Introduction

Mosquitoes are the most important insects worldwide in terms of public health importance. Mosquito-borne diseases (such as malaria, filariasis, yellow fever, dengue fever and viral encephalitis) amount to a large proportion of health problems in developing countries (James 1992). Mosquitoes are also important as human pests in Europe, since their bite causes a local skin reaction and, in some cases, serious allergic and systemic reactions such as angioderma and urticaria (Peng et al. 1999). Moreover, after the introduction in Europe of new dangerous species, such as Aedes albopictus Skuse, many cases of chikungunya (a very severe disease transmitted by Aedes spp.) were recently reported in France and Italy (Angelini et al. 2007; Ledrans et al. 2007).

It is well known that one way to reduce the mosquito populations is targeting larvae with organophosphate applications and with insect growth regulators, such as diflubenzuron and methoprene (Yang et al. 2002). Repeated use of these synthetic insecticides can lead to the development of resistance or to undesirable effects on non-target organisms or to human health (Brown 1986; Severini et al. 1993). Treatments with Bacillus thuringiensis (var israeliensis) can be a solution, but in several cases they are not suitable against A. albopictus (Kamgang et al. 2011). For these reasons, there is a worldwide need to find alternatives to synthetic insecticides. Botanical pesticides are effective, environmentally friendly, easily biodegradable and often less expensive than the synthetic ones (Govindarajan et al. 2011 and references therein). Among these products, essential oils (EOs) are well known for their antibacterial, antifungal and insecticidal activities (Cheng et al. 2003). Many active ingredients isolated and identified from plant extracts can exert toxic activity against mosquito larvae (Cheng et al. 2004; Rahuman et al. 2008; Mathew et al. 2009; Conti et al. 2010; Hafeez et al. 2011). Moreover, they can be used as ovicidal, oviposition deterrents, growth and reproduction inhibitors (Rajkumar and Jebanesan 2005; Pushpanathan et al. 2006) or adult repellents (Gleiser et al. 2011 and references therein). It is known that plants contain various chemicals endowed with unique biological activities (Farnsworth and Bingel 1977), caused by secondary metabolites, who act as attractants or deterrents (Fisher 1991). The number of EOs showing repellent properties against mosquitoes continues to grow (Amer and Mehlhorn 2006; Gillij et al. 2008; Gleiser and Zygadlo 2009; Maheswaran and Ignacimuthu 2011). In some cases, the repellent activity of these compounds is higher or it has longer duration than synthetic chemicals (Moore et al. 2002; Omolo et al. 2004).

Lamiaceae have traditionally been used in developing countries for their insecticidal and repellent properties against several insect species (Ngamo et al. 2007). Rosmarinus officinalis L. and Lavandula angustifolia Milller (Lamiaceae) EOs showed moderate larvicidal activity (Conti et al. 2010) but a noticeable repellent and ovicidal effect against several mosquito species (Prajapati et al. 2005), possibly caused by α-terpinene, carvacrol and thymol (Choi et al. 2002). Lamiaceae species of the Hyptis genus—which included more than 400 species—are highly aromatic and grow in tropical regions, mainly in Africa and America. Several studies have shown that Hyptis suaveolens (L.) Poiteau EO has useful insecticidal properties against mosquitoes (Amusan et al. 2005; Jaenson et al. 2006) and many stored products pests (Peerzada 1997; Othira et al. 2009; Conti et al. 2011). Moreover, its chemical composition and biological activity change as a function of plants origin and their collecting period (Tchoumbougang et al. 2005; Noudjou et al. 2007).

As further studies are important to improve the knowledge of new plant extracts and their pure constituents for their use against mosquito species, this study investigates the chemical composition of H. suaveolens EO, extracted from plants cultivated in Tuscany (Italy), and its larvicidal and repellent activity against the Culicidae mosquito A. albopictus. Furthermore, the effectiveness of its main EO constituents as larvicides was measured.

