Introduction

Anopheles stephensi Liston is responsible for causing malaria in India and other western countries (Burfield and Reekie 2005). Pyrethrin-based products have been widely used to protect people from mosquito bites through their repellent and killing effects. However, these synthetic products do not provide complete protection, and their cost is also prohibitive for low socioeconomic groups. Therefore, efforts are continuing to seek natural repellents or insecticides as safer alternatives to synthetic insecticides which are nonbiodegradable, with high mammalian toxicity, and also faced vector resistance (WHO 1987).

Several phytochemicals extracted from various botanical sources have detrimental effects on mosquitoes (Syamala Devi and Vasudevan 1995). Essential oils provide a rich source of biologically active monoterpenes and are well documented for bioactivities against insect pests. Some of the essential oils with promising mosquito control potential are plant from genus Tagetes spp. (Vasudevan et al. 1997), Ocimum spp. (Bhatnagar et al. 1993), Cymbopogon spp. (Ansari and Razdan 1995), and Mentha spp. (Ansari et al. 2000) etc. Further, essential oils of cassia, camphor, wintergreen, pine, and eucalyptus are already being used in several commercial products for mosquito control (Ansari and Razdan 1994). The essential oils are generally considered nontoxic to human beings (Bagvan et al. 2008) apart from their uses in flavoring, pharmaceuticals, and confectionary industries.

Seeds of Trachyspermum ammi (Apiaceae) commonly known as ajowan, has widespread use as culinary spice and contains thymol as a major constituent.

Thymol has been reported as the major constituent of other medicinal plants like Carum copticum L. and Semenovia tragioides (Boiss.) Manden (Masoudi et al. 2002), Satureja pilosa Velen (Konakchiev and Tsankova 2002), Nigella sativa L. (Enomoto et al. 2001), Oliveria decumbens (Amin et al. 2005), Thymus species (Meshkatalsadat et al. 2007), Ocimum gratissimum (Martins et al. 1999; Koba et al. 2007), and Aeollanthus pubescens Benth. (Koba et al. 2007; Sonda et al. 1999) etc.

Bioactivities of thymol has been documented: acaricidal properties against mites, Acarapis woodi, Tyrophagus putrescentiae (Schrank) and Varrora jacobsoni (Calderone et al. 1997; Ellis and Baxendale 1997; Kuwahara 1982); insecticidal against beetles, Tetranychus urticae (El-Gengaihi et al. 1996), and Acanthoscelides obtectus (Say) (Regnault-Roger and Hamroui 1995); nematicidal toward, Caenorrhabditis elegans (Tsao and Yu 2000); molluscicidal against Lymnacea accuminata (Singh et al. 1999); antibacterial against bacteria, Escherichia coli (Calcuttawalla et al. 2002; Helander et al. 1998); toxic to slug, Deroceras reticulatum (Powell and Bowen 1996); fungitoxic toward Macrophomina phaseolina (Tassi) (Gold.) (Dwivedi and Singh 1998); and genotoxic toward Drosophila spp. (Karpouhtsis et al. 1998).

However, the essential oil of T. ammi seed and its major constituent, thymol has not been evaluated against insect-pests of public health importance. For the first time, the present investigation is aimed to study the efficacy of T. ammi oil and thymol against malarial vector, Anopheles stephensi as larvicidal, oviposition-deterrent, vapor toxicant, and repellent.

Materials and methods

Essential oil extraction

Seeds (500 g) of T. ammi were steam distilled for 4–5 h in a clevenger type apparatus to extract the oil (Senthilkumar et al. 2008). The gas chromatography of the essential oil was done on a Varian Gas Chromatogram, model CX-3400, under the conditions: carrier gas hydrogen, injector (detector FID) temperatures, 220°C and 225°C, respectively, capillary column (Supelcowax –10, 30 m × 0.32 mm, film thickness 0.25 μm); and temperature programmed from 2 min at 40°C to 270°C at 5°C/min. The area percentage was obtained on Varian 4400 integrator. The identity of the component was assigned by comparing their retention time with those of authentic samples. Thymol (99.5 % purity) was purchased from M/s SIGMA Co., USA.

Test organism

Malarial vector mosquito, A. stephensi was reared in the laboratory. The larvae were fed on 5% yeast suspension. Adults were provided with 10% sucrose solution and rabbit for blood meal. Gravid females were used to obtain egg, larva, and adult. All the bioassays were conducted at 28 ± 2°C, 70–88% relative humidity, with a photoperiod of 12:12 (L/D).

Bioassay

Larvicidal test

Essential oil of T. ammi and thymol were evaluated at the level of 0.0, 25.0, 50.0, 75.0, 100.0, 125.0, and 150.0 μg/ml in tap water. Tween-80 was used as emulsifier at a concentration of 0.001%. Tap water mixed with Tween-80 was used as control. Standard WHO test (WHO 1981) was employed with slight modification in the test procedure. A single fourth-instar larva of A. stephensi was put into each of 20 vials containing 5.0 ml of the test solution of each concentration. Observation on larval mortality was recorded after 24 h. Larvae were considered dead, when they did not react to touching with a needle. Data recorded on larval mortality were analyzed statistically for LC50 and LC95 values.

