Introduction

Arthropods are dangerous vectors of important life-threatening and debilitating diseases. Among them, mosquitoes (Diptera: Culicidae) belonging to the genera Anopheles, Aedes, and Culex act as vectors of pathogens and parasites causing malaria, filariasis, Japanese encephalitis, dengue and dengue hemorrhagic fever, yellow fever, chikungunya, and, very recently, Zika virus (Benelli and Mehlhorn 2016; Ward and Benelli 2017). Several efforts have been made to improve the control of mosquito vectors. Besides the efficacy at low doses, the use of synthetic insecticides can lead to high costs, concerns for environmental sustainability, harmful effects on human health and other non-target populations, and development of insecticide resistance in the targeted pests (Benelli 2015a; Naqqash et al. 2016).

While chemical insecticides are usually based on a single active ingredient, plant-derived pesticides consist of a combination of molecules which can act concertedly on both behavioral and physiological processes (Jain et al. 2001; Pavela 2015). Thus, there is very little chance of resistance development in the treated arthropods. In this scenario, the identification of effective and eco-friendly biopesticides is crucial for the successful management of arthropod vectors (Lucia et al. 2007; Cheng et al. 2003, 2004, 2008, 2009a; Govindarajan and Benelli 2016a, b).

Essential oils (EOs) are complex mixtures of volatile organic compounds produced as secondary metabolites in plants. They include blends of terpenes, sesquiterpenes, and oxygenated compounds, such as alcohols, esters, ethers, aldehydes, ketones, lactones, phenols, and phenol ethers (Chericoni et al. 2004; Astani et al. 2010; Almeida et al. 2011). Plant EOs and their constituents have been proposed as effective pesticides, since they are able to evoke acute toxicity and oviposition and feeding deterrence, as well as repellency (Barnard 1999). The insecticidal properties of EO are widely documented (e.g., Pathak and Dixit 1988; Araujo et al. 2003; Traboulsi et al. 2002; Ansari et al. 2000a, b, 2005; Koul et al. 2008; Rattan 2010; Pavela 2008a, b; Urzúa et al. 2010; Benelli 2015b). To the best of our knowledge, any development of resistance to EOs and their constituents has been reported (Sharma et al. 1992; Cavalcanti et al. 2004; Pavela and Benelli 2016).

A growing number of researchers focused on the effectiveness of plant EOs against young mosquito instars (Pavela 2015). Good examples include Lippia sidoides (Carvalho et al. 2003), Tagetes patula (Dharmagadda et al. 2005), Pinus kesiya (Govindarajan et al. 2016a), Cordia leucomalloides and C. curassavica (Santos et al. 2006), Blumea mollis (Senthilkumar et al. 2008), Piper klotzschianum (Do Nascimento et al. 2013), Tetradium glabrifolium (Liu et al. 2015), Chloroxylon swietenia (Kiran et al. 2006), Origanum majorana (El-Akhal et al. 2014), Thymus vulgaris (El-Akhal et al. 2015), T. magnus (Park et al. 2012), T. transcaspicus (Dargahi et al. 2014), Cinnamomum osmophloeum (Cheng et al. 2009b), Clausena excavata (Cheng et al. 2009c), Toddalia asiatica (Liu et al. 2013), Saussurea lappa (Liu et al. 2012), Ipomoea carica (Thomas et al. 2004), and Zingiber officinalis (Pushpanathan et al. 2008). Pitasawata et al. (2007) and Champakaew et al. (2007) reported that Curcuma zedoaria EO showed larvicidal toxicity on Aedes aegypti, while the Zingiber zerumbet rhizome EO had larvicidal and pupicidal activity on anopheline mosquitoes (Tewtrakul et al. 1998). Sutthanont et al. (2010) recommended the use of this EO as mosquito larvicide. Kamaraj et al. (2010) reported that the hexane extract of Z. zerumbet had larval toxicity against Culex quinquefasciatus.

