Larvicidal & ovicidal efficacy of Pithecellobium dulce (Roxb.) Benth. (Fabaceae) against Anopheles stephensi Liston & Aedes aegypti Linn. (Diptera: Culicidae)

Mosquitoes are the major vector for the transmission of malaria, dengue fever, yellow fever, chikungunya, filariasis, schistosomiasis, and Japanese encephalitis. Mosquitoes also cause allergic responses that include local skin and systemic reactions such as angioedema in humans1. Aedes aegypti (L.) is generally known as a vector for an arbovirus responsible for dengue fever, which is endemic to Southeast Asia, the Pacific island area, Africa, and the Americas, and also for yellow fever in Central and South America and West Africa2. In the past decade, chikungunya - a virus transmitted by Aedes spp mosquitoes - has re-emerged in Africa, southern and southeastern Asia, and the Indian Ocean Islands as the cause of large outbreaks of human disease3. Malaria is a protozoan infection of erythrocytes caused in human beings by five species of the genus Plasmodium (P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi). In most cases, malaria is transmitted via the bite of an infected female anopheline mosquito, but congenital malaria and acquisition through infected blood transfusion are well described4. More than 40 per cent of the world's population - approximately 3 billion people - are exposed to malaria in 108 endemic countries5. About one million cases of malaria are reported in India every year. In 2010 an estimated 219 million (range 154-289 million) cases occurred worldwide and 660,000 people died (range 610 000-971 000), mostly in children under five years of age6. Presently, organochlorine, organophosphate, and synthetic pyrethroid insecticides are being used for mosquito control. Successive changes in the insecticides result in multiple insecticide resistant malaria vectors. Malaria vectors in India are resistant to several insecticides (DDT, HCH, malathion, and deltamethrin)7.

There has been an increasing interest in anti-mosquito products derived from natural origin because the continued applications of synthetic compounds have some drawbacks, including the widespread development of insecticide resistance8. Another drawback with the use of chemical insecticides is that these are non-selective and could be harmful to other organisms in the environment. The toxicity problem, together with the growing incidence of insect resistance, has called attention to the need for novel insecticides, and for more detailed studies of naturally-occurring insecticides9. It is, therefore, necessary to develop new materials for controlling mosquitoes in an environmentally safe way, using biodegradable and target-specific insecticides against them10.

The larvicidal activity of crude acetone, hexane, ethyl acetate, methanol, and petroleum ether extracts of the leaf of Centella asiatica, Datura metal, Mukia scabrella, Toddalia asiatica, extracts of whole plant of Citrullus colocynthis and Sphaeranthus indicus11; the leaf benzene, chloroform, ethyl acetate, and methanol extracts of Acalypha indica12; the methanol extracts of leaves of Dysoxylum malabaricum have been tested against mature and immature stages of An. stephensi under laboratory conditions13; root bark extracts of Turraea wakefieldii and Turraea floribunda against third-instar larvae14 and extracts of Pelargonium citrosa leaf have been tested for their biological, larvicidal, pupicidal, adulticidal, antiovipositional activity, repellency, and biting deterrency15 against An. stephensi. Hexane extract obtained from leaves of Eucalyptus citriodora was tested against larvae of An. stephensi, Culex quinquefasciatus, and Ae. aegypti to assess its toxicity and growth-inhibiting activity16.

Pithecellobium dulce Benth. (Fabaceae) is a small to medium sized, evergreen, spiny tree up to 18 m height, native of tropical America and cultivated throughout the plains of India (Vilayati babul). The presence of steroids, saponins, lipids, phospholipids, glycosides, glycolipids and polysaccharides has been reported in the seeds. The bark contains 37 per cent of tannins of catechol type. Quericitin, kaempferol, dulcitol and afezilin have been reported from the leaves17. The plant has been shown to have potential in treating a number of ailments where the free radicals have been reported to be the major factors contributing to the disorders. However, no information was available on the ovicidal and larvicidal activities of this plant species against An. stephensi and Ae. aegypti. Therefore, the aim of this study was to investigate the mosquito ovicidal and larvicidal activities of the different solvent extracts of P. dulce.

