Background

Mosquitoes are one of the most medically significant vectors, and they transmits parasites and pathogens, which continue to have devastating impact on human beings; the vector-borne disease caused by mosquitoes are one of the major health problems in many countries. Malaria, dengue, yellow fever, and filariasis are few of the deadly disease spread by mosquitoes (Maheswaran, Kingsley, & Ignacimuthu, 2008). The mosquito is the principal vector of the vector-borne disease affecting human beings and other animals; several mosquito species including Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus are vectors for the pathogens of various diseases (Karthikeyan, Sivakumar, Aishwarya, & Mohanasundram, 2012).

An. stephensi (Liston) is the primary vector of malaria in India and other West Asian countries; malaria remains one of the most prevalent diseases in the tropical world. With 200 million to 450 million infections annually worldwide, it causes up to 2.7 million deaths (World Health Organization, 2010).

C. quinquefasciatus (Say.) acts as a vector for filariasis in India. Lymphatic filariasis caused by Wuchereria bancrofti and transmitted by mosquito C. quinquefasciatus is found to be more endemic in the Indian subcontinent. It is reported that C. quinquefasciatus infects more than 100 million individuals worldwide annually (Govindarajan, Mathivanan, Elumalai, Krishnappa, & Anandan, 2011).

The control of mosquito larvae worldwide depends primarily on continued applications of synthetic insecticides including organophosphates and chlorinated hydrocarbons (e.g., DDT); however, heavy wide use of these insecticides has caused several environmental and health concerons (Chen, Chao, Ligang, & Zhi, 2013).

Botanical pesticides have the advantage of providing novel modes of actions against insects that can reduce the risk of cross resistance as well as offering new designs for specific molecule targets. During the screening program for new agrochemicals from Chinese medical herbs and wild plants, essential oil of Ageratum conyzoides L. aerial parts at flowering stage was found to possess strong insecticidal toxicity against the Asian tiger mosquito Aedes albopictus (Liu & Liu, 2014).

Essential oils play important role in controlling several mosquito species. In general, essential oils from plants have been considered important natural resources to act as insecticides (Gbolade, Dyedele, Sosan, Adewayin, & Soylea, 2012); they are effective, environmentally friendly, and easily biodegradable in nature. It is suggested that many compounds derived from various essential oils can cause toxic activity against mosquito species (Bhat & Kempraj, 2009).

The use of herbal products is one of the best alternatives for mosquito control. Many researchers have been reported on the larvicidal properties of plant essential oils against Anopheles mosquitoes. Essential oils extracted from Azadirachta indica (Okumu, Knols, & Fillinger, 2007) and leaves and rhizomes of Curcuma longa (Molodchik, 2013). Plectranthus amboinicus, Zanthoxylum aratum, Eucalyptus tereticornis, and Tagetes patula demonstrated larvicidal activity against Anopheles stephensi (Dharmagadda, Naik, Mittal, & Asudevan, 2014). Larvicidal activity of essential oils from Blumea mollis and Zingifer officinalis (Pushpanathan, Jebanesan, & Govindarajan, 2008) has been reported against C. quinquefasciatus.

Larvicidal activity of essential oils from Melaleuca leucadendron, Litsea cubeba and Listea salicifolia, Ocimum suave, and O. kilimandshricum (Kweka, Mosha, Lowassa, Mahande, Kitau, Matowo, Mahande, Massenga, Tenu, Feston, Lyatuu, Mboya, Meneme, Chuwa, & Temu, 2008) have been reported against Anopheles arabiensis, An. gambiae, and C. quinquefasciatus. Larvicidal activity of essential oils from Zanthoxylum armatum (Tiwary, Naik, Tewary, Mittal, & Yadav, 2007) and Ocimum canum (Singh, Kumari, & Chauhan, 2003) have been reported against C. quinquefasciatus, Ae. aegypti, and An. stephensi. Essential oils derived from various plants not only exhibit inhibitory activity against bacteria, fungi, and termites but also show strong mosquito repellent larvicidal activities; the present study was aimed to assess the larvicidal and knockdown activities of the essential oils from various plants against C. quinquefasciatus, Ae. aegypti, and An. stephensi (Cheng, Liu, Tsai, Chen, & Chang, 2004).

