The mosquitoes, Aedes aegypti L. and Culex pipiens pallens Coquillett (Diptera: Culicidae) are two worldwide insects causing dreadful nuisance and transmitting many dangerous diseases [1,2]. In China, A. aegypti is considered one of the major vectors for the transmission of dengue fever (common clinical characteristics as fever, headache, chilly, joint pain) and C. pipiens pallens is the primary vector of wuchereriasis and epidemic encephalitis B (clinical symptoms included high fever, nausea and vomiting, altered consciousness) [1,2]. Currently, synthetic insecticides and insect growth regulators are widely applied to control larval mosquitoes [3]. However, repeated and injudicious application of these synthetic insecticides have caused disrupt of the natural biological control systems and sometimes resulted in the widespread development of resistance as well as undesirable effects on non-target organisms, toxic residues in food, workers’ safety, and high cost of procurement [4]. Essential oils and their constituents have been suggested as alternative sources for conventional mosquito larvicides [5-7]. During the present author’s mass screening program for new agrochemicals from wild plants and Chinese medicinal herbs, the essential oil of Dipsacus asper Wallich ex Candolle (Family: Dipsacaceae) roots, was found to possess larvicidal activity against the larvae of A. aegypti and C. pipiens pallens.

Himalayan teasel (D. asper) is a perennial herb distributed widely in the southwest of China (Chongqing, Guangdong, Guangxi, Guizhou, Hubei, Sichuan, Xizang, and Yunnan province) and India as well as Myanmar [8]. The roots of D. asper has been used in traditional Chinese medicine for hundreds of years as an antiosteoporosis, tonic and antiaging agent for the therapy of low back pain, traumatic hematoma, threatened abortion and bone fractures [9,10]. In the previous studies, dozens of chemical constituents, including triterpenoids, triterpene saponins, iridoids, iridoid glucosides, lignans, phenolics, and alkaloids, have been identified from the roots of D. asper [9-15]. However, a literature survey has shown that there is no report on chemical composition of essential oil of D. asper and larvicidal activity of D. asper essential oil against mosquitoes. Hence, the objective of the present study was to investigate the chemical constituents and larvicidal activity of the essential oil of the plant against two species of mosquitoes.

Plant collection and identification

Dried roots of D. asper (10 kg, harvested from Hubei Province, China) were purchased from Anguo Herb Market (Anguo, Hebei Province). The species was identified by Dr. Liu, Q.R., College of Life Sciences, Beijing Normal University, Beijing 100875 and a voucher specimen of D. asper (Dipsacaceae-Chuanxuduan-Hubei-07) was deposited at the museum of Department of Entomology, China Agricultural University.

Oil extraction

The sample was chopped to small pieces and immersed in water at a ratio of 1:4 (w/v) for 1 h, and subjected to hydro distillation using a modified Clevenger-type apparatus for 6 h. The essential oil was extracted from the distillate with n-hexane and then dried over anhydrous Na₂SO₄. After filtration, the solvent was removed by distillation under reduced pressure in a rotary evaporator at 35 °C and the pure oil was kept in a refrigerator (4 °C) pending subsequent experiments.

