Introduction

Mosquitoes (Diptera: Culicidae) are vectors of important pathogens and parasites, such as malaria, lymphatic filariasis, Japanese encephalitis and yellow and dengue fevers which cause morbidity, mortality, economic loss, and social disruption (Mehlhorn et al. 2012; Benelli 2015a). The repeated use of synthetic insecticides for mosquito control has disrupted natural biological control systems and led to resurgences in mosquito populations. It also resulted in the development of resistance (Brown 1986), undesirable effects on non-target organisms, and fostered environmental and human health concern (Thomas et al. 2004). Culicidae eggs, larvae, and pupae are usually targeted using organophosphates, insect growth regulators, and microbial control agents. Indoor residual spraying and insecticide-treated bed nets are also employed to reduce transmission of malaria in tropical countries (Benelli 2015a). However, synthetic chemicals have strong negative effects on human health and the environment and induce resistance in a number of mosquito species (Wattanachai and Tintanon 1999; Hemingway and Ranson 2000).

Eco-friendly control tools are urgently needed. In the latest years, extensive research has been carried out to investigate the efficacy of botanical products against mosquito vectors (Benelli 2015b; Pavela 2015a, 2015b). People entering into regions where dengue, malaria, or yellow fever risks exist may protect themselves using plant-derived repellents (Mehlhorn et al. 2005, 2011, 2012; Amer and Mehlhorn 2006a, 2006b). On the other hand, people living in endemic regions have to protect themselves using several strategies at the same time, since infection rates of mosquitoes may be extremely high (Pushpanathan et al. 2006; Amer and Mehlhorn 2006c, 2006d; Semmler et al. 2009; Benelli et al. 2015a, 2015b, 2015c; Govindarajan and Benelli 2015; Pavela 2015a; Benelli 2015b). In this framework, recent research tested essential oils obtained from various plants from India, including Origanum vulgare (Govindarajan et al. 2016), Plectranthus barbatus (Govindarajan et al. 2015), Coleus aromaticus (Govindarajan et al. 2013a), Ocimum basilicum (Govindarajan et al. 2013b), Clausena anisata (Govindarajan 2010), and Mentha spicata (Govindarajan et al. 2012), against larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus.

Furthermore, repellency plays an important role in preventing the vector-borne diseases by reducing man–vector contact. However, some repellents of synthetic origin may cause skin irritation and affect the dermis (Das et al. 2000). The majority of commercial repellents are prepared by using chemicals like allethrin, N,N-diethyl-meta-toluamide (DEET), dimethyl phthalate (DMP), and N,N-diethyl mendelic acid amide (DEM). It has been reported that these chemical repellents are not safe for public use (Ronald et al. 1985). Because of unpleasant smell, oily feeling to some users, and potential toxicity, some prefer to use natural insect repellent products (Robbins and Cherniack 1986). Repellents of plant origin do not pose hazards of toxicity to human and domestic animals and are easily biodegradable. Natural products are safe for humans when compared to that of synthetic compounds (Sharma and Ansari 1994).

The genus Zingiber has about 85 species of aromatic herbs mostly distributed in East Asia and tropical Australia (Mabberley 1990). Plants belonging to Zingiberaceae are known for a number of medicinal properties (Basu 2002; Prajapathi et al. 2005). The term “Zingiber” is derived from the Sanskrit word “shringavera,” owing to their “horn shaped” rhizomes. Zingiber species are rich in volatile oils and are commonly used in traditional medicine and as spices. Zingiberaceae plants have significant medicinal properties (Kumar et al. 2006). They are having insecticidal, repellant (Millar 1998; Chane-Ming et al. 2003), anti-inflammatory, and chemopreventive activities (Kirana et al. 2003; Nakamura et al. 2004).

Zingiber nimmonii (J. Graham) Dalzell is an endemic species from the Western Ghats in South India, which grows both at low and high altitudes, in moist areas under the shades of trees (Sabu 2003). Z. nimmonii rhizome oil is a unique natural product with 69.9 % of isomeric caryophyllenes, viz. β-caryophyllene (42.2 %) and α-caryophyllene (27.7 %), along with traces of isocaryophyllene (0.03 %) in it. The major constituents of the rhizome oil of Z. nimmonii varied from the rhizome oils of Zingiber zerumbet and Zingiber officinale. The oil showed significant activities against the human pathogenic fungi, Candida glabrata, Candida albicans, and Aspergillus niger (Baby et al. 2006). To the best of our knowledge, the biotoxicity of Z. nimmonii essential oil (EO) against mosquito vectors is unknown.

