Bioprospecting and development of a biopesticide for use against mosquitoes from selected plants in Msambweni subcounty, Kenya

Abstract

Diseases transmitted by mosquitoes affect many people globally with significant numbers of deaths. They are an impediment to economic development in countries where they occur. More than half of the world’s inhabitants are threatened with malaria and dengue infections. Another mosquito borne disease, yellow fever lacks cure. Half of those infected by it die and its remedy is personal protection and vaccination. Some mosquito control methods are expensive and toxic. It is difficult to eliminate all aquatic habitats while mosquito predators prey on non target organisms. Mosquitoes have developed resistance to most of insecticides in current use. Plant based insecticides are simple, cost-effective, readily available and culturally acceptable. The plants studied were selected with regard to ethnobotanical surveys carried on the area in addition to systematic reviews of literature on plants used in Msambweni sub county that have been reported to have various activities against mosquitoes. They were Plectranthus barbatus A, Adansonia digitata Linn, Tagetes minuta L., Ocimum suave, Azadirachta indica A. Juss. and Lantana camara L. They were investigated for phytochemical composition, skin and eye irritability, genotoxicity, mosquito repellency and larvicidal activity. Finally, a mosquito repellent formulation was developed. Phytochemical studies were performed using established procedures to test for the following secondary metabolites: alkaloids, tannins, saponins, flavonoids, sterols, terpenoids and glycosides. Assessment of possible genotoxic activity was by the Allium cepa test. Repellency tests were performed according to protocol of world health organization (WHO) using Aedes aegypti mosquitoes on human volunteers.Approval for use of human subjects was obtained from the Kenyatta National Hospital / University of Nairobi, ethical review committee (KNH/UoN ERC). The WHO bioassay method was used to determine knockdown effects of the extracts against mosquitoes. Acute dermal and ocular irritation potential was assessed using New Zealand white rabbits. Thereafter, the plants’ extracts were made into formulations of 10% and 20% which were also subjected to repellency and knockdown tests as outlined above. A. digitata and P. barbatus had alkaloids, tannins, saponins, sterols and flavonoids. O. suave had alkaloids, tannins, saponins and sterols. A. indica extracts had saponins, sterols and flavonoids while T. minuta had flavonoids, alkaloids, saponins and tannins. L. camara had alkaloids, saponins, flavonoids, glycosides while tannins, sterols, terpenoids, and were absent. Larvicidal activity was tested on 4th instar larva of A. aegypti. A hundred percent larval mortality was achieved by all extracts except aqueous extract of L. camara that killed 90% of the larvae. Acetone extracts of T. minuta and hexane extract of was O. suave were most active as they caused 100% larval mortality at all tested concentrations thus LD50 and LD95 could not be determined for these extracts. Mitotic inhibition was dose dependent. The most active was A. Indica causing significant mitotic inhibition at 67.95±0.72 for aqueous extracts, 70.36±0.68 for hexane extracts and 71.53±1.18 for acetone extracts while the least active was aqueous extract of A. Digitata at 1.42±0.68. A. indica produced binucleate and ghost cells. Regarding repellency activity, percent protection for L. camara was 98.33±1.67, 94.86±0.53 and 77.63±1.04 for acetone hexane and aqueous extracts respectively, P<0.001. In T. minuta, percent protection was 98.33±1.67 for acetone extracts, 98.33±1.67 for hexane extracts and 94.86±0.53 for aqueous extract, P=0.182. Percent protection for A. indica acetone extract was 98.33±1.67 for hexane 94.86±0.53 and aqueous 94.86±0.53 and P= 0.070. A. digitata had percent protection of 84.58±1.58, 75.27±4.07 and 80.00±4.71 for acetone, hexane and aqueous extracts with P=0.259. O. suave extracts had percent protection of 100.00±0.00 for acetone extracts, 94.86±0.5394 for the hexane extracts and 86±0.53 for aqueous extracts, P<0.001. The percent protection for P. barbatus was 84.47±2.70 for acetone extracts, 74.14±3.59 for hexane extracts and 80.53±2.81 for aqueous extracts, P=0.107. The positive control was N, N-diethyl-m-toluamide (DEET) and its percent protection was 98.33±1.67. Extracts of acetone had knockdown effects like citronella oil. In acute dermal and ocular irritation tests, the extracts did not cause any toxicity even after 14 days and 21 days of observation on the skin and eyes respectively. The two formulated products exhibited a repellency effect greater than or similar to DEET. When compared to Ballet® mosquito repellent jelly (which consists of paraffinum liquidum, cera microcrystalline, paraffin, tacromanthus camphoratus, pelargonium graveolens, isopropyl myristate, glycerol, myristyl myristate, citronellal, tocopherol acetate, cymbopogon nardus, geraniol, eucalyptus globulus, rosemarinus officinalis, citral, benzophenone-3, linalool and limonene) they showed a better repellency and knockdown activity P<0.001. In conclusion, all the plants in this study caused appreciable mortality on Aedes aegypti larvae after twenty four hours and did not produce dermal or eye irritation. The two formulated products can offer suitable substitutes to most non-naturally used agents for repelling mosquitoes. Further research is needed to evaluate the activity of the formulated products in field trials. These plants should be evaluated for development of efficacious and environment friendly larvicides. Conservation measures should be put in place to curb over-exploitation of the plants.