Analysis of the essential oil

Gas chromatographic analysis was performed using Hewlett–Packard 5890 gas chromatograph equipped with a flame ionization detector and fused silica capillary column HP-5MS (5 % diphenyl and 95 % dimethylpolysyloxane, 30 m × 0.25 mm, 0.25 μm film thickness), operated at a flow rate of 1 mL min⁻¹. Column temperature was initially 60 °C for 1 min, then gradually increased to 180 °C at 10 °C min⁻¹, and finally increased to 280 °C at 20 °C min⁻¹. The components of the essential oil were separated and identified by gas chromatography–mass spectrometry (GC - MS). (Agilent 6890N gas chromatography coupled to Agilent 5973N mass selective detector). The system was equipped with a flame ionization detector and capillary column with HP-5MS (30 m × 0.25 mm × 0.25 μm). GC settings were as follows: the initial oven temperature was maintained at 60 °C for 1 min and increased at 10 °C min⁻¹ to 180 °C where it was held for 1 min, and then ramped at 20 °C min⁻¹ to 280 °C and kept there for 15 min. The injector temperature was maintained at 270 °C. The essential oil was diluted 100:1 (v/v) with acetone and the diluted samples (1 μL) were injected automatically in splitless mode. The carrier gas was helium at a flow rate of 1.0 mL min⁻¹. Spectra were detected over the scan range 20 to 550 m/z at 2 scans s^-1. Most constituents were identified by gas chromatography and comparison of their retention indices with those published in the literature or with those of authentic compounds available in our laboratories. Retention index was measured in relation to a homologous series of n-alkanes (C₈ – C₂₄) under the same operating conditions. Further identification was taken by comparison of their mass spectra with those stored in NIST 05 and Wiley 275 libraries or with mass spectra from literature [16]. Relative contents of the oil components were calculated based on GC peak areas without applying correction factors.

Insect cultures and rearing conditions

Mosquito eggs of A. aegypti and egg masses of C. pipiens pallens utilized in bioassays were obtained from a laboratory colony maintained in the Department of Vector Biology and Control, Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The eggs of A. aegypti and egg rafts of C. pipiens pallens were collected from Nanjing, Jiangsu province, China in 1997. The adults were maintained in a cage (60 × 30 × 30 cm) at 28 – 30 °C and 75 – 85 % RH. The female adults were fed with rat blood every alternate day whereas the males were fed with 10 % glucose solution soaked on cotton pad, which were hung in the middle of the cage. A beaker with strips of moistened filter paper was kept in the cage for oviposition of A. aegypti. The eggs laid on paper strips were kept wet for 24 h and then dehydrated (air-dried) at room temperature. The dehydrated eggs were put into plastic tray containing tap water in our laboratory at 26 – 28 °C and natural summer photoperiod (L14:D10) for hatching and yeast pellets provided as food for the emerging larvae. However, C. pipiens pallens deposited in tap water and the egg masses were transferred to a white porcelain basin containing tap water for hatching. Larvae were daily observed until they reached the fourth instar, when they were employed for bioassays (within 12 h).

Larvicidal bioassay

Range-finding studies were performed to determine the appropriate testing concentrations. Concentrations of 200, 100, 50, 25, and 12.5 μg/mL of essential oil were assessed. The larval mortality bioassays were carried out according to the test method of larval susceptibility as recommend by WHO [17]. Twenty larvae were put in a glass beaker with 250 mL of aqueous suspension of tested material at various concentrations. Five replicates were run simultaneously per concentration and with each experiment, a set of control and untreated sets of larvae in tap water, were also run for comparison. Commercial rotenone (purchased from Aladdin Industrial Inc., Shanghai, China) was utilized as a positive control. The assays were placed in a growth chamber (L16:D9, 26 – 28 ^oC, 78 – 80 % RH). Mortality was detected after 24 h of exposure.

Statistical analysis

Percent mortality was corrected for control mortality using Abbott’s formula [18]. Results from all replicates for the pure compounds/oil were subjected to probit analysis using PriProbit Program V1.6.3 (http://ars.usda.gov/Services/docs.htm?docid =11284) to estimate LC₅₀ values and their 95 % confidence intervals [19]. Samples for which the 95 % fiducial limits did not overlap were regarded as significantly different.

The yield of essential oil from 10 kg of dried D. asper roots was 8.5 ml while its density was 0.87 g/ml. A total of 34 components of the essential oil of D. asper roots were identified (). The principal compounds of the essential oil were caryophyllene oxide (13.29 %), caryophyllene (9.14 %), cubebene (7.87 %), β-gurjunene (6.43 %), carvone (5.38 %), 1,8-cineole (5.29 %) and calamenene (5.05 %) (). Sesquiter-penoids represented 18 of the 34 constituents, corresponding to 62.80 % of the essential oil of D. asper roots while 13 of the 34 compounds were monoterpenoids, corresponding only to 32.81 % of the whole essential oil.