In the present study, we investigated the larvicidal and repellent activity of the essential oil extracted from the rhizome of Z. nimmonii against the malaria vector An. stephensi, the dengue vector Ae. aegypti and the filariasis vector Cx. quinquefasciatus. The EO obtained from hydro-distillation was analyzed by gas chromatography–mass spectrometry (GC-MS), in order to identify its major constituents. Furthermore, the toxicity of this EO was assessed against two non-target species sharing the same ecological niche of mosquito larvae, Diplonychus indicus and Gambusia affinis.

Materials and methods

Plant material and extraction of essential oil

Z. nimmonii was collected from Nilgiris, Western Ghats (11° 10ʹ N to 11° 45ʹ N latitude and 76° 14ʹ E to 77° 2ʹ E longitude), Tamil Nadu, India. The plant was authenticated at the Department of Botany, Annamalai University. Vouchers specimens are deposited at the herbarium of Plant Phytochemistry Division, Department of Zoology, Annamalai University. The EO was obtained by the hydro-distillation of 3 kg of rhizomes in a Clevenger apparatus for 8 h. The oil layer was separated from the aqueous phase using a separating funnel. The resulting EO was dried over anhydrous sodium sulfate. The essential oil was stored in the dark at 4 °C until the testing phase.

Gas chromatography

Gas chromatography (GC) was carried on a Varian gas chromatograph equipped with a flame ionization detector and a BPI (100 % dimethyl polysiloxane) capillary column. Helium at a flow rate of 1.0 ml min−1 and 8 psi inlet pressure was employed as a carrier gas. Temperature was programmed from 60 to 220 °C at 5 °C min−1 with a final hold time of 6 min. The injector and detector temperatures were maintained at 250 and 300 °C, respectively. The sample (0.2 μl) was injected with 1:20 split ratio.

Gas chromatography–mass spectrometry

Gas chromatography–mass spectrometry (GC-MS) was performed using an Agilent 6890 GC equipped with 5973 N mass selective detector and an HP-5(5 % phenyl methyl polysiloxane) capillary column. The oven temperature was programmed from 50 to 280 °C at the rate of 4 °C min−1 and held at this temperature for 5 min. The inlet and interface temperatures were 250 and 280 °C, respectively. The carrier gas was helium at a flow rate of 1.0 ml min−1 (constant flow). The sample (0.2 μl) was injected with a split of 20:1. Electron impact mass spectrometry was carried out at 70 eV. Ion source and quadrupole temperatures ware maintained at 230 and 150 °C, respectively. The identification of EO compounds was based on the comparison of their retention indices and mass spectra with those in commercial libraries NIST 98.1 and Mass Finder 3.1. The concentration of each EO component was calculated from the integration area of the chromatographer.

Mosquito rearing

Laboratory-bred pathogen-free strains of the three mosquito vectors tested in this study were reared in the Vector Control Laboratory, Department of Zoology, Annamalai University. At the time of adult feeding, these mosquitoes were 3–4 days old after emergences (maintained on raisins and water) and were starved for 12 h before feeding. Each time, 500 mosquitoes per cage were fed on blood using a feeding unit fitted with Parafilm as membrane for 4 h. Ae. aegypti feeding was done from 12 noon to 4.00 p.m. and An. stephensi and Cx. quinquefasciatus were fed during 6.00 p.m. to 10.00 p.m. A membrane feeder with the bottom end fitted with Parafilm was placed with 2.0 ml of the blood sample (obtained from a slaughterhouse by collecting in a heparinized vial and stored at 4 °C) and kept over a netted cage of mosquitoes. The blood was stirred continuously using an automated stirring device, and a constant temperature of 37 °C were maintained using a water jacket circulating system. After feeding, the fully engorged females were separated and maintained on raisins. Mosquitoes were held at 28 ± 2 °C, 70–85 % R.H., with a photoperiod of 12-h light and 12-h dark.

Larvicidal activity

Larvicidal activity of the Z. nimmonii EO was evaluated following World Health Organization (2005). EO was tested at 20, 40, 60, 80, and 100 μg/ml. EO was dissolved in 1 ml DMSO, and then diluted in 249 ml of filtered tap water to obtain each of the desired concentrations. The control was prepared using 1 ml of DMSO in 249 ml of water. Twenty early third instar larvae were introduced into each solution. For each concentration, five replicates were performed. Larval mortality was recorded at 24 h after exposure, during which no food was given to the larvae.