Materials and methods

H. suaveolens cultivation

Plants were grown at the Department of Agronomy and Agroecosystem Management (University of Pisa) (Fig. 1). Seeds of H. suaveolens (from Nepal) were placed on filter paper moistened in Petri dishes, placed in a climatic chamber [alternating temperature of 20–30°C, photoperiod 8:16 (L:D)] and left to germinate, between February and April 2010. Seedlings (germination 83%) were transferred to nurseries and then placed in a cold greenhouse for ca. 40 days. The young plants were transplanted in June 2010, at a density of 4.5 plant m−2 in a silt–loam soil (sand, 15.5%; silt, 65.5%; clay, 18.0%; organic matter, 1.15%; pH 8.1), with a rather shallow water table, above a depth of 120 cm. Urea (50 kg ha−1 of N), 100 kg ha−1 of triple superphosphate (P2O5) and 100 kg ha−1 of potassium sulphate (K2O) were used as fertilisers. Irrigation and mechanical weed control were used for the entire cultivation period. The biomass was collected at the beginning of October 2010.

Fig. 1
figure 1

Plants of Hyptis suaveolens (L.) Poiteau (Lamiaceae)

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Essential oil extraction and analysis

Leaves were dried in the shade, at room temperature until constant weight, and then coarsely ground and hydrodistilled in a Clevenger-type apparatus for 2 h. Gas chromatography (GC) analyses were carried out with an HP-5890 series II instruments equipped with HP-WAX and HP-5 capillary columns (30 m × 0.25 mm, 0.25 μm film thickness), working with this temperature program, 60°C for 10 min, ramp of 5°C/min up to 220°C; injector and detector temperatures 250°C; carrier gas was nitrogen (2 ml/min); detector dual FID; split ratio 1:30; and injection of 0.5 μl. Component identification was carried out, for both columns, by comparing their retention times with those of pure authentic samples and by means of their linear retention index (LRI), relative to the series of n-hydrocarbons.

Gas chromatography/electron impact mass spectroscopy (GC/EIMS) analyses were performed with a Varian CP-3800 gas chromatograph, equipped with an HP-5 capillary column (30 m × 0.25 mm; coating thickness 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions are as follows: injector and transfer line temperatures 220°C and 240°C, respectively; oven temperature programmed from 60°C to 240°C at 3°C/min; carrier gas helium at 1 ml/min; injection of 0.2 μl (10% hexane solution); and split ratio 1:30. Constituent identification was based on the comparison of retention times with those of authentic samples; this implied comparing their LRIs with the series of n-hydrocarbons and using computer matching against commercial (NIST 98 and ADAMS) and home-made library mass spectra (built up from pure substances and components of known oils and MS literature data, refer to Stenhagen et al. 1974; Massada 1976; Jennings and Shibamoto 1980; Swigar and Silverstein 1981; Davies 1990; Adams 1995). Moreover, molecular weights of all identified substances were confirmed by GC/CIMS, using methanol as the chemical ionization gas.

Mosquitoes rearing conditions

Mosquito larvae and adults of A. albopictus originated from field-collected eggs, deposited by wild females on a bar of masonite placed outdoors in a dark vase containing water. Egg batches were collected daily and kept moist for 24 h; then they were placed in laboratory conditions [25 ± 1°C, 65 ± 5% relative humidity (R.H.), natural summer photoperiod] in 100-cc glass tubes and submerged in mineral water for hatching. Newly emerged larvae were isolated in groups of five specimens in 100-cc glass tubes, with mineral water and a small amount of cat food. The larvae were examined daily, until they reached the fourth instar; they were then used for bioassays (within 12 h) or to obtain adults. Adults were stocked in cages (300 specimens/cage, sex ratio 1:1), held at 25 ± 1°C, 65 ± 5% R.H., natural summer photoperiod and supplied with 10% sucrose solution on a cotton wick.

Larvicidal activity

Three groups of 20 fourth instar larvae were isolated in 250-ml beakers and exposed to dosages of 50, 100, 150, 200, 250, 300, 350, 400 and 450 ppm of EO in mineral water with 0.1% of Tween® 80 for 24 h (WHO 1981); 250-ml beakers with the same number of larvae (for three replicates) and mineral water with 0.1% of Tween® 80 were used as control. Mortality was recorded after 24 h, at the end of the test, during which no food was given to the larvae. Larval mortality was reported as an average of three replicates; mortality percentage rates were corrected using Abbott’s formula (Abbott 1925) and they were used to calculate the LC50 values.

