Adult emergence inhibition and adulticidal activity of leaf crude extracts against Japanese encephalitis vector, Culex tritaeniorhynchus

1 Introduction

Japanese encephalitis (JE) outbreaks occur frequently in 14 Asian countries with about 3,060 million people at the risk of infection (Sabesan, 2003). JE is a major public health concern due to its high epidemic potential, high case fatality, and neuropsychiatric sequel among survivors. The annual incidence and mortality estimates for JE are 30,000–50,000 and 10,000, respectively, (Solomon, 2004). Approximately 2 billion people live in countries where JE presents a significant risk to humans and animals, particularly in China and India, with at least 700 million potentially susceptible children (Gould et al., 2008) and it is highly endemic in few districts of Tamil Nadu, Southern India (Ravi et al., 1989; Reuben and Gajanana, 1997). C. tritaeniorhynchus is a primary vector of Japanese encephalitis virus, with a distribution throughout Southeast and South Asia. The larval habitat is mainly rice fields and shallow marshes. Mosquito control represents an important strategy for prevention of disease transmission and epidemic outbreaks. However, a high level of insecticide resistance has developed through chemical control of the vector and pests threatening the control strategies. To overcome these problems, it is necessary to search for alternative methods of vector control. The failure of chemical insecticides to control the insect and growing public concern for safe food and a healthy environment have catalyzed the search for more environmentally benign control methods for the management of the vectors. Biopesticides provide an alternative to synthetic pesticides because of their generally low environmental pollution, low toxicity to humans and other advantages (Liu et al., 2000).

The present study was carried out on A. marmelos, an Indian plant with religious and medicinal importance and the stem bark ethyl acetate extracts exhibited moderate insecticidal activity against Phaedon cochleariae and Musca domestica (Samarasekera et al., 2004). Zaridah et al. (2001) have reported that the aqueous extract from the dried leaves of A. paniculata showed the strongest activity against adult worms of Brugia malayi. Elango et al. (2010) reported that the hexane extracts of A. marmelos and A. paniculata served as a potential repellent, ovicidal, and oviposition deterrent against C. tritaeniorhynchus. C. hirsutus is a widely growing plant found in the plains of India in dry localities and the methanol extract showed effective oviposition repellency against Anopheles subpictus (Elango et al., 2009a). E. prostrata, a member of the Asteraceae family and commonly known as False Daisy and the ethyl acetate extract of E. prostrata and leaf hexane extract of A. paniculata have the potential to be used against the fourth-instar larvae of A. subpictus and C. tritaeniorhynchus (Elango et al., 2009b). Macêdo et al. (1997) have reported that the ethanol extract of E. paniculata and Tagetes minuta were significantly active against the larvae of Aedes fluviatilis.

The callus tissue of T. erecta which showed the presence of insecticidal pyrethrin mixture was screened against Tribolium spp. Immediate ‘knock down’ effect was observed (Sarin, 2004), and the steam-distilled oils of T. patula, T. erecta and T. minuta were tested for larvicidal activity toward third instar A. aegypti and the results suggest a potential utilization of oil of T. minuta or its components for the control of A. aegypti and other species of mosquitoes (Green et al., 1991). The larvicidal potential of the essential oil of T. patula against A. aegypti, A. stephensi, and C. quinquefasciatus was evaluated (Dharmagadda et al., 2005); the compounds, 4 thiophenes, 5-(but-3-ene-1-ynyl)-2,2′-bithiophene,5-(but-3-ene-1-ynyl)-5′-methyl-2,2′-bithiophene,2,2′,5′,2″- terthiophene, and 5-methyl-2,2′,5′,2″-terthiophene isolated from the floral extract of T. minuta were largely responsible for the toxicity exhibited against the adults of A. aegypti and A. stephensi (Perich et al., 1995).

The bioactive constituents of these plants could be either a single substance or a mixture of substances. The separation of the mixture is neither practical nor advantageous in the insect economic control strategies. The aim of this study was to investigate the activity of different solvent extracts of six plant species from Tamil Nadu, South India. As far as our literature survey could ascertain, no information was available on the adult emergence inhibition and adulticidal activities of the experimental plant species given here. Therefore, the present study was an attempt to assess the adult emergence inhibition and adulticidal activity leaf extracts against C. tritaeniorhynchus. This is the first report of the solvent extracts of selected plants.