Repellent activity

The EO was applied on a membrane used for membrane feeding of unfed mosquitoes in a 1-ft cage. About 50 unfed 3–4-day-old laboratory-reared pathogen-free strains of Cx. quinquefasciatus, Ae. aegypti, and An. stephensi was introduced in a 1-ft cage fitted with a membrane with blood for feeding with temperature maintained at 37 °C through circulating water bath maintained at 40–45 °C. The time taken for the first feeding in the cage containing the membrane treated with repellent needs to be observed at 30-min intervals, and each observation was made for 60 s. The experiment was repeated at this application rate for five times to confirm reproducible results. The time taken for feeding is considered as the protection time (in hours). Each test included one membrane feeding unit as control without applying any repellent. The testing period was 6:00 a.m. to 2:00 p.m. for Ae. aegypti and 6:00 p.m. to 2:00 a.m. for An. stephensi and Cx. quinquefasciatus.

If more than 4 h are taken at 2 mg/cm2 application, the EO was considered to exhibit promising repellency. If the protection time is <1–2 h at 2 mg/cm2 application rate, the EO may be discarded for repellence testing. ‘The percentage of repellency was calculated by the following formula:

$$ \%\ \mathrm{Repellency} = \left[\left({T}_a\hbox{--}\ {T}_b\right)/{T}_a\right] \times 100 $$

Where T a is the number of mosquitoes in the control group and T b is the number of mosquitoes in the treated group.

Acute toxicity on non-target organisms

The acute toxicity of Z. nimmonii EO to non-target organisms was assessed following the method by Sivagnaname and Kalyanasundaram (2004). The effect of EO was tested against non-target organisms D. indicus and G. affinis. The species were field collected and separately maintained in cement tanks (85-cm diameter and 30-cm depth) containing water at 27 ± 3 ° C and relative humidity 85 %. The Z. nimmonii EO was also tested at a concentration of even 50 times higher than the lethal concentration (LC)50 dose for mosquito larvae. Ten replicates were performed for each concentration along with four replicates of untreated controls. The non-target organisms were observed for mortality and other abnormalities such as sluggishness and reduced swimming activity after 48-h exposure. The exposed non-target organisms were also observed continuously for 10 days to understand the post treatment effect of this extract on survival and swimming activity.

Data analysis

Mortality data were subjected to probit analysis. LC50 and LC90 were calculated using the method by Finney (1971). Repellency data were analyzed using two-way ANOVA followed by Tukey’s honest significant difference (HSD) test (P < 0.05). In experiments evaluating toxicity against non-target organisms, the Suitability Index (SI) was calculated for each non-target species using the following formula (Deo et al. 1988)

$$ \mathrm{S}\mathrm{I}=\frac{{\mathrm{LC}}_{50}\kern0.5em \mathrm{o}\mathrm{f}\ \mathrm{n}\mathrm{o}\mathrm{n}\hbox{-} \mathrm{target}\ \mathrm{o}\mathrm{rganisms}}{{\mathrm{LC}}_{50}\kern0.5em \mathrm{o}\mathrm{f}\ \mathrm{target}\ \mathrm{vector}\ \mathrm{species}} $$

All data were analyzed using the SPSS Statistical Software Package version 16.0. A probability level of P < 0.05 was used for the significance of differences between values.

Results

Chemical composition of the essential oil

The yield of Z. nimmonii EO was 16.9 ml/kg fresh weight. Table 1 showed the constituents of the EO, their percentage composition, and their Kovats Index (KI) values listed in order of elution. A total of 33 compounds were identified, representing 97.3 % of the EO. The major constituents were myrcene (5.1 %), β-caryophyllene (26.9 %), α-humulene (19.6), and α-cadinol (5.2). Chemical structures of four major constituents were reported in Fig. 1. The percentage compositions of the remaining 29 compounds ranged from 0.7 to 2.8 %.

Table 1 Chemical composition of Zingiber nimmonii essential oil
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Fig. 1
figure 1

Chemical structure of the four major constituents of Zingiber nimmonii essential oil

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Mosquito larvicidal and repellent activity

The toxicity of EO from Z. nimmonii against early third larvae of mosquito vectors An. stephensi, Ae. aegypti, and Cx. quinquefasciatus, were presented in Table 2. The EO from the rhizome of Z. nimmonii exhibited significant larvicidal activity, with the LC50 and LC90 values of 41, 44, and 48 and 80, 85, 90 ppm, respectively. The EO of Z. nimmonii shows significant repellency against An. stephensi, Ae. aegypti, and Cx. quinquefasciatus (Table 3). Repellency depended on the strength of the EO concentration. A higher concentration of 5.0 mg/cm2 provided 100 % protection up to 180, 150, and 120 min, respectively.

