Introduction

Alternatives to synthetic products against malaria vector Anopheles gambiae are being sort as the available products are less effective due to resistance by the vector (Djogbénou et al., 2011; Ranson et al., 2011; Tiwari et al. 2010) and that their residues persist in the environment and harm human and other non target animals (Cartilla and De la Cruz, 2012). Lately there has been intense search for effective phytochemicals to replace synthetic insecticides. The reason, they are target specific, eco-friendly, biodegradability, cost-effectiveness and possess a complex chemistry that limits development of resistance by target organisms or pests (Miresmailli et al., 2006).

Since the identification of pyrethrum, rotenone, neem, and essential oils (Isman, 2006) for the control of insect vectors, other phytochemical extracts have since been added to the list (Govindarajan and Karuppannan, 2011, Govindarajan, 2010). Some of these have included crude hexane, ethyl acetate, benzene, chloroform, and methanol extracts of leaf of Eclipta alba and A. paniculata against Cx. Quinquefasciatus and Ae. aegypti (Govindarajan and Sivakumar, 2012), Delonix elata (Family: Fabaceae) against dengue vector, Aedes aegypti (Mohan and Marimuthu, 2014) and ethanol extracts of citrus oil against Aedes albopictus (Faisal et al., 2010).

Extracts from Endod has also been used and found potent against snails (Abebe et al., 2005; Erko et al., 2002) and An. gambiae larvae (Were, 2008) but has not been tested against adult An. gambiae mosquitoes. This study reports on the test and activities of crude ethanol extracts of Endod on laboratory reared and field sourced An. gambiae adults on modeled surfaces in a laboratory set up.

1 Results

The experiment was done for a period of twelve days and a total of 22,400 laboratories and 2,520 fields sourced but laboratory cultured An. gambiae mosquitoes used. On Whatman No.1 filter papers, extracts of mature green fruits of Endod sourced from the highlands killed more than 60% and 30% of laboratory and field sourced female An. gambiae mosquitoes respectively. Extracts of Endod from the highlands were more toxic that those from the lowlands for both mature fruits and leaves. Extracts of Neem leaves killed less than 35% of both laboratory and field sourced mosquitoes. A threshold of >80% mortality of exposed mosquitoes was demonstrated for deltamethrin for laboratory sourced mosquitoes only (Table 1).

Table 1 Percent mortality of laboratory cultured and wild An. gambiae mosquitoes exposed to different crude ethanol extracts of Endod

Extracts of Endod leaves were slightly more toxic than extracts from mature green fruits, killing more than 61% of exposed mosquitoes as opposed to 55% for mature green fruits on cement surfaces. High mortalities where observed on modeled cement followed by plain and then mud smeared wall surfaces for all exposures. None of the used concentrations of Endod extracts met the WHO threshold of >80% for adulticidal effectiveness regardless of part of plant, geographical source or nature of surface used. Extracts of Neem met the WHO threshold on cement surfaces only and deltamethrin met the condition on all surfaces (Table 2).

Table 2 Percent mortality of exposed laboratory cultured An. gambiae mosquitoes exposed to treatments on modeled wall surfaces

2 Discussion

This study found crude ethanol extracts of Endod to have potential as adulticide against An. gambiae. Although the mortalities of exposed female mosquitoes were below the WHO threshold of > 80% mortality per exposure (WHO, 2006), the fact that these were exposures on crude extracts on improvised surfaces shows great potential. Interestingly a higher percent of exposed adult of An. gambiae mosquitoes died from exposure to extracts from Endod than Neem for both laboratory reared and wild mosquitoes. Neem has demonstrated effectiveness against mosquitoes (Shaalan et al., 2005) and other environmental pest of medical importance (Ogbuewu et al., 2011) and the fact that observed mortality of exposed mosquitoes to extracts of Endod were higher than those of Neem shows great promise on the potential of extracts of Endod as botanical adulticide.

When used on modeled wall surfaces, mortalities of both laboratories reared and wild sourced mosquitoes were higher for extracts of Endod leaves than fruits. In this study, percent mortalities of exposed mosquitoes were higher on cement surfaces followed by plain surfaces and lastly mud smeared. This finding was contrary to an earlier finding that found plain wooden surfaces to absorb more toxic components of bendiocarb WP, than either mud smeared or cement modeled surfaces but similar in the order of activity when deltamethrin WG was used (Etang et al., 2011).

Mortality of exposed mosquitoes was higher when extracts from Neem leaves were used on the modeled surfaces than extracts from either mature green fruits or leaves of Endod. In all cases the modeled wall surfaces were found to contribute significantly to the mortality of the exposed mosquitoes (p<0.05) when they were interrogated using Duncan’s regression procedure.

In conclusion, it was observed that ethanol extracts from mature green fruits and leaves of Endod were toxic against mature stages of Anopheles gambiae mosquitoes. With pure samples, we envisage extracts of Endod being used as alternative source of insecticide against malaria vector in future.

3 Materials and Methods

3.1 Experimental mosquitoes and study area

Field sourced and laboratory reared (pink eye) An. gambiae were used in these experiments. Field mosquitoes were sourced from Ahero (Kenya) rice fields where larvae were collected using dippers and modified two litre plastic water pails, processed and transported to the laboratory at the Centre for Global Health Research/Kenya Medical Research Institute (CGHR/KEMRI) for further processing and culturing. The mosquitoes were then reared following standard techniques (Das et al., 2007). The rearing and experiments were conducted at the Entomology insectary and laboratory of CGHR/KEMRI. The temperatures within the insectary and the laboratory were maintained at 28-30℃, relative humidity at 70-80% and photoperiod of 12h light alternating with 12h darkness.

