4 oils (CQ, HN, SD and GS), with the highest from GS oil. The mean % of repellent longevity from seven mugwort essential oils were presented in Table 2. The essential oil extracted from leaves collected in GS provided almost 66% of protection against mosquitoes at the 65th minutes post application, which was similar to the effectiveness found from 10% DEET.

Figure 1
figure 1

The first 5-min observed landing rates of starved An. sinensis on human skins treated with seven Ar. argyi essential oils extracted from different geographical areas. The significances between each treatment and control group are less than 0.0001 (p < 0.0001). Different letters on each treatment bars indicate significantly different between among the seven essential oils tested and the positive control, DEET (ANOVA).

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Table 2 The percentages of repellencies of seven Ar. argyi essential oils and DEET against the four-day old An. sinensis adults during different exposed times.
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Fumigant toxicity of distilled Ar. argyi essential oils

The median knockdown time (KT50) ranged from as short as 4.7 min from essential oils from GS, to over 50 min of oils from YN province (Table 3). Similar trends appeared also in the median lethal dose (LD50), which oils of GS showed the strongest toxicity against the female adults of An. sinensis, and the least from oils of YN. The essential oil from GS has the highest fumigant toxicity with a LD50 value at 9.40 μL/L (Table 4).

Table 3 Median lethal knockdown activity (mins) of the essential oils extracted from seven geographical regions of Ar. argyi against female adults of An. sinensis.
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Table 4 Fumigation toxicity of the essential oils extracted from seven geographical regions of Ar. argyi in China against female adults of An. sinensis.
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Chemical compositions of Artemisia argyi essential oils

In total, 56–87 chemical constituents were identified from Ar. argyi essential oils from seven geographical origins, with the highest number of compounds (87) found in the oil from Gansu province (Table 5). Four chemical compounds, β-caryophyllene, caryophyllene oxide, eucalyptol and phytol were the most abundant and detected from all seven oils. There were significant differences in compositions of various compounds among oils extracted from seven different regions. Eucalyptol was found over 20% in GS essential oils, but only 5.5% from YN province. Only one oil from HB province contained over 16% phytol, with less than 10% from oils of the rest of regions. Another monoterpenoid, thujone, was detected over 16% from the SC oil.

Table 5 GC–MS analyses of compositional compounds of the essential oils extracted from seven regions of Ar. argyi.
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Larvicidal and fumigant toxicity of the 4 dominant compositional compounds

All four individual compounds showed significant toxicity against both larvae and adults of An. sinensis (Tables 6 and 7). Phytol was the strongest larvicidal compound, with the LC50 at 16.03 μg/mL. Significant larvicidal activity was also found from caryophyllene oxide (LC50 = 39.09 μg/mL), relative to those of eucalyptol and β-caryophyllene (Table 6). Eucalyptol exhibited the highest fumigant lethal activity against adult mosquitoes among the four tested compounds with the shortest time to kill at the LT50 value of 5.48 μL/L.

Table 6 Larvicidal activity of the four individual compounds mostly found from the seven essential oils of Ar. argyi in China against the 4th instar larva of An. sinensis.
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Table 7 Fumigation toxicity of the four individual compounds mostly found from the seven essential oils of Ar. argyi in China against the female adults of An. sinensis.
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Discussion

It is not surprising that the essential oils of the mugwort plant leaves have shown strong larvicidal activity against An. sinensis, as Wang30 have previously reported that Ar. argyi essential oil exhibiting larvicidal activity against Culex mosquitoes. Interestingly, significant differences in larvicidal activity have been demonstrated among the Ar. argyi oils from 7 different geographic regions, which may result from various quantities of some terpene and monoterpenoid compounds as their major compositional constituents31. Eucalyptol, β-caryophyllene, caryophyllene oxide and phytol, the four most common compounds found from all distilled oils in seven geographic origins, have been reported with moderate to strong larvicidal activity against various mosquito larvae. For instance, phytol has been reported with larvicidal activity against Aedes and Culex mosquitoes as one of compositional compounds from several essential oils32,33,34,35. From the distilled mugwort leaf essential oils, phytol is also one of the most dominant compositional compounds (6.3–16.3%). Eucalyptol is another most abundant compound ranged from 5–22% among all identified constituents. The essential oil from HB displays the highest larvicidal activity with over 52% of eucalyptol and phytol presence. Eucalyptol also shows larvicidal activity against larvae of An. anthropophagous, LC50 values from 45–50 μg/mL36. However, eucalyptol has displayed a relatively weaker larvicidal activity at LC50 of 155 ug/mL, which is one third of what reported from above mentioned studies and ten times less than that of phytol (LC50 = 16 μg/mL) in the present study. Interestingly, a relatively strong activity of caryophyllene has been demonstrated (LC50 = 39 μg/mL). Similar findings of weak larvicidal activity from eucalyptol identified from oil of Ar. gilyescens have been reported against An. sinesis with LC50 > 200 μg/mL37. Although we have shown that β-caryophyllene is a much weaker larvicide than that of caryophyllene oxide against An. sinesis in the present study, Govindarajan et al.38 found that β-caryophyllene from Plectranthus barbatus essential oil performed a strong larvicidal activity against Aedes and Culex mosquito larvae, and larvae of An. subpictus. Furthermore, Zhu and Tian37 have reported a strong larvicidal activity against An. anthropophagus, with only 5% of caryophyllene oxide constituently from the essential oil of Ar. gilvescene. Obviously, synergistic effects may play additional roles to enhance the larvicidal activity from various essential oil constituent compounds.