Table 2 Larvicidal activity of essential oil from Zingiber nimmonii against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus
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Table 3 Repellent activity of essential oil of Zingiber nimmonii against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus
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Effect on non-target aquatic organisms

The acute toxicity of the Z. nimmonii EO towards non-target organisms D. indicus and G. affinis were presented in Table 4. Interestingly, LC50 values were 3241.53 and 16,670.30 μg/ml. G. affinis was less susceptible to the Z. nimmonii EO when compared to D. indicus. Overall, SI/PSF indicated that the Z. nimmonii EO was less harmful to the non-target organism if compared to the targeted mosquito species (Table 5). Focal observations conducted during the testing phase also showed that the survival and swimming activity of the non-target species were not altered during the exposure concentrations of target species.

Table 4 Biotoxicity of Zingiber nimmonii essential oil against two non-target organisms sharing the same ecological niche of Anopheles, Aedes and Culex mosquito vectors
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Table 5 Suitability index of non-target organisms over young instars of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus exposed to Zingiber nimmonii essential oil
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Discussion

Different parts of plants contain a complex of chemicals with unique biological activity (Farnsworth and Bingel 1977; Benelli, 2015b; Pavela 2015a) which is thought to be due to toxins and secondary metabolites, which act as attractants or deterrents (Fisher 1991). Our result showed that EO from the rhizome of Z. nimmonii has significant larvicidal as well as repellent activity against several mosquito vectors of economic importance. This result is also comparable to earlier research by Singh et al. (2003) who observed the larvicidal activity of Ocimum canum oil against vector mosquitoes Ae. aegypti and Cx. quinquefasciatus (LC50 301 ppm) and An. stephensi (234 ppm). Traboulsi et al. (2005) reported that the larvicidal activity of EO of Citrus sinensis, Eucalyptus spp., Ferula hermonis, Laurus nobilis, and Pinus pinea against Cx. pipiens. LC50 values were 60.0, 120.0, 44.0, 117.0, and 75.0 ppm, respectively. The EO of Z. nimmonii exhibited higher toxic action, if compared to the reported plants. Also, the EO of Tagetes minuta, providing a repellency of 90 % protection for 2 h was observed by Tyagi et al. (1994). EO obtained from Vitex negundo leaves shows repellency ranged from 1 to 3 h (Hebbalkar et al. 1992).

In the present study, Z. nimmonii rhizome extracted essential oil exhibited larvicidal activity LC50 = 41.19, 44.46, and 48.26 ppm against An. stephensi, Ae. aegypti, and Cx. quinquefasciatus, respectively. These values are higher or comparable to other recent reports. For instance, Pushpanathan et al. (2008) have reported larvicidal activity of Z. officinalis oil as LC50 = 50.78 ppm against Cx. quinquefasciatus, while in the present work, it was 48.26 ppm. This variation may be also due to the difference in strains of Cx. quinquefasciatus, since it has been shown that there is a vast difference between two strains of same species in respect of bioactivity (Tare et al. 2004). Pushpanathan et al. (2008) also reported that Z. officinalis oil offers 100 % protection for 2 h against Cx. quinquefasciatus at 4 mg/cm2, while we have obtained 2-h protection at 0.5 mg/cm2.

Monoterpenes such as α-pinene, cineole, eugenol, limonene, terpinolene, citronellol, citronellal, camphor, and thymol are common constituents of a number of EOs described in the literature as mosquito repellents (Yang et al. 2004; Park et al. 2005; Jaenson et al. 2006). Among sesquiterpenes, β-caryophyllene is highly cited as a strong repellent against Ae. aegypti (Gillij et al. 2008). Although repellent properties of several EOs regularly appear to be associated with the presence of monoterpenoids and sesquiterpenes (Jaenson et al. 2006), Odalo et al. (2005) also showed that phytol, a linear diterpene alcohol, has high repellent activity against Anopheles gambiae. Notably, the oxygenated compounds phenylethyl alcohol, β-citronellol, cinnamyl alcohol, geraniol, and α-pinene, isolated from the essential oil of Dianthus caryophyllum, also showed strong repellent activities against ticks (Ixodes ricinus) (Tunón et al. 2006).