Main constituents (Table 1) identified in H. suaveolens EO (sabinene, α-pinene and β-pinene, limonene, terpinolene, β-caryophyllene and 4-terpineol) were purchased from Sigma-Aldrich® and tested—singularly or in blend—for larvicidal activity at the same dosage contained in 450 ppm of EO, at which 100% of larval mortality was previously obtained. Compound solutions were prepared in 250-ml beakers with mineral water containing 0.1% Tween® 80. Three groups of 20 fourth instar larvae were used for the test and 250-ml beakers with 20 larvae (three replicates) and mineral water with 0.1% of Tween® 80 were used as control.

Table 1 Composition of the essential oil of Hyptis suaveolens used in the biological assays
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Repellent activity

H. suaveolens EO repellency was evaluated using the human bait technique to simulate the condition of human skin on which repellents will be applied, as reported by Schreck and Mc Govern (1989), Gleiser et al. (2011) and Kamsuk et al. (2006). Tests were conducted during the summer of 2011. Groups of 150 nulliparous, nonblood-fed, starved female of A. albopictus (7–10 days old) were placed, in order to facilitate viewing, into Plexiglass cylindrical laboratory cages (diameter, 35 cm; length, 60 cm). Each cage had a cotton stockinet access sleeve on the front. A. albopictus is a day-biting mosquito; therefore, testing period was between 08:00 and 16:00 hours. Ten volunteers were chosen amongst susceptible to mosquito bites and non-allergic subjects. They had no contact with lotions, perfumes, oils or perfumed soaps on the day of the bioassay. After cleaning their hands in distilled water, they protected their forearms with a thick fabric sleeve and wore a latex surgical glove, in which a dorsal square area 5 × 5 cm was cut open.

Mosquito-exposed skin was firstly treated with 100 μl of ethanol, as negative control, and then with 100 μl of EO in ethanol solution (dosages ranging from 0.0375 to 0.750 μg/cm2, refer to Table 5). All concentrations were replicated five times. Firstly, the control hand was exposed in the cage for 3 min, during which the number of probing mosquitoes was recorded. Immediately after, the hand was withdrawn and treated with repellent formulation; then it was re-exposed to mosquitoes in the same test cage. The number of probing mosquitoes in a 3-min exposure period was recorded. The percentage of repellency obtained from five replicates—expressed as percentage protective efficacy (PE%)—was calculated at each dosage using this formula: PE% = [(number probing untreated hand − number probing treated hand)/number probing untreated hand] × 100 (Fradin and Day 2002).

To calculate the full protection time (PT), the test was repeated every 5 min for the lowest concentration and every 10 min for the other concentrations, until either two bites occurred in a single exposure period or one bite occurred in each of two consecutive exposure periods. The period of time, between repellent application and the first two bites in a single exposure or two bites in successive observations, was recorded as the complete protection time. Each PT is an average of five replicates. Each concentration has been tested for a total of 150 min. During the tests, the control and the treated hands were regularly interchanged, to verify the mosquitoes’ readiness to bite. On rare occasions, when no mosquito attempted to bite the untreated hand, trial was discarded and test was repeated with a new mosquito cage, to ensure that the lack of bites was due to repellency and not to mosquitoes being unwilling to have a blood meal at the time. To calculate the RD50 values, the EO was tested at dosages of 9.370, 5.622, 3.748, 1.874, 0.3748, 0.1874 and 0.0937 10−3 μg cm−2 (WHO 2009).

Statistical analysis

EO larvicidal activity data were transformed into arcsine square root percentage values, before statistical analysis. Data were processed by JMP®, using a general linear model (GLM) with one factor, the concentration: y j  = μ + C j  + e j , in which y j is the observation, μ is the overall mean, C j is the dosage (j = 1–9) and e j the residual error. Averages were separated by Tukey–Kramer HSD test (Sokal and Rohlf 1981). Only probability level P < 0.05 was used, for the significance of differences between means, to simplify statistical analysis. Data on larvicidal activity of pure compounds were transformed into arcsine square root percentage values and analysed using a GLM as described above, in which C j is the compound (j = 1–7). Repellency data were analysed using a GLM (JMP® SAS, 1999) with two factors with interactions, dosage and time: y j  = μ + C j  + T j  + C j  × T j  + e j in which y j is the observation, μ is the overall mean, C j the dosage (j = 1–5), T j the time (j = 1–5), C j  × T j the interaction between dosage and time and e j the residual error in the interaction between oil and dosage.