The moxibustion therapy of using Ar. argyi has been considered as a form of complementary or alternative medicine existing in the holistic system of health care and healing in many parts of Southeast Asia39. Burning of Artemisia dried leaves has been widely used to repel mosquitoes by minority people living in remote areas in southern provinces of China40. Among the four most dominant constituent compounds, eucalyptol has been reported acting as a strong repellent against various species of adult mosquitoes41. In the present study, the essential oil extracted from Gansu province has demonstrated a high level of protection as found from DEET, which could be contributed by containing over 22% of eucalyptol among other compositional compounds. Eucalyptol, also named 1,8-cineole, has been reported as a strong mosquito repellent identified from many species of Eucalyptus, Ocimum and Lippia15,31. Fumigant test with individual compound (eucalyptol) alone has further supported its strongest toxicity against adult An. sinensis mosquitoes. Phytol, a linear dipterpene alcohol, has been reported with high repellent activity against An. gambiae and Ae. aegypti42,43. β-Caryophyllene identified from essential oils of seven different provinces only provides low level of repellent activity and fumigant toxicity, which similar results have been reported from essential oils of Nepeta cataria, Lippia camara and Lippia cheraliera etc.44,45. A detailed literature review by Mathew and Thoppil32 has shown that caryophyllene oxide is detected from many essential oils with probable cause for high mosquito larvicidal activity. For instance, caryophyllene oxide is a major constituent compound in essential oils of Lippia gracilis and Hyptis pectinate with the potent insecticidal activity against Aedes aegypti larvae46. Strong larvicidal activity was also demonstrated from the essential oil of Ar. argyi.

The potential of using essential oils or their constituents as fumigants needs to be explored more thoroughly. In southeastern Asia remote areas and Latin American rural regions, the use of various fumigations for insect repelling and disease vector control has been widely reported47,48. Many monoterpenes (eucalyptol), diterpenes (phytol), sesquiterpenes (caryophyllene) as discovered in Ar. argyi essential oil have been reported possessing fumigant toxicity49. All of these are supported by the strong fumigant toxicity and the shortest knockdown time found from Ar. argyi essential oils extracted from Gansu province. Three other species of Artemisia essential oils also have the fumigant activity against mosquitoes, including Ar. scoparia, Ar. capillaries and Ar. carvifolia50.

The chemical composition and content of essential oils of plants are related to the diverse climate51. In addition, various elements in the soil also contribute to the differences in the chemical composition of Ar. argyi essential oil. For instance, the content of calcium, magnesium, manganese and nickel in Hubei Ar. argyi essential oil were higher, the elements of Nickel, cobalt, chromium and zinc in Sichuan Ar. argyi essential oil were relatively abundant, and the content of copper and cobalt in Henan Ar. argyi essential oil were also higher52. It was found that phosphorus content in soil affected plant growth, quality and chemical composition content53. The content of Angelica sinensis volatile oil was 0.65% from Gansu, 0.59% from Yunnan and 0.29% from Sichuan, which were directly related to the longitude and latitude of different regions54. In addition to the reasons above, the altitude and water quality may also be another key factor of the chemical composition and content of Ar. argyi essential oil55, which need further discussion.