The essential oil of D. asper exhibited larvicidal activity against the 4^th instar larvae of A. aegypti and C. pipiens pallens with LC₅₀ values of 56.29 μg/mL and 47.59 μg/mL, respectively.

The main constituents of D. asper essential oil were caryophyllene oxide, caryophyllene, cubebene, β-gurjunene, carvone, 1,8-cineole, and calamenene. This is the first time to report chemical composition of D. asper roots essential oil. The results are quite different from the essential oils of other Chinese species in Dipsacus [20,21]. For example, the essential oil of D. asperoides roots mainly contained carvotanaceton (8.54 %), 2,4,6-tri-t-butyl-phenol (5.46 %), 3-enthyl-5-methyl-phenol (4.15 %) and 4-methyl-phenol (3.98) and the essential oil of D. asperoides had remarked effect against Staphyloccus aureus [20]. However, the main constituents of the essential oil of the aerial parts of D. japonicus at the flowering stage were linalool (11.78​ %)​, trans-​geraniol (8.58​ %)​, 1,​8-​cineole (7.91​ %)​, β-​caryophyllene (5.58​ %)​, α-​terpineol (5.32​ %)​, β-​selinene (5.15 ​%)​, and spathulenol (5.04​ %) and the essential oil of D. japonicus exhibited contact and fumigant toxicity against two grain storage insects (Sitophilus zeamais and Tribolium castaneum) [21]​.

The essential oil of D. asper roots possessed strong larvicidal activity against the 4^th instar larvae of A. aegypti and C. pipiens pallens. The commercial insecticide, rotenone showed larvicidal activity against the two species of mosquitoes with LC₅₀ values of 3.75 μg/mL and 1.88 μg/mL, respectively.

However, compared with the other essential oils/extracts in the literature, the essential oil of D. asper exhibited the same level of or stronger larvicidal activity against A. aegypti larvae, e.g., essential oil of Eucalyptus urophylla (LC₅₀ = 95.5 μg/mL) [22]; essential oils from four Guarea species (G. scabra leaves, LC₅₀ = 98.6 μg/mL; G. silvatica leaves, LC₅₀ = 117.9 μg/ml and G. convergens branches 145.1 μg/mL) [23] and leaf essential oil of Cryptomeria japonica (LC₅₀ = 56.8 μg/mL) [24]. However, the essential oil of D. asper was less toxic than essential oils of Salvia plebeian aerial parts (LC₅₀ = 46.26 μg/mL) [25], Isodon japonicus var. glaucocalyx aerial parts (LC₅₀ = 40.82 μg/mL) [27] and Illicium difengpi stem bark (LC₅₀ = 31.68 μg/mL) [26]. Considering that the currently used larvicides are synthetic insecticides, larvicidal activity of the essential oil of D. asper roots is quite promising and it shows its potential for use in the control of A. aegypti and C. pipiens pallens larvae and could be useful in the search for new, safer and more effective natural compounds as larvicides.

For the actual use of D. asper essential oil as a novel larvicide or insecticide to be realized, further research is needed to establish their human safety and environmental safety. In traditional Chinese medicine, the plants are used as a tonic and antiaging agent for the therapy of low back pain, traumatic hematoma and threatened abortion and bone fractures [12,13]. It appears to be safe for human consumption. However, no experimental data on toxicity of the essential oil to human is available, to the best of our knowledge. Additionally, their larvicide modes of action have to be established, and formulations for improving larvicidal potency and stability need to be developed. Furthermore, field evaluation and further investigation of the effects of the essential oil on non-target organisms are necessary.

The essential oil of D. asper roots demonstrates some activity against Aedes aegypti and Culex pipiens pallens mosquito larva but needs to be further evaluated for safety in humans and to enhance its activity.