Toloza et al. (2008) evaluated the repellent activity of 16 essential oils from native and exotic Argentine plants and 21 isolated metabolites; three alcohols (benzyl alcohol, menthol and thymol) were found as the most effective towards Pediculus humanus capitis. Omolo et al. (2004) and Odalo et al. (2005) evaluated the repellent activities of 12 Kenyan plants from different genus against An. gambiae, and some pure metabolites extracted from them. The most effective repelling chemicals were perillyl alcohol, cisverbenol, cis-carveol, geraniol, citronellal, perillaldehyde, caryophyllene oxide, carvacrol, 4-isopropyl benzene methanol, thymol, 3-carene, and myrcene. These belong to different structural types such as sesquiterpenoid, diterpenoid and acyclic, monocyclic, and bicyclic monoterpenoids.

Panneerselvam and Murugan (2013) observed the repellent activity of An. stephensi the hexane, ethyl acetate, benzene, aqueous, and methanol extract of Andrographis paniculata, Cassia occidentalis, and Euphorbia hirta plants at three different concentrations of 1.0, 3.0, and 6.0 mg/cm2. Five different concentrations, 5, 10, 15, 20, and 25 % (v/v), were prepared from each extract stock. Topical application of the extract concentrations on human volunteers revealed that 20 and 25 % repelled mosquitoes for at least 2 and 5 h, respectively. The methanol extract of Ervatamia coronaria was found to be more repellent than Caesalpinia pulcherrima extract. A higher concentration of 5.0 mg/cm2 provided 100 % protection up to 150, 180, and 210 min against Cx. quinquefasciatus, Ae. aegypti, and An. stephensi, respectively (Govindarajan et al. 2011). Karunamoorthi et al. (2008) have reported that the leaves of Echinops sp. (92.47 %), Ostostegia integrifolia (90.10 %), and Olea europaea (79.78 %) were effective and efficient to drive away mosquitoes, and the roots of Silene macroserene (93.61 %), leaves of Echinops sp. (92.47 %), Os. integrifolia (90.10 %), and Ol. europaea (79.78 %) exhibited a significant repellency by direct burning.

Choi et al. (2002) tested the repellent activity of Lavandula officinalis and Rosmarinus officinalis EOs against Culex pipiens pallens, showing an effective repellent effect mainly due to adult mosquitoes due to α-terpinene, carvacrol, and thymol. Tawatsin et al. (2001) have reported repellent activity against Ae. aegypti, Anopheles dirus, and Cx. quinquefasciatus, which is due to 5 % vanillin, which has been added to the EO of Curcuma longa. Neem products are good mosquito repellents, showing from 90 to 100 % protection against malaria vectors and about 70 % against Cx. quinquefasciatus (Ansari and Razdan 1994). Autran et al. (2009) have reported that the EO from leaves and stems of Piper marginatum exhibited an oviposition deterrent effect against Ae. aegypti at 50 and 100 ppm, since significantly lower numbers of eggs (<50 %) were laid in glass vessels containing the test solutions compared with the control. The oviposition deterrent properties against An. stephensi have been observed for various plant extracts including the methanol extract of Pelargonium citrosa, which exhibited 56 and 92 % inhibition of oviposition at 1 and 4 ppm, respectively (Jeyabalan et al. 2003).

Conclusions

Overall, this research adds knowledge to develop newer and safer natural larvicides and repellent against malaria, dengue, and lymphatic filariasis mosquito vectors. The plant tested in the study, Z. nimmonii, is available in large quantities in India and other Asian countries. The cost involved in the preparation of the Z. nimmonii EO is minimal. In addition, natural products are generally preferred in vector control measures due to their less deleterious effect on non-target organisms and their innate biodegradability (Pavela 2015a). In the context of resistance developed by the mosquito larvae against chemical insecticides, it is worthwhile to identify new larvicidal compounds from natural products against mosquitoes (Benelli 2016a, b). The results reported here open the possibility of further investigations of efficacy on their larvicidal properties of natural product extracts. In particular, Z. nimmonii EO may be utilized by local people for controlling mosquito larvae in small breeding places like water coolers, tree holes, abandoned wells, drums, and containers in and around the rural/suburban dwellings. Such practice would not only reduce environmental pollution but also promote sustainable utilization of locally available bio-resources by marginalized rural communities.