2 Materials and methods

3 Results

The activity of crude plant extracts is often attributed to the complex mixture of active compounds. The preliminary screening is a good means of evaluation of the potential of plants adult emergence inhibition and adulticidal activity of crude plant extracts is often attributed to the different solvent extracts of six plants which are noted and presented in Table 1 and Fig 1. Among the crude extracts tested, the present results showed that emergence inhibition of leaf methanol extract of A. marmelos, A.paniculata, T. erecta and chloroform extract of E. prostrata (EI50 = 141.94, 214.17, 166.43, and 184.58 ppm; EI90 = 590.26, 882.34, 532.00 and 571.81 ppm), and the maximum adulticidal activity observed in acetone extract of A. marmelos, hexane extract of A. lineata, ethyl acetate extract of A. paniculata, methanol extract of C. hirsutus, E. prostrata, and T. erecta (LD50 = 139.05, 251.24, 205.06, 222.10, 166.73, and 232.74 ppm; LD90 = 426.19, 837.09, 813.59, 794.42, 579.43 and 807.41 ppm), respectively against C. tritaeniorhynchus. The chi-square values calculated during the analysis did not show any heterogeneity of the response. Table 2 and 3 and Fig. 2 and 3.

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Figure 1

Adult emergence inhibition and adulticidal activity of different plant extracts against C. tritaeniorhynchus at 1000 ppm.

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Figure 2

The adult emergence inhibition activity of leaves extract against C. tritaeniorhynchus expressed as EI₅₀ and EI₉₀.

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Figure 3

Adulticidal activity of different plant extracts against C. tritaeniorhynchus expressed as LD₅₀ and LD₉₀.

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4 Discussion

In this study it was observed that, six plant leaves extract of different solvents have tested adult emergence inhibition and adulticidal activity against C. tritaeniorhynchus. The biological activity of the experimental plant extracts may be due to various compounds, including phenolics, terpenoides, flavonoids and alkaloids present in plants, of these compounds may jointly or independently contribute to produce adult emergence inhibition, and adulticidal effect against Japanese encephalitis vector, C. tritaeniorhynchus. Undoubtedly, plant-derived toxicants are a valuable source of potential insecticides. These and other naturally occurring insecticides play a prominent role in mosquito control programs in the future (Mordue and Blackwell, 1993). The results of this study may contribute to a great reduction in the application of synthetic insecticides, which in turn increase the opportunity for natural control of various medicinally important vectors by botanical chemicals. Since these are often active against a limited number of species including specific target insects, less expensive, easily biodegradable to non-toxic products, and potentially suitable for use in mosquito control programme (Alkofahi et al., 1989), they could lead to the development of new classes of possible safer insect control agents. Plant allelochemicals may be quite useful in increasing the efficacy of biological control agents because plants produce a large variety of compounds that increase their resistance to insect attack (Berenbaum, 1988; Murugan et al., 1996; Senthil Nathan et al., 2005).

Our results agree with some previous studies, such as the aqueous leaves extract of Calotropis procera reported that the 50% of adult emergence inhibition (EI50) was shown at 277.90 and 183.65 ppm for An. arabiensis and Cx. quinquefasciatus, respectively, and the pupal stage was not affected till a concentration of 5000 ppm (Elimam et al., 2009b). Similar study was conducted by Elimam et al. (2009a) and they reported that aqueous extracts from leaves of Ricinus communis showed 50% of adult emergence inhibition (EI50) was 374.97 and 1180.32 ppm against 3rd instar larvae of Anopheles arabiensis and Cx. quinquefasciatus and the extract showed oviposition deterrent effect against both species. The larvicidal, growth inhibitor and repellent actions of Dalbergia sissoo oil was evaluated against A. stephensi, A. aegypti and C. quinquefasciatus under laboratory conditions and observed no adult emergence was observed at 4 ml/m² (Ansari et al., 2000). Deformities produced in pupae further reduced adult emergence from 78% in the control to 75% at 6.25 ppm, 72% at 12.5 ppm, 70% at 25 and 50 ppm, and 66% at 100 ppm and the successful adult emergence from eggs treated was 78% in the control, which was reduced to 45%, 40%, 22%, 17%, and 10% at 6.25, 12.5, 25, 50, and 100 ppm, respectively was observed in methanol extract of Azadirachta indica on the development and growth index of C. quinquefasciatus (Sharma et al., 2009).

The effects of the tested extract, adult emergence and adulticidal activity of the mosquitoes are remarkably greater than those reported for other plant extracts in the literature. For example at the highest concentration, 50% inhibition of the emergence of the adult mosquitoes was observed by the use of the ethyl acetate fractions of Calophyllum inophyllum seed and leaf, Solanum suratense and Samadera indica leaf extracts and the petrol ether fraction of Rhinocanthus nasutus leaf extract on C. quinquefasciatus, A. stephensi and A. aegypti (Muthukrishnan and Puspalatha, 2001). Similarly 88% of the adult mortality was observed by the use of Pelargonium citrosa leaf extracts at 2% concentration against A. stephensi (Jeyabalan et al., 2003).