Leucas aspera (Wild.) Link of the family Lamiaceae is an annual, branched herb that commonly grows in grassland. It is distributed throughout India from the Himalaya to Ceylon. In traditional medicine, this plant is used as an antipyretic and insecticide, and diverse biological activities such as antioxidant, antimicrobial, hepatoprotective, antinociceptive cytotoxic, and anthelminthic have been reported. The major volatile constituents are alpha-farnesene, alpha-thujene, and menthol leaves, while namely propionate and isoamyl propionate from flowers of L. aspera have been reported from India (Mangathayatu, Amitabha, Rajeev, & Kaushik, 2006). In another report from Nepal, the main constituents from the essential oil of aerial parts of L. aspera were identified as1-octen-3-ol, caryophyllene, and caryophyllene oxide; the essential oil of the seeds of L. aspera has larvicidal properties against the mosquito Aedes aegypti (Joshi 2013). In the present study, the larvicidal activity of the oil extract of L. aspera leaves were investigated against Ae. aegypti, An. stephensi, and C. quinquefasciatus.

Methods

Collection of plant

Leucas aspera plant leaves were collected from Kancheepuram District, Tamil Nadu, India during the month of January, 2016. L. aspera (Fig. 1) was identified by (Voucher. No. 2110; Flora of South India by G.S. Gamble—Volume-II) Prof. P. Jayaraman, Plant Anatomy Research Centre (PARC), West Tambaram, Chennai-600045.

Fig. 1
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Leucas aspera

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Distillation of essential oils

Fresh leaves of L. aspera were subjected to hydrodistillation using a modified clevenger type apparatus for 3 h (Cheng, Liu, Tsai, Chen, & Chang, 2004). The yield was averaged over four experiments and calculated according to dry weight of the plant material. Essential oil was stored in an air-tight container prior to analysis by gas chromatography mass spectrometry (GC-MS).

Gas chromatography mass spectrometry analysis

The composition of the essential oil was determined using an Agilent 7890 GC-MS instrument. Oxygen-free nitrogen was used as a carrier gas and hydrogen was used for the flame. The GC conditions used were as follows: capillary column: fused silica (polydimethylsiloxane 0.25 μm film thickness); temperature program: 70 °C (2 min1), 70–230 °C (3 min1), 230–240 °C (5 min1), 270 °C (5 min1); carrier gas, held at 5 bar, linear velocity of 20 cm min1; injection port splitless at 250 °C; injection volume, 0.1 μL. The MS conditions were as follows: ionization EIat 70 eV; m/z range, 30–300 °C; scan rate 1 s1; ionization chamber at 180 °C; and transfer line at 280 °C. The identification of the essential oil constituents was done based on a comparison of their retention times and these constituents were further identified and authenticated using mass spectrophotometry (MS) data compared to the NIST mass spectral library.

Selection and identification of mosquito species

The important vector species of mosquitoes such as Ae. aegypti, An. stephensi, and C. quinquefasciatus were selected and identified in the Zonal Entomological Research Centre, Vellore, Tamil Nadu, India. An. stephensi is vector of malaria in India and larvae of these species are generally found in distinctly different habitat. Ae. aegypti is a vector for transmitting several tropical fevers such as dengue fever, chikungunya, yellow fever, and other diseases. C. quinquefasciatus (Say.) acts as a vector for filariasis in India. C. quinquefasciatus is the vector of West Nile which causes encephalitis or meningitis affecting the brain tissue resulting in permanent neurological damage.

Bioassays and larval mortality

Fourth instar larvae of Ae. aegypti, An. stephensi, and C. quinquefasciatus were exposed to test concentrations of 5, 10, 15, 20, and 25 ppm of essential oil for 24 h according to standard methods described by the World Health Organization (WHO, 1981). In the control setup, ethanol was applied in the water (1%) and the numbers of dead larvae were counted after 24 h of exposure and the percentage of mortality were analyzed from the average of five replicates. The lethal concentration (LC50 and LC90) were calculated by probit analysis (Finney, 1971).

Statistical analysis

The average larval mortality data were subjected to probit analysis for calculating LC50, LC90, and other statistics at 95% confidence limits of upper confidence limit and lower confidence limit and Chi-square values were calculated using the SPSS 11.5 (Statistical Package of Social Sciences) software. Results with P < 0.05 were considered to be statistically significant.