Oviposition-deterrence

The effect of T. ammi seed oil and thymol on oviposition and subsequent egg hatching by A. stephensi were studied by introducing 20 gravid females (fed on rabbit blood) and unlimited number of males from a laboratory colony in a \( 25 \times 25 \times 25\;{\text{cm}} \) oviposition cages under choice conditions. The cages contained seven 100-ml glass dishes with 0.0, 10.0, 25.0, 50.0, 75.0, 100.0, and 125.0 μg/ml concentrations of the oil and 0.0, 5.0, 10.0, 20.0, 40.0, 80.0, and 100.0 μg/ml of the compound. Each cage had a control glass dish having only tap water with 0.001% of Tween-80. The test was replicated four times. The number of eggs was counted for 7 days. The laid eggs were observed for hatching and subsequent survival up to second larval instar only. Oviposition inhibition percentage was calculated according to Mulla et al. (1974).

Vapor toxicity

The essential oil of T. ammi and thymol were dissolved in acetone to make desired concentrations. Aliquot (0.50 ml) of the test solution was dispensed over a cardboard sheet (mat) of size (22 × 35 mm) and thickness (2.5 mm) equal to commercially available mosquito mats, so that amount of the oil and thymol received per mat were 0, 50, 100, 200 300, 400, and 500 mg and 0, 25, 50, 100, 150, 200, and 250 mg, respectively. The solvent was allowed to evaporate at room temperature. The treated cardboard sheet was placed on a mosquito mat machine, and machine was kept on for 15 min. The cardboard used in the control was dispensed with acetone only. Vapor toxicity was evaluated in a specially designed apparatus (Tripathi et al. 2004).

Fifty 6- to 8-day-old females of A. stephensi were used. Cardboard mat treated with essential oil of T. ammi along with mat machine was kept at the corner in the cage. Observations on adult mortality at varying dosages were recorded at 1 h after the treatment. The experiment was repeated three times. Before the start of every experiment, all the chambers were thoroughly washed with soap water and detergent, and dried properly. Data recorded on adult mortality were analyzed statistically for LC50 and LC95 values.

Repellency

The essential oil of T. ammi and thymol were dissolved in acetone to make desired concentrations. Repellency was also evaluated in a specially designed apparatus (Tripathi et al. 2004) as mentioned in vapor toxicity assay, but with further attachments. For repellency studies, the criteria was migration of mosquitoes from one chamber to another connected by tunnel after 1 h for assessment of true repellency. A rabbit (anaesthetized) was placed on the copper wire mesh surface of chamber. Thirty adult female mosquitoes were used for the test.

Aliquot (0.50 ml) of the test solution of the oil and compound were dispensed separately over a cardboard sheet (mat) of size (22 × 35 mm) and thickness (2.5 mm) equal to commercially available mosquito mats so that each mat received 0, 5, 15, 25, 35, 45, and 55 mg and 0, 2, 5, 10, 15, 20, and 25 mg of the oil and thymol, respectively. The solvent was allowed to evaporate at room temperature. The treated cardboard sheet was placed on a mosquito mat machine, and the machine was kept on for 15 min. The cardboard used in control was dispensed with acetone only. After 1 h experimental duration, the number of mosquitoes present in both two chambers was counted, and percent repellency was calculated as:

$$ \% \;{\text{repellency}}\; = \frac{{\% \;{\text{mosquitoes}}\;{\text{observed}}\;{\text{in}}\;{\text{chamber}}\;\prime{\text{C}}\prime - \% \;{\text{mosquitoes}}\;{\text{observed}}\;{\text{in}}\;{\text{chamber}}\;\prime{\text{B}}\prime}}{{100 - \% \;{\text{mosquitoes}}\;{\text{observed}}\;{\text{in}}\;{\text{chamber}}\;\prime{\text{B}}\prime}} \times 100 $$

Statistical analysis

Probit analysis (Finney 1971) was used to analyze lethal doses (LC50/LD50 and LC99/LD99) of the oil and thymol. Linear regression was used to describe the relationship between dosage–mortality (SPSS 1999).

Results

Oil extraction

GC analysis of the oil revealed thymol (66.96%) as major constituent (Table 1).

Table 1 GC analysis of the essential oil of T. ammi used for bioassay studies
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Larvicidal

T. ammi seed oil and thymol showed LD50 values of 80.77 and 48.88 µg/ml, respectively, whereas LD99 values were observed as 172.12 and 105.49 µg/ml, respectively (Fig. 1). Thus, thymol was 1.65-fold more toxic than the oil itself toward the fourth-instar larvae of A. stephensi. At the dose of 100.0 µg/ml, the thymol gave 100.0% mortality, whereas T. ammi seed oil resulted into 63.0% larval mortality only at the same dose. Further, regression analysis of the data also showed significant (F = 38.90, df = 6, P < 0.01; F = 281.24, df = 6, P < 0.01) dose-dependent toxicity toward the fourth-instar larvae exposed to thymol and T. ammi seed oil, respectively (Fig. 1).