Material & Methods

Collection of plants: Fully developed leaves and seeds of the P. dulce were collected from Thanjavur district, Tamil Nadu, India. It was authenticated by a plant taxonomist from the Department of Botany, Annamalai University, Annamalainagar. A voucher specimen was deposited at the herbarium of plant phytochemistry division, Department of Zoology, Annamalai University.

Extraction: The healthy leaves and seeds were washed with tap water, shade-dried, and finely ground. The finely ground plant leaf and seed powder (1.0 kg/solvent) was loaded in Soxhlet extraction apparatus and was extracted with five different solvents, namely, hexane, benzene, chloroform, ethyl acetate and methanol, individually. The solvents from the extracts were removed using a rotary vacuum evaporator to collect the crude extract. Standard stock solutions were prepared at 1 per cent by dissolving the residues in ethanol. From this stock solution, different concentrations were prepared and these solutions were used for larvicidal and ovicidal bioassays.

Test organisms: The laboratory-bred pathogen free strains of mosquitoes were reared in the vector control laboratory, Department of Zoology, Annamalai University. The larvae were fed on dog biscuits and yeast powder in the 3:1 ratio. 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 1200 to 1600 h, and An. stephensi were fed during 1800 to 2200 h. A membrane feeder with the bottom end fitted with parafilm was placed with 2.0 ml of the blood sample (obtained from a slaughter house 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 per cent relative humidity, with a photo period of 12-h light and 12-h dark.

Larvicidal bioassay: The larvicidal activity of the plant crude extracts was evaluated as per the method recommended by World Health Organization18. Batches of 25 third instar larvae were transferred to a small disposable paper cups, each containing 200 ml of water. The appropriate volume of dilution was added to 200 ml water in the cups to obtain the desired target dosage, starting with the lowest concentration (60-450 mg/l). Four replicates were set up for each concentration, and an equal number of controls were set up simultaneously using tap water. To this, 1 ml of ethanol was added. The LC₅₀ (lethal concentration that kills 50 per cent of the exposed larvae) and LC₉₀ (lethal concentration that kills 90 per cent of the exposed larvae) values were calculated after 24 h by probit analysis19.

Ovicidal activity: For ovicidal activity, slightly modified method of Su and Mulla20 was performed. An. stephensi and Ae. aegypti eggs were collected from vector control laboratory, Department of Zoology, Annamalai University. The leaf and seed extracts were diluted in the ethanol to achieve various concentrations ranging from 100 to 750 mg/l. Eggs of these mosquito species (100) were exposed to each concentration of leaf and seed extracts. After 24 h treatment, the eggs from each concentration were individually transferred to distilled water cups for hatching assessment after counting the eggs under microscope. Each experiment was replicated six times along with appropriate control. The hatch rates were assessed 48 h post treatment by following formula:

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 (UCL) and lower confidence limit (LCL) values were calculated using the SPSS12.0 (Statistical Package of Social Sciences Inc., USA) software.

Results

The results of the larvicidal activity of crude hexane, benzene, chloroform, ethyl acetate, and methanol solvent leaf and seed extracts of P. dulce against the larvae of two important vector mosquitoes, viz. An. stephensi and Ae. aegypti are presented in Table I. Among the extracts tested, the highest larvicidal activity was observed in leaf methanol extract against An. stephensi followed by against Ae. aegypti with the LC₅₀ and LC₉₀ values were 145.43, 155.78 mg/l and 251.23, 279.73 mg/l, respectively. Compared to leaf extracts seed have low potency against two mosquitoes. The 95% confidence limits LC₅₀ (LCL-UCL) and LC₉₀ (LCL-UCL) were also calculated. The mean per cent egg hatchabilities of An. stephensi and Ae. aegypti were tested with five different solvents at different concentrations of P. dulce leaves and seed extracts, and the results are listed in Table II. The per cent hatchability was inversely proportional to the concentration of extract and directly proportional to the eggs. Zero hatchability of An. stephensi (Ae. aegypti) eggs was attained at the concentration of 400 mg/l (400 mg/l) for leaf extract and 500 mg/l (625 mg/l) for seed extract of P. dulce. Control eggs showed the 100 per cent hatchability. The leaf extract of P. dulce was found to be most effective than seed against larvae and eggs of the two vector mosquitoes.