Median lethal concentration and median repellent dosage (LC50 and RD50) were calculated by using SigmaPlot© software (Systat Software Inc., CA, USA). Bottom and top parameters were fixed to 0 (0% mortality and no repellence, respectively) and 100 (100% mortality and full repellence, respectively).

Results and discussion

H. suaveolens cultivation

H. suaveolens plants have reached an average height of 85 cm in mid-October. At the end of August, they have reached a height of about 80 cm. Biomass obtained after about 4 months from transplanting was 2,184 gpt−1. Dry matter produced was 431.3 gpt−1. In our environment, H. suaveolens did not reach the flowering stage.

Chemical composition of H. suaveolens EO

H. suaveolens is polymorphic in its essential oil composition because many different compositions have been reported (Grassi et al. 2008). Several chemotypes have been described, such as β-caryophyllene type from Nigeria (Iwu et al. 1990), 1,8-cineole/sabinene from India (Mallavarapu et al. 1993), three 1,8-cineole, α-terpinolene and fenchone/fenchol types from El Salvador (Grassi et al. 2008).

The essential oil obtained from the leaves of plants grown in Pisa (Table 1) cannot be ascribed to a precise chemotype, as it contains high percentages of sabinene (21.9%), β-caryophyllene (16.1%), terpinolene (9.6%) and 4-terpineol (7.3%). Globally, 52 constituents were identified, accounting for 99.1% of the whole essential oil. From a chemical classification point of view (Table 2), monoterpene hydrocarbons were the most represented volatiles (55.2%), followed by sesquiterpene hydrocarbons (24.1%) and oxygenated monoterpenes (10.5%). In addition, smaller amounts of oxygenated sesquiterpenes (7.2%) and diterpenes (1.9%) were detected. Non-terpene derivatives were present in very small amounts (0.2%).

Table 2 Mean percentages (%) of main chemical classes of the H. suaveolens essential oil volatiles
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Larvicidal activity

It has been acknowledged that ethanolic extracts of H. suaveolens caused high mortality rate (80%) on yellow fever mosquito Aedes aegypti (L.) larvae, at concentrations of 0.9 and 0.3 ppm (Amusan et al. 2005). By contrast, H. suaveolens petroleum leaf extracts did not show any effective larvicidal activity against Culex spp. mosquito larvae (Okigbo et al. 2010). Data reported by Tamprasit and Indrapichate (2004) showed that H. suaveolens crude extract manifested a larvicidal activity higher than the one from Lantana camara crude extract, with a synergic effect if these extracts were mixed. To our knowledge, the larvicidal activity of H. suaveolens EO against mosquito species was not evaluated. Our results clearly demonstrated that H. suaveolens EO had insecticidal activity against A. albopictus larvae, thus widening the extraction methods of its larvicidal botanical components. Larval mortality was dosage dependent. It was found that there are significant differences in mortality rates, as a function of EO concentration (F = 43.68, df = 8, P < 0.0001) (Table 3). At the highest dosages of 450 and 400 ppm, there were no significant differences on larval mortality, with mortality percentage rates ranging from 98.33% and 93.33%, respectively. At dosages ranging from 250 to 350 ppm, mortality rates were lower and not significantly different from each other.

Table 3 Mortality obtained in larvicidal test conducted with Hyptis suaveolens essential oil against fourth instar larvae of Aedes albopictus
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LC50 value of H. suaveolens EO was 240.3 ppm (Table 3). Tests with A. aegypti and EOs from different species of Hyptis showed that LC50 of Hyptis fruticosa and Hyptis pectinata were 502 and 366 ppm, respectively (Silva et al. 2008). It must be noted that these values were relatively higher, with respect to those obtained in our study. Moreover, H. suaveolens ethanolic extracts showed a high toxicity against A. aegypti larvae, with LD50 = 0.60 ppm and LD90 = 1.45 ppm (Amusan et al. 2005).

Table 4 illustrates the larvicidal activity of α-pinene and β-pinene, β-caryophillene, sabinene, terpinolene, limonene and 4-terpineol—tested on its own or blended—in the same concentration at which they were detected in 450 ppm of the EO. There was a significant larvicidal effect of the whole blend, with respect to the control and to α-pinene and β-pinene, β-caryophillene, sabinene, limonene and 4-terpineol. On the contrary, it was evident that terpinolene toxicity was not statistically different from the one of the whole blend (43.33% and 55%, respectively). Terpinolene toxicity has been well acknowledged with other insect species, such as Sitophilus zeamais Motschulsky, Tribolium castaneum (Herbst) (Wang et al. 2009), Callosobruchus chinensis (L.) and Sitophilus oryzae (L.) (Park et al. 2003). We suppose that the blend larvicidal activity is probably due to the sum of individual toxicities of the main constituents.