The family of Zingiberaceae represents a key source of herbal preparations and phytoconstituents of interest for current pharmacology, parasitology, and entomology (Burkill 1966; Negi et al. 1999; Scartezzini and Speroni 2000; Youko et al. 2000; Patricia et al. 2003; Jirovetz et al. 2003; Bendjeddou et al. 2003; Nguefack et al. 2004; Govindarajan et al. 2016b). The Zingiber officinale rhizome EO is one of the most studied Zingiberaceae EOs. It contains monoterpenoids and sesquiterpenoids. Main molecules are α-zingiberene, α-curcumene, β-bisabolene, and β-sesquiphellandrene (Menon 2007; Rana et al. 2008; Padmakumari et al. 2009), while Z. zerumbet EO shows a high content of the monocyclic sesquiterpene ketone zerumbone, as well as an oxygenated humulene derivative (Srivastava et al. 2000; Bhuiyan et al. 2009).

Zingiber cernuum Dalzell (Zingiberaceae), commonly known as curved-stem ginger, is widely found in the evergreen forests of Western Ghats, India. It is a large perennial herb, 1–2 m tall, with curved stem. The leaves are 15–30 cm in length, narrow-elliptic, and long-pointed. The flowers are borne in spikes 5–10 cm long, directly from the rootstock, rising just above the ground. Bracts are 2–3 cm long, greenish-yellow. The sepal cup is shortly three-lobed. Stamen is single, with a short filament. The style is threadlike. The capsules are 1 cm long, smooth with red, channeled seeds. The flowers are yellow colored, variegated with red, with the lib broad and three-lobed (Sanjay 2015). From a phytochemical point of view, Z. cernuum is extremely scarcely studied. Kasarkar and Kulkarni (2011) recently identified flavonoids and tannins in Z. cernuum extracts, which showed antioxidant activity (Sanjay 2015). This species is rich in iron and manganese and showed low amounts of molybdenum, sulfur, and nitrate in rhizome and leaves (Kasarkar and Kulkarni 2011). To the best of our knowledge, the composition and mosquitocidal bioactivity of Z. cernuum EO have not been explored.

In this research, we analyzed the chemical composition of Z. cernuum EO using gas chromatography-mass spectroscopy analysis. Furthermore, we studied the larvicidal and oviposition deterrent activity of the Z. cernuum EO on six mosquito species, the malaria vectors Anopheles stephensi and Anopheles subpictus; the dengue and Zika virus vectors Aedes aegypti and Aedes albopictus; the filariasis, West Nile virus, and St. Louis encephalitis vector Culex quinquefasciatus; and the Japanese encephalitis vector C. tritaeniorhynchus. In addition, to assess the biosafety of Z. cernuum EO-based treatments in the aquatic environment, we investigated the toxicity of Z. cernuum EO on four non-target enemies of mosquito young instars, the insects Anisops bouvieri and Diplonychus indicus and the fishes Poecilia reticulata and Gambusia affinis.

Materials and methods

Extraction and GC-MS analysis of the Z. cernuum essential oil

Fresh rhizomes of Z. cernuum were collected during May 2016 in the Munnar mountains (India 10° 05′ 21″ N 77° 03′ 35″ E, 1700 m a.s.l.). Four hundred grams of fresh rhizomes of Z. cernuum was hydrodistillated for 3 h using a modified Clevenger-type apparatus; then, the Z. cernuum EO was dried over anhydrous sodium sulfate and stored in the dark at 5 °C. GC and GC-MS analyses were carried out as recently described by Govindarajan and Benelli (2016a). The constituents of the Z. cernuum EO were identified by comparison of their mass spectra and retention indices (Table 1) with the ones indexed in the Wiley library, as well as those available in the literature (Adams 2007).