This research demonstrates the strong larvicidal activity, fumigant toxicity and repellent effect of Ar. argyi essential oils of seven different geographic origins in southern provinces of China against larvae and adult An. sinensis mosquitoes. Eucalyptol and phytol are the most abundant constituent compounds and most active compounds among others. Additional minor compounds detected from these oils may enhance insecticidal, repellent and fumigant effects as well, which further studies need to be carried out. Essential oils that constitute an important alternative to conventional insecticides are largely due to their selectivity (high toxicity for mosquitoes but not for other co-existing organisms) and their minimal environmental effects56. Essential oils and their constituents, such as monoterpenes, have been widely used as fragrances in cosmetics, food additives, household products and medicine. Many of them are generally recognized as safe by the U.S. Food and Drug Administration (FDA). The outcomes from this study have provided new insights in use of phytochemicals derived from various botanical sources for more targeted mosquito control.

Materials and methods

Chemicals

Caryophyllene oxide (> 90%) was obtained from Shanghai Yuanye Technology Co., Ltd. of China. N,N-Diethyl-3-methylbenzamide (DEET, 99%), eucalyptol (99%), β-caryophyllene (> 80%) and phytol (90%) were all purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). The alkanes analytic standards (C5–C25 Hc,), reagent brand: o2si were purchased from ANPEL Laboratory Technologies (Shanghai, China), with over 99% purity.

Mosquitoes

Anopheles sinensis was originally collected from Jiangsu province in China, and reared in the Institute of Insect and Molecular Biology, Chongqing Normal University, for more than 40 generations. Mosquitoes were reared at a 12:12 light/dark photoperiod, 65 ± 5% relative humidity under 27 ± 1 °C, which larvae were fed with fish food and adults were provided with 10% glucose solution. The 4th instar of larvae and the four-day old adult mosquitoes were used for larvicidal, fumigant and repellent activity assays in this study. All experimental methods of this study are carried out in accordance with relevant guidelines and regulations, and The Ethical Committee of Chongqing Normal University approved the hunan-bait assays performed in the study.

Plant material collections and essential oil extraction from Artemisia argyi

The plant Ar. argyi is widely distributed in field in China. All experimental materials of Ar. argyi were collected on public lands in field at each location of seven provinces in China from September to October in 2018, and the plant material collecting did not require permission or other relevant certificates. The wet weights of collected aerial branches and leaves were 70 kg from 25 m2 of wasteland in Chongqing, 65 kg from 100 m2 of wasteland in Sichuan, 46 kg from 200 m2 of mountain area in Yunnan, 40 kg from 100 m2 of agriculrural farm in Hebei, 78 kg from 35 m2 of agricultural field in Henan, 50 kg from 150 m2 of mountain area in Shangdong, and 55 kg from 150 m2 of mountain area in Gansu, respectively. These seven provinces could represent the main distribution of the Ar. argyi plant in China in term of geography and climate. The collected Ar. argyi was dried in the collecting locations, and then sent to laboratory at Chongqing Normal University. The samples of Ar. argyi plants from each location were identified by the plant taxonomist Prof. Hai He at the Chongqing Normal University where we work, and the specimens are stored in the Herbarium of Chongqing Normal University with following information as attached label: collecting time, collecting location, collector's name, specimen number, species name, and identifier’s name. The dried Ar. argyi was cut to pieces at a size of about 10 cm in diameter by guillotine, and the essential oil of Ar. argyi was extracted using 6 kg of dried material per location. The extraction lasted for six hours using a steam distillation apparatus, and oil–water mixture was separated by a separatory funnel with an addition of NaCl as described in Joshi et al.57. The upper layer from the separated essential oils were taken, and then dried with added anhydrous sodium sulfate58. The purified oils were stored in brown reagent bottles at 4 °C prior to bioassays and chemical analysis.

Larvicidal activity assay

The larvicidal activity of Ar. argyi essential oils and their four dominant compounds against An. sinensis larvae was tested according to the method introduced by Nyamoita59. The range of lethal doses for the investigated samples was determined using the doses at 20, 40, 60, 80, 100 and 120 μg/mL, respectively, and a total of three replicates were conducted. Ethanol was used as the control. Different dosages of samples were added to a 50-mL cup containing a total of 20 mL of dechlorinated water. Twenty 4th instar larvae were then gently moved to the cup and the bioassay was performed under room temperature at 27 ± 1 °C with an 80 ± 10% relative humidity. The number of larvae with no movement (after touching) were considered as dead and were counted after 24 h of exposure.