The present study results agree with Howard et al. (2009) who had reported that 50% inhibition of adult emergence (IE50) of all larval instars obtained with <0.4 g of neem bark chippings of A. indica in 1 L of distilled water. The hexane extract obtained from leaves of Eucalyptus citriodora tested at lowest concentration viz. 10 ppm, 73% larvae of An. stephensi failed to emerge as adult mosquito while in Cx. quinquefasciatus and Ae. aegypti only 10% and 6% larvae failed to emerge (Singh et al., 2007). Wiesman and Chapagain (2006) reported that the high total saponins compound isolated from methanol extracts of fruit mesocarp of Balanites aegyptiaca showed inhibition of 50% of the larval population from emerging adults (EC50 at 0.0014% (w/v) against A. aegypti. The neem formulation, Neem Azal produced an overall mortality or inhibition of emergence of 90% (EI(90), when 3(rd) instar larvae were treated) at 0.046, 0.208 and 0.866 ppm in An. stephensi, Cx. quinquefasciatus and Ae. aegypti, respectively (Gunasekaran et al., 2009). The extracts of Syzygium aromaticum were less toxic to the larvae; however their influence on development was remarkable, causing complete inhibition of adult emergence at 200 and 600 ppm concentrations of the methanol and ether extracts, respectively against Culex pipiens (El Hag et al., 1999). Three limonoids, namely limonin, nomilin and obacunone, isolated from the seeds of Citrus reticulata showed that the EC50 for inhibition of adult emergence were 6.31, 26.61 and 59.57 ppm for obacunone, nomilin and limonin, respectively on 4th instar larvae of mosquito C. quinquefasciatus (Jayaprakasha et al., 1997). Similar result was obtained in the root extract of Valeriana jatamansi which exhibited adulticidal activity of 90% lethal concentration against adult An. stephensi, An. culicifacies, Ae. aegypti, Anopheles albopictus, and Cx. quinquefasciatus and were 0.14, 0.16, 0.09, 0.08, and 0.17 and 0.24, 0.34, 0.25, 0.21, and 0.28 mg/cm2, respectively (Dua et al., 2008). Nathan et al. (2005) considered pure limonoids of neem seed, testing for biological, larvicidal, pupicidal, adulticidal, and antiovipositional activity, A. stephensi and the larval mortality was dose-dependent with the highest dose of 1 ppm azadirachtin evoking almost 100% mortality, affecting pupicidal and adulticidal activity and significantly decreased fecundity and longevity of A. stephensi. Choochote et al. (2005) also reported that the in testing for adulticidal activity, the hexane-extracted Curcuma aromatica (LC₅₀: 1.60 microg/mg female) was found to be slightly more effective against female A. aegypti than volatile oil (LC₅₀: 2.86 microg/mg female). The highest adulticidal effect was established from Piper sarmentosum, followed by Piper ribesoides and Piper longum, with LD₅₀ values of 0.14, 0.15 and 0.26 microg/female, respectively (Choochote et al., 2006). In testing for adulticidal activity, the crude seed extract of celery, Apium graveolens, exhibited a slightly adulticidal potency on A. aegypti with LD₅₀ and LD₉₅ values of 6.6 and 66.4 mg/cm², respectively, (Choochote et al., 2004). The unsaponifiable portion and volatile oil of Thymus capitatus showed the highest adulticidal potency (LC₅₀ = 0.0070 and 0.0076 mg/cm², respectively against Culex pipiens (Mansour et al., 2000). This result is also comparable to earlier reports of Dua et al. (2010) who observed that the adulticidal activity of the essential oil of Lantana camara was evaluated against different mosquitoes species on 0.208 mg/cm² impregnated papers, and the KDT50 and KDT90 values of the essential oil were 20, 18, 15, 12 and 14 min and 35, 28, 25, 18 and 23 min against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluvialitis and An. stephensi with their percent mortality of 93.3%, 95.2%, 100%, 100% and 100%, respectively. Also the result agrees with the finding of Halim (2008) who have reported the insecticidal activity of Zingiber officinale against the adult emergency of A. pharoensis 3rd stage was evaluated at the concentrations of 100%, 70%, 50%, 25%, 5%, 2%, 1%, 0.9%, 0.7%, 0.5% and 0.3% showed 100% larval mortality rate and at 0.2% and 0.1% caused mortality of 66.7%, respectively.

All the toxins used in vector control pose some hazards to the user and also to the aquatic environment (Kreutzweiser, 1997). Hence this research is mainly focused on finding newer insecticides which will be more effective, biodegradable and also easily available at low cost. In our observation, acetone extract of A. marmelos, hexane, extracts of A. lineata, the methanol of C. hirsutus, E. prostrata, and T. erecta possessed higher EI and adulticidal activity than the other solvent extracts.

In conclusion, the present study clearly proved that the efficacy of plant leaves extracts of A. lineata and T. erecta can be suggested as an adult emergence inhibition and adulticidal activity against C. tritaeniorhynchus activity. The results reported that the efficacy for controlling mosquitoes and mortality properties of natural product extracts. Since it is considered environmentally safe, the isolation and purification of crude extract of leaf hexane of A. lineata and methanol of T. erecta are in progress.