Table 1 Chemical composition of Zingiber cernuum essential oil
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Larvicidal and oviposition deterrence assays

The six mosquito species were reared as described by Govindarajan and Benelli (2016a). Early third instar larvae and adults were used to evaluate the larvicidal potential and oviposition deterrence, respectively. The larvicidal activity of the Z. cernuum EO was studied following the method by WHO (2005). Various doses of the Z. cernuum EO were dissolved in 1 ml dimethyl sulfoxide (DMSO) and then diluted in 249 ml of filtered tap water. Control was 1 ml of DMSO diluted in 249 ml of water. Within each replicate, 20 early third instar larvae were tested (WHO 2005); n = 5 per each dose.

In oviposition deterrent experiments, Z. cernuum EO was evaluated at a dose range of 40–250 μg/ml in DMSO. DMSO diluted in water served as a control. We followed the method by Xue et al. (2001). Twenty gravid females (5–7 days old) of each mosquito species were released in the bioassay cage (60 × 60 × 45 cm). After 24 h, the number of eggs laid in treated and control bowls was counted using a stereomicroscope (Olympus, Japan).

Toxicity on non-target organisms

Toxicity on the four predators was assessed following Sivagnaname and Kalyanasundaram (2004) with minor modifications by Govindarajan and Benelli (2016a). The Z. cernuum EO was evaluated at doses of even 50 × LC50 values calculated for mosquito larvae studied in the paragraph above, 10 replicates per each dose, plus 4 control replicates (where no EO was added to the water). The mortality of non-target species was assessed 48 h post-treatment.

Data analysis

All data were analyzed using the SPSS Statistical Software Package version 16.0. LC50 and LC90 were estimated following the method by Finney (1971). The oviposition activity index (OAI) was calculated as indicated by Kramer and Mulla (1979):

$$ \mathrm{OAI}=\left(\mathrm{NT}-\mathrm{NC}\right)/\left(\mathrm{NT}+\mathrm{NC}\right) $$

Effective repellency (ER %) due to Z. cernuum EO was estimated following Xue et al. (2001). In non-target assays, the suitability index (SI) was calculated as described by Deo et al. (1988).

Results

GC and GC-MS of Z. cernuum essential oil

The yield of the Z. cernuum rhizome EO was 1.8 ml/kg of rhizome fresh weight. Table 1 showed a total of 28 compounds representing 96.2% of the Z. cernuum EO. The major constituents of Z. cernuum EO were δ-3-carene, trans-caryophyllene, and α-humulene (Fig. 1). The other 25 compounds ranged from 0.8 to 5.8%.

Fig. 1
figure 1

Chemical structures of the three major constituents of Zingiber cernuum essential oil. a δ-3-Carene. b trans-Caryophyllene. c α-Humulene

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Larvicidal and oviposition deterrent activity

The Z. cernuum EO showed acute toxicity on third instar larvae of Anopheles stephensi, Aedes aegypti, Culex quinquefasciatus, Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus, with LC50 of 41.34, 44.88, 48.44, 51.42, 55.84, and 60.20 μg/ml, respectively (Table 2). No mortality was detected in the control.

Table 2 Larvicidal activity of the essential oil from Zingiber cernuum on six mosquito vectors
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The results obtained from the oviposition deterrence experiments testing Z. cernuum EO on the six mosquito species are reported in Table 3. The mean number of eggs laid in sites treated with the Z. cernuum EO tested at the highest doses (i.e., 200–250 μg/ml) was 44.5, 41.5, and 36.7 eggs per bowl for Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus, respectively, and 56.2, 52.8, and 47.5 eggs per bowl for Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus, respectively. Significant differences (P < 0.05; P < 0.01) were detected comparing these values to the respective controls (Table 3). The range of OAI achieved by Z. cernuum EO tested against the six mosquito vectors at 200 and 250 μg/ml ranged from −0.79 to −0.84 (Table 3).