Table 4 Mortality obtained in larvicidal test conducted with the seven main constituents of the Hyptis suaveolens essential oil tested at the concentration detected in 450 ppm of the oil
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Repellent activity

To our knowledge, there is no reported data in literature on H. suaveolens EO repellent activity against A. albopictus. Table 5 summarises the results of repellency tests—expressed as PE%—at different dosages of H. suaveolens EO, during 150 min of observation. The results indicated that the EO had a significant repellent activity (RD50 = 0.00035 μg cm−2; RD90 = 0.00048 μg cm−2, Table 6), with relevant differences in repellency rates, as a function of both concentration (F = 119.55, df = 4, P < 0.0001) and time of observation (F = 151.62, df = 5, P < 0.0001). At lower dosage, the EO gives a PE of 94.2% in the first 15 min of observation. At dosage of 0.1874 μg/cm2, H. suaveolens EO offered a PE of 100% for at least 30 min, and after 60 min, it reached 91.2%. At a medium dosage of 0.3748 μg/cm2, H. suaveolens EO reached a similar value (94.2% of PE) after 90 min. Two higher dosages (0.5622 and 0.7496 μg/cm2 of skin) gave almost complete protection (99.6% and 100% PE, respectively) for 90 min. Table 7 shows protection times for the five concentrations of the H. suaveolens EO tested. The results indicated a significant effect of the EO concentration (F = 51.01, df = 4, P < 0.0001); protection time ranged between 16 and 135 min. Our observations of H. suaveolens EO efficacy as repellent improve previous evidence from several studies, in which the repellent activity of H. suaveolens was proven through different application methods (Table 8). In fact, it is known that placing H. suaveolens branches or whole plants in houses was one of the most effective methods, in western Kenya, to repel malaria vector Anopheles gambiae s.s. Giles (Seyoum et al. 2002b). By contrast—in semi-field conditions—it was observed that H. suaveolens potted plants did not significantly repel A. gambiae mosquitoes (Seyoum et al. 2002a). Moreover, studies performed in Guinea Bissau, West Africa showed that smoke produced by burning whole plants of H. suaveolens, indoors at night, significantly repelled mosquitoes (Pålsson and Jaenson 1999a, b). Pålsson and Jaenson (1999b) reported that fresh or smouldering whole plants of H. suaveolens were used in Guinea Bissau, to reduce the number of mosquitoes indoors at night, with a repellent activity ranging from 85.4% to 66.5% (for smouldering and fresh plants, respectively). Similar results were obtained with the same method in western Kenya, against A. gambiae (Seyoum et al. 2002b). However, any significant repellent effect was recorded against this latter mosquito species, when H. suaveolens flowers and leaves were tested through thermal expulsion method (Seyoum et al. 2002b). Finally, ethyl acetate extracts of H. suaveolens from Guinea Bissau strongly reduced the probing activity of A. aegypti (Jaenson et al. 2006).

Table 5 Efficacy protection of Hyptis suaveolens essential oil at different dosages against Aedes albopictus, during 150 min of observations
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Table 6 Repellent activity of the Hyptis suaveolens essential oil
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Table 7 Protection time (in minutes) for the tested five concentrations of the Hyptis suaveolens essential oil
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Table 8 Repellent activity against mosquitoes, through different application methods of Hyptis suaveolens (L.) Poiteau (Lamiaceae)
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Conclusions

The present article improves the knowledge about the composition and the main constituents of the EO of an important tropical Lamiacea, such as H. suaveolens. Our data, compared with those reported in literature, confirm that its EO can have different chemical content as a function of the habitat where plants are grown. Investigation on larvicidal activity against A. albopictus demonstrated that H. suaveolens EO had insecticidal properties, thus widening its spectrum of action. Moreover, the study on EO repellency improved previous evidence from several studies, in which the repellent activity of H. suaveolens was proven through different application methods. Its insecticidal and/or repellent activity could be used for the development of new and safer products against A. albopictus larvae and adults.