Table I Larvicidal activity of different solvent leaf and seed extracts of P. dulce against Ae. aegypti and An. stephensi

Table II Ovicidal activity of P. dulce plant leaf and seed extracts against An. stephensi and Ae. aegypti

Discussion

Phytochemicals may serve as suitable alternatives to synthetic insecticides in future as these are relatively safe, inexpensive, and are readily available in many areas of the world. Different parts of plants contain a complex of chemicals with unique biological activity which is thought to be due to toxins and secondary metabolites, which act as mosquitocidal agents. Furthermore, the crude extracts may be more effective compared to the individual active compounds, due to natural synergism that discourages the development of resistance in the vectors. Our results showed that the crude hexane, benzene, chloroform, ethyl acetate, and methanol solvent extracts of leaf and seed of P. dulce were effective against the eggs and larvae of two important vector mosquitoes, viz. An. stephensi and Ae. aegypti. Chowdhury et al21 have reported that the chloroform and methanol extracts of mature leaves of Solanum villosum showed the LC₅₀ value for all instars between 24.20 and 33.73 mg/l after 24 h and between 23.47 and 30.63 mg/l after 48 h of exposure period against An. subpictus. The efficacy of 11 commonly available medicinal plants and compare its efficacy in relation to larvicidal activity against of An. stephensi. All the medicinal plants and the mixture were effective against larvae of A. stephensi as evidenced by low lethal concentration and lethal time22. The n-hexane, ethyl acetate and methanol extracts of Combretum nigricans, Jatropha curcas and Datura innoxia, Strophantus hispidus, Securidaca longepedunculata and Sapium grahamii exhibited 100 per cent mortality at 250 μg/ml concentrations against fourth instar larvae of Ochlerotatus triseriatus23.

Mullai and Jebanesan24 have reported that the ethyl acetate, petroleum ether and methanol leaf extracts of C. colocynthis and Cucurbita maxima had LC₅₀ values 47.58, 66.92 and 118.74 mg/l and 75.91, 117.73 and 171.64 mg/l, respectively, against Cx. quinquefasciatus larvae. Govindarajan25 evaluated larvicidal activity of crude extract of Sida acuta against three important mosquitoes with LC₅₀ values ranging between 38 and 48 mg/l. The crude extract had strong repellent action against the three species of mosquitoes as it provided 100 per cent protection against An. stephensi for 180 min followed by Ae. aegypti (150 min) and Cx. quinquefasciatus (120 min). The methanol extracts of Euphorbia tirucalli latex and stem bark were evaluated for larvicidal activity against laboratory-reared larvae of Cx. quinquefasciatus with LC₅₀ values of 177.14 and 513.387 mg/l, respectively26. The aqueous extract of Piperretrofractum showed LC₅₀ values of 5,124 and 9,681 mg/l against Cx. quinquefasciatus and Ae. aegypti, respectively27. The water extract of citrus-seeds showed LC₅₀ values of 135, 319.40 and 127, 411.88 mg/l against the larvae of Ae. aegypti and Cx. quinquefasciatus28. A crude chloroform extract of seeds of Millettia dura showed high activity (LC₅₀=3.5 μg/ml at 24 h) against second-instar larvae of Ae. aegypti29.

In conclusion, our findings showed that the plant P. dulce exhibits larvicidal and ovicidal activity against two important vector mosquitoes. These results could encourage the search for new active natural compounds offering an alternative to synthetic insecticides from other medicinal plants. P. dulce extracts may contribute greatly to save environment and to an overall reduction in the population density of two significant vectors (An. stephensi and Ae. aegypti). The results of this study also demonstrate the potential of new alternative sources of mosquito larvicides and ovicides which are generally free of adverse effects. The isolation and purification of these extracts are in progress and evaluation of these compounds will be needed to identify the active component.