Table 3 Oviposition deterrent activity of the Zingiber cernuum essential oil on six mosquito vectors
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Toxicity on non-target predators

Z. cernuum EO toxicity on A. bouvieri, D. indicus, P. reticulata, and G. affinis was reported in Table 4 . LC50 values were 3119, 5273, 10,363, and 11,233 μg/ml, respectively. PSF indicated that the Z. cernuum EO showed scarce toxicity on A. bouvieri, D. indicus, P. reticulata, and G. affinis (Table 5). Survival and swimming activity of the non-target water bugs and fishes were not affected by the exposure to Z. cernuum EO LC50 and LC90 estimated on the six mosquito species.

Table 4 Toxicity of the Zingiber cernuum essential oil on non-target mosquito predators
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Table 5 Predator safety factor calculated on four mosquito predators and mosquito young instars post-treatment with Zingiber cernuum essential oil
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Discussion

GC and GC-MS of Z. cernuum essential oil

Our results showed that 28 compounds were identified in the Z. cernuum EO, with δ-3-carene, trans-caryophyllene, and α-humulene as main components. This highlighted a quite surprising composition, if compared to EOs extracted from other Zingiber species. Indeed, several studies have been conducted on the EOs from other Zingiber species, such as Z. officinale (Foko et al. 2011), Z. cassumunar (Jantan et al. 2003), Z. zerumbet (Tewtrakul et al. 1998), Z. piperitum (Kamsuk et al. 2006), Z. limonella (Somanabandhu et al. 1992), Z. armatum (Tiwary et al. 2007), and Z. monophyllum (Pavela and Govindarajan 2017). Campbell et al. (2011) pointed out that several mono- and sesquiterpenes, including trans-caryophyllene, α-terpineol, β-pinene, germacrene-D, limonene, and α-zingiberene, present in the EOs evoke responses in Aedes aegypti antennae. Sesquiterpenes α-curcumene, β-sesquiphellandrene, zingiberene, and β-bisabolene from Z. officinale EO (Campbell 2009), as well as trans-caryophyllene and Ocimum forskolei (Dekker et al. 2011), also induced antennal responses by the antennae of A. aegypti females.

Larvicidal and oviposition deterrent potential

Essential oils from plants can represent an alternative source of eco-friendly and biodegradable mosquito ovicides (Benelli 2015b), larvicides (Pavela 2015) and adult repellents (Barnard 1999). A growing number of researches concentrated on the effectiveness of plant EOs against mosquito young instars, with special reference to larvae (Sukumar et al. 1991; Benelli 2015b; Pavela 2015). According to Tawatsin et al. (2001), the bioactivity of EOs depends on various factors, including the plant species and cultivar, the growing conditions, the harvesting time, the storage conditions, and the extraction method (see also Pavela and Benelli 2016).

In our assays, the EO extracted from the rhizome of Z. cernuum showed high toxicity against third instar larvae of Anopheles stephensi (LC50 = 41.34 μg/ml), Aedes aegypti (LC50 = 44.88 μg/ml), Culex quinquefasciatus (LC50 = 48.44 μg/ml), Anopheles subpictus (LC50 = 51.42 μg/ml), Aedes albopictus (LC50 = 55.84 μg/ml), and Culex tritaeniorhynchus (LC50 = 60.20 μg/ml). Concerning the bioactivity of other EOs and extracts from the Zingiberaceae family, Rahuman et al. (2008) evaluated the larvicidal activity of 4-gingerol from Z. officinalis, against A. aegypti (4.25 ppm) and C. quinquefasciatus (5.52 ppm). Sutthanont et al. (2010) reported that Z. zerumbet and Kaempferia galanga EOs are effective on A. aegypti, with LC50 of 48.88 and 53.64 ppm, respectively. Tewtrakul et al. (1998) showed the toxicity of Z. zerumbet ethanol extract on anopheline larvae, with LD50 of 18.9 μg/ml. The Z. cassumunar EO is effective against A. aegypti larvae (LT50 = 1.4 min) (Jantan et al. 2003). Pitasawata et al. (2007) and Champakaew et al. (2007) noted that the C. zedoaria EO showed larvicidal activity on A. aegypti, with LC50 of 33.45 ppm. Z . zerumbet EO also showed larvicidal toxicity on A. aegypti and A. nuneztovari, with LC50 of 89.8 and 62.8 μg/ml, respectively (Tewtrakul et al. 1998). Other studies reported that Z. officinale EO tested at 20 mg/ml and 700 μl/ml effectively repelled stored product pests, such as Sitophilus zeamais and Prostephanus truncatus adults (Ogbonna et al. 2014).