Fumigant toxicity assay

The fumigant activity of Ar. argyi essential oils and their four dominant compounds against An. sinensis adults was investigated using methods described in Jiang et al.60, with some changes. Twenty female An. sinensis adults at the age of 4-day old were released into a 250 mL of Erlenmeyer flask sealed using a rubber stopper with a predrilled hole (diameter = 0.4 cm) for a 15 min of acclimatization. A piece of filter paper (1 × 3 cm) was then placed into the flask. The filter papers were applied testing materials at doses of 2, 4, 6, 8, 10, 12 μL of essential oils. After one hour of exposures, mosquitoes were then transferred to cages for 24-h provided with cotton balls containing 10% glucose. The number of dead mosquitoes was counted. For the fumigant knockdown test, the dose of testing material was 2 μL61, the knockdown number were recorded every five minutes until reaching the first hour. The time of mosquitoes observed falling down at the bottom were considered as the knocked down time, no any movement was recorded as the lethal time. A total of three replicates were performed in each trial. The experiments were carried out at a room temperature of 26 ± 1 °C. Probit analysis was used to calculate LC50 and KT50 values62.

Repellent activity assay

The repellent assay was carried out using a human-bait technique reported by Kaliyaperumal et al.63 with a few modifications. Before the experiment, both hands and forearms of the three volunteers were asked to be washed with scent free soaps, then dried. Gloves wore to cover the entire hand and forearm except for an area of 25 cm2 (5 × 5 cm) on the dorsal sides were cut by a pair of scissors. One hundred 24-h starved 3-day-old female An. sinensis were released into a screen cage (300 mm × 300 mm × 300 mm) prior to the experiment. About 37.5 µL of testing extracted Ar. argyi oils were evenly applied on top of the exposed skin area, then inserted into the testing mosquito cage. 10% DEET repellent formulation was used as positive control. The number of mosquitoes landing on exposed area of skin was counted every fifteen minutes up to the 65th min. Ethanol used as the negative control by applying the same amount onto another hand. Both hands were exposed to starved mosquitoes starting at the same time. Each extracted mugwort oil and 4 individual compounds was repeated three times in random orders. The time intervals of every new volunteers were about 2 h. The percentage of repellency was calculated by using the formula64:

$$ {\text{Percentage}}\;{\text{of}}\;{\text{repellency}} = [\left( {{\text{N}}_{{\text{c}}} - {\text{N}}_{{\text{t}}} } \right)/{\text{N}}_{{\text{c}}} \times 100 $$

where Nc and Nt is the number of mosquito landings on hand of negative control and test samples, respectively. The testing protocol was approved by the Committee of Laboratory Animal Experimentation at Chongqing Normal University (Approval No. Zhao-20200416-01), and all volunteers were provided with the official written informed consent form for testing.

Chemical analysis of the Ar. argyi essential oils

The chemical constituents of Ar. argyi essential oils were analyzed using a gas chromatography-mass spectrometer (Thermo Scientific TSQ XLS GC–MS with a model TRACE 1310 GC), which was equipped with an HP-5MS capillary column (30 m in length, 0.25 mm internal diameter, and 0.25 μm stationary phase film thick nesses). We followed the method as described by Yuan et al.65. The GC oven temperature was programmed at 60 °C for 1 min, then increased to 128 °C at a rate of 2.5 °C/min and maintained for 2 min, and at 5 °C/min to 250 °C (held for 6 min). High purity helium gas was used as mobile phase with a constant flow of 1 mL/min. The parameters for mass spectrometer were set as: 280 °C for both GC injector and MS transfer line, 300 °C for ion source. Mass spectra were recorded in the electron impact ionization (EI) at 70 eV. The MS scan range was from 50–550 m/z. The total ion chromatogram peak areas were used to calculate the percentages of the compositional compounds in essential oils. Chemical identification of each compositional compounds was based on their retention indices determined by reference to a homologous series of n-alkanes (C5-C25:Hc), and by comparisons of their mass spectral fragmentation patterns with those reported in the literature66, and their referenced mass spectra from the TSQ mass spectral library.

Statistical analyses

Log-probit analysis (SPSS 24) was used to determine the median lethal concentration (LC50), the median knockdown concentration (KT50), regression equation, chi-square value and the 95% confidence limit. The numbers of mosquito landings and biting, on negative control, positive control and chemical-treated hands, were compared using an unpaired T-test. Further repellency data among treatments were analyzed using two-way ANOVA followed by Tukey’s honest significant difference (HSD) test (p < 0.05).