Result and discussion

The regression equations of the oil extract against fourth instar larvae of Ae. aegypti, An. stephensi, and C. quinquefasciatus after 24 h of exposure is represented in (Table 1). The results clearly indicate that the leaf oil extracts of L. aspera at very low concentration was toxic against all the three mosquito species tested. The oil extract was found to be potent against Ae. aegypti with LC50 and LC90 value of 15.59 ppm and 46.77 ppm when compared to An. stephensi (17.10 ppm and 51.20 ppm) and C. quinquefasciatus with LC50 and LC90 (16.19 ppm and 47.79 ppm) respectively. The essential oils were found to be relatively more toxic to the larvae of mosquitoes. Earlier studies involving the essential oils obtained from various plants, viz. Ocimum lamiifolium, Chenopodium ambrosioides, Mentha spicata, Eucalyptus globules, and Azadirachta indica (neem), showed larvicidal activity against the larvae of the Anopheles gambiae mosquito (Massebo, Tadesse, Bekele, Balkew, & Michael, 2009). The use of plant essential oils in insect control is an alternative pest control method for minimizing the noxious effects of some pesticides compounds on the environment (Fatope, Ibrahim, & Takeda, 1993).

Table 1 Larvicidal activity of oil extract of Leucas aspera against malaria, dengue, and filariasis vectors
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The results of GC-MS characterization of L. aspera are presented in (Table 2). In the essential oil of L. aspera, 30 components are present. Some major components observed are Longifolene, 1,4,7,-caryophyllene oxide, 1,6,-hexadecanoic acid, 8-heptadecene, naphthalene, heptacosane,1-chloro, phytol, 5-eicosene, IH-Cyclopropa naphthalene, pentadecanal, etc. GC-MS analysis shows the presence of 30 components (Table 3, Fig. 2). The essential oil from the leaf extract was found to be potent against Ae. aegypti when compared to An. stephensi and C. quinquefasciatus. Extracts of Lantana aculeata against Plutella xylostells and Spodoptera litura larvae showed antifeeding and repellant effect on tea mosquito bug (Deka & Handique, 1998). Essential oil of Ocimum americanus and Ocimum ratissium contains Caryophyllene as main constituent which possessed larvicidal activity against Ae. aegypti (Cavalcanti, Demorais, Lima, & Santana, 2004).

Table 2 Gas chromatography mass spectrometry of essential oil from the leaves of L. aspera
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Table 3 Activity of phyto-components identified in medicine of essential oil from the leaves of L. aspera
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Fig. 2
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GC-MS analysis of oil leaf extract of L. aspera

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Major constituents from the Tagetes patella essential oil such as limonene, β-ocimene, and β-caryophyllene possessed potent larvicidal activity (Rana & Rana, 2012). Similar compound such as limonene and β-caryophyllene present in L. aspera may be responsible for the potent larvicidal activity; these phytocompounds may be responsible for ecdysal failure and mortality (Hemalatha, Elumalai, Vignesh, Murugesan, & Kaleena, 2014). Besides toxic and repellent properties, essential oils have been shown to have a pronounced effect on the developmental period, growth, adult emergence, fecundity, fertility, and egg hatching of insects (Elango, Rahuman, Kamaraj, Zahir, & Bagavan, 2010).

All terpenoids, alcohols, ketones, and carboxylic esters showed toxicity to mosquito species. Monoterpene alcohols have been reported to be toxic against mosquito species Tiwary, Naik, Tewary, Mittal and Yadav (2007) reported larvicidal activity of the essential oil extracted from the seeds of Zanthoxylum armatum against three species of mosquito vectors, Ae. aegypti, An. stephensi, and C. quinquefasciatus. Sutthanont, Choochote, Tuetun, Junkum, Jitpakdi and Chaithong (2010)) investigated the chemical compositions and larvicidal potential of Citrus hystrix, Citrus reticulate, Zingiber zerumbet, Kaempferia galanya, and Syzygium aromaticum against mosquito vectors. They suggested the use of these essential oils from edible herbs as a potentially alternative source for developing novel larvicides to be used in controlling vectors of mosquito-borne diseases. Active compounds of L. aspera oil extracts may be responsible for the larvicidal activity. It is evident from the present study that plant oil extracts might have promising larvicidal efficacy and could be useful in producing newer, safer, and more effective natural compounds as larvicides.

Conclusion

Plant products are emerging as a potential source for mosquito control. From the present study, it is evident that the essential oil leaf extracts of L. aspera have promising larvicidal efficacy. Leaf oil extracts of the plant could be used in stagnant water bodies, which are the breeding grounds for the mosquitoes. The mode of action and larvicidal efficiency of the L. aspera oil extract under the field conditions should be scrutinized and determined. Besides, further investigation regarding the effect on non-target organism is extremely important and imperative in the near future.