The Z. cernuum EO tested in this study was mainly composed of δ-3-carene, trans-caryophyllene, and α-humulene. Recently, several effective mosquitocidal molecules have been identified in the EOs of other Indian plants. For example, Govindarajan and Benelli (2016a) investigated the toxicity of α-humulene and β-elemene from Syzygium zeylanicum EO on A. albopictus (LC50 = 6.86 and 11.15 μg/ml), C. tritaeniorhynchus (LC50 = 7.39 and 12.05 μg/ml), and A. subpictus (LC50 values were 6.19 and 10.26 μg/ml). Further research focusing on potential synergic larvicidal effects occurring among the abovementioned molecules is ongoing.

Concerning the oviposition deterrent potential, we observed that the range of OAI achieved by the Z. cernuum EO tested at 200 and 250 μg/ml compared with controls ranged from −0.7 to −0.8. Recently, a growing number of studies focused on the oviposition deterrent activity of plant extracts and EOs against mosquito vectors of economic importance (Elango et al. 2009). However, few of them investigated the oviposition deterrent potential of Zingiber species. Coria et al. (2008) reported 100% oviposition deterrent effect obtained with Melia azedarach leaf extract tested at 1 g/l concentration against Aedes aegypti. Prajapati et al. (2005) noted that the bark EO of Cinnamomum zeylanicum reduced the oviposition rates of A. aegypti to 50% when tested at 33.5 ppm. Autran et al. (2009) recorded the oviposition deterrent effect of EO obtained from leaves, inflorescences, and stems of Piper marginatum; the EOs from leaves and stems of P. marginatum exhibited oviposition deterrent effect on A. aegypti females at 50 and 100 ppm concentrations and the number of eggs laid was significantly lower (<50%), if compared to control.

Biotoxicity on mosquito predators

It is worthy to note that the toxicity of Z. cernuum EO on the mosquito predators A. bouvieri, D. indicus, G. affinis, and P. reticulata was very low, with LC50 values always higher than 3000 μg/ml. EOs have been recently recognized as novel and reliable biopesticides, which do not induce resistance and have few toxic effects on human health and non-target species. For example, scarce toxicity of P. kesiya EO on A. bouvieri, D. indicus, and G. affinis was noted, with LC50 from 4135 to 8390 mg/ml, and in agreement with the present results, G. affinis has been found less susceptible to EO, if compared to A. bouvieri and D. indicus (Govindarajan et al. 2016a). S. zeylanicum EO tested on G. affinis showed LC50 = 20,374.26 μg/ml (Govindarajan and Benelli 2016a). Moreover, Heracleum sprengelianum EO, lavandulyl acetate and bicyclogermacrene, tested on A. bouvieri, D. indicus, and G. affinis, led to LC50 ranging from 414 to 4219 μg/ml (Govindarajan and Benelli 2016b). Taken together, the data reported above underline the environmental-friendly nature of botanicals from selected Asian plant species, which can be further considered for as larvicides and oviposition deterrents in urban and peri-urban areas.

Conclusions

Overall, the present research sheds light on the chemical composition of the EO of Z. cernuum, as well as on its larvicidal and oviposition deterrent activity on six important mosquito species. Notably, really limited non-target effects of Z. cernuum EO were found on four important mosquito predators. Therefore, the results from this study supported our hypothesis to consider the Z. cernuum EO for the development of effective and eco-friendly larvicides and oviposition deterrents effective against a broad range of mosquito vector species.