Fig. 1
figure 1

Dose–response relationships of toxicity of T. ammi seed oil and thymol toward mortality of the fourth-instar larvae of A. stephensi

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Oviposition-deterrence

Thymol and T. ammi seed oil both significantly (F = 29.49, df = 6, P < 0.01; F = 341.56, df = 6, P < 0.01, respectively) reduced egg laying (oviposition) by A. stephensi as concentration increased (Table 2). At the dose of 100 µg/ml, thymol-exposed A. stephensi female adults laid only <5.2 eggs, whereas T. ammi seed oil exposed adults laid 25.8 times less number of eggs compared to control. Thus, both the compound and oil reduced oviposition by A. stephensi adults significantly (F = 29.37, df = 6, P < 0.01; F = 358.0, df = 6, P < 0.01, respectively) as evidenced from percent oviposition-deterrence values (Table 2). Similarly, both the compound and oil significantly (F = 18.61, df = 6, P < 0.01; F = 99.80, df = 6, P < 0.01, respectively) reduced the viability of eggs laid. Further, both the compound and oil significantly (F = 3.35, df = 6, P < 0.01; F = 8.83, df = 6, P < 0.01, respectively) suppressed survival of larvae emerged from laid eggs (Table 2). Thymol caused 37.20% egg hatching at a dose of 20.0 µg/ml and T. ammi seed oil caused 24.0% egg hatching at a dose of 75.0 µg/ml, but the emerged larvae could not survive up to the second instar at these doses.

Table 2 Effect of T. ammi seed oil and thymol on fecundity and fertility of Anopheles stephensi
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Vapor toxicity

Thymol was highly toxic toward the adults of A. stephensi in vapor toxicity assay, as it provided complete adult mortality at two times less dose rate compared to T. ammi seed oil (Fig. 2). LC50 values of thymol and T. ammi seed oil were found to be 79.5 and 185.4 mg/mat, respectively, whereas LD99 values observed were 203.41 and 453.85 mg/mat, respectively. Thus, the lethal toxicity of thymol was 2.3 times more to that of T. ammi seed oil (Fig. 2). Further, regression analysis of the data also showed significant (F = 62.57, df = 6, P < 0.01; F = 86.63, df = 6, P < 0.01) dose-dependent mortality toward adults exposed to thymol and T. ammi seed oil, respectively (Fig. 3).

Fig. 2
figure 2

Dose–response relationships of vapor toxicity of T. ammi seed oil and thymol toward adults mortality of A. stephensi

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Fig. 3
figure 3

Repellent responses of adults of A. stephensi to the doses of T. ammi seed oil and thymol

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Repellency

Exposure of thymol to A. stephensi adults showed complete repellency at 2.2 times less dose to that of T. ammi seed oil after 1 h duration (Fig. 3). At the dose of 25.0 mg/mat, thymol provided complete repellency, whereas T. ammi seed oil could achieve a repellency of 45.0% only (Fig. 3).The repellent doses (RD50) observed were 25.02 and 11.63 mg/mat for T. ammi seed oil and thymol, respectively.

Discussion

The results of the present investigation showed that pure constituent thymol was twofold more active than essential oil of T. ammi seed in vapor toxicity and repellent assays against adults of A. stephensi. However, as a larvicidal, thymol was only 1.65-fold more active than the T. ammi oil. The importance of toxic and growth-retarding influence of the thymol may have better practical significance, if such effects can also be observed when applied to natural habitat like larval breeding sites. Present investigation revealed the promising potential of thymol as larvicidal, oviposition-deterrent, adulticidal, and repellent against A. stephensi. Toxic and growth-retarding activities of thymol makes its wide applications both in larval breeding nitches and household conditions. Thymol has also been reported to be highly toxic (LD50 = 25.4 µg/larva) toward the larva of Spodoptera litura (Hummelbrunner and Isman 2001) and tracheal mites, Acarapis woodi with a LC50 value of 0.90 µg/ml (Ellis and Baxendale 1997), whereas it gave LD50 value of 48.8 µg/ml toward fourth-instar larvae of A. stephensi in our studies. The difference in LD50 values may be attributed to the mode of application of the compound. In the case of A. stephensi larvae, thymol was mixed in water, whereas in the case of S. litura larvae, it was applied topically.

The dose–response relationships reported in this study provide a foundation for future investigations of thymol and T. ammi seed oil as vapor toxicant and repellent against adults of A. stephensi. Length of exposure, temperature, and humidity are factors that can influence the activity of the test chemicals as vapor toxicants under household conditions and merit further investigations. Likewise, mode of action studies can provide insight on to how best to use T. ammi seed oil and thymol. The potential use of these selective and fully biodegradable materials in management of malarial vector is encouraging.