3.2 Modeling experimental wall surfaces

Standard WHO procedures (WHO, 2006) with slight modification were used to model wall surfaces to represent common residential wall surfaces to which adult mosquitoes rest. The surfaces were created from plywood measuring 26 cm × 26 cm. The surfaces were either left plain to depict wooden wall, smeared with cement to depict permanent or semi permanent houses with cement plaster walls or smeared with a mixture of mud and cow dung to depict mud walled houses as described elsewhere (Yugi et al., 2014).

3.3 Plant extract and deltamethrin (KOTab 1-2-3^®) preparation

Leaves (shoot and midsection) and mature green fruits of Endod and leaves of Neem were used to obtain plant extracts. Endod plants were obtained from the highlands (Eldoret) and lowlands (Nyando) while Neem leaves were obtained from the lowlands (Nyando). The plants were prepared and extracted using standard methods (Parekh et al., 2005) and the extract kept in airtight glass bottles to serve as stock quantity. Deltamethrin (KOTab 1-2-3^®) tablet weighing 1.6g and containing 0.4g deltamethrin (pyrethroid (250g/kg)) was bought from a local chemist, ground to powder before aliquoting.

Eighty milligrams of freeze-dried stock of leaf and fruit extracts and deltamethrin powder were weighed and serially diluted to obtain different concentrations of 80, 40, 20, 10, 5 and 2.5 mg as described elsewhere (Yugi et al., 2014).

3.4 Adulticidal assays

A serial diluant of a particular preparation (Leaf or fruit extract of Endod, leaf extract of Neem or deltamethrin) was applied to a modeled surface and left to stand in a shade for an hour to dry before use. WHO exposure cones were then clipped on the surfaces and then ten three to five day old female An. gambiae mosquitoes introduced in the confinement and left exposed for 5 minutes. After exposure, the mosquitoes were transferred to clean, dry holding paper cups and provided with 10% sugar solution for energy supplement. Mortality was recorded after 24 hours and WHO threshold of >80% used as a measure of effectiveness of toxicants (WHO, 2006).

3.5 Data analysis

Data obtained from the bioassays was entered in excel spreadsheets and then corrected for control mortality using Abbott’s formula (Abbott, 1925). The relationship between mortality and the effect of the extracts on source (part of Endod plant used) and surface applied on was determined using one way analysis of variance (ANOVA). The means were separated using LSD and differences between them considered significant at P<0.05. All statistical analysis was performed using SAS statistical package version 20.

Authors’ contributions

YJO conceived the concept, conducted the experiments and wrote the manuscript. YJO and O-OJB designed the experiments and sourced for funds. YJO and AR sourced for the wild mosquito larvae and cultured the mosquitoes.

Acknowledgements

We thank Richard Amito and Jessica Anyango for processing and culturing the experimental mosquitoes, Kisumu Polytechnic for providing equipments for the extractions of the phytochemicals from the plants, Centre for Global Health Research/Kenya Medical Research Institute (CGHR/KEMRI) for laboratory space, mosquitoes and equipments for conducting the experiments and VIRED International for providing transportation and logistics for sourcing for Endod and Neem. This project was funded by the National Commission for Science Technology and Innovation (NACOSTI).

References

Abbott W.S., 1925, A method for computing the effectiveness of an insecticide, Journal of Economic Entomology, 18: 265–267

http://dx.doi.org/10.1093/jee/18.2.265a

Abebe F., Erko B., Gemetchu T., and Gundersen S.G., 2005, Control of Biomphalaria pfeifferi population and schistosomiasis transmission in Ethiopia using the soap berry endod (Phytolacca dodecandra), with special emphasis on application methods, Transactions of Royal society of Tropical Medicine and Hygiene, 99(10): 787-794

http://dx.doi.org/10.1016/j.trstmh.2005.04.013

Cartilla P., and De la Cruz J., 2012, Termiticidal Potential of Stachytarpheta Jamaicensis (L.), Vahl, 1: 1-5

Das N.G., Goswami D., and Rabha B., 2007, Preliminary evaluation of mosquito larvicidal efficacy of plant extracts, Journal of Vector Borne Diseases, 44: 145-148

Djogbénou L., Pasteur N., Akogbéto M., Weill M., and Chandre F., 2011, Insecticide resistance in the Anopheles gambiae complex in Benin: a nationwide survey, Medical Veterinary Entomology, 25: 256–267

http://dx.doi.org/10.1111/j.1365-2915.2010.00925.x

Erko B., Abebe F., Berhe N., Medhin G., Gebre-Michael T., Gemetchu T., and Gundersen S.G., 2002, Control of Schistosoma mansoni by the soapberry Endod (Phytolacca dodecandra) in Wollo, Northeastern Ethiopia: post-intervention prevalence, East African Medical Journal, 79(4): 198-201

http://dx.doi.org/10.4314/eamj.v79i4.8878

Etang J., Nwane P., Mbida J.A., Piameu M., Manga B., Souop D., and Awono-Ambene P., 2011, Variations of insecticide residual bio-efficacy on different types of walls: results from a community-based trial in south Cameroon, Malaria Journal, 10: 333

http://dx.doi.org/10.1186/1475-2875-10-333

Faisal H., Waseem A., Anjum S., and Muhammad A.K., 2010, Adulticidal action of ten citrus oils against Aedes albopictus (Diptera: Culicidae), Pakistani Journal of Agricultural Sciences, 47(3): 241-244

Govindarajan M., and Karuppannan P., 2011, Mosquito larvicidal and ovicidal properties of Eclipta alba (L.) Hassk (Asteraceae) against chikungunya vector, Aedes aegypti (Linn.) (Diptera: Culicidae), Asian Pacific Journal of Tropical Medicine, 4(1): 24-28

http://dx.doi.org/10.1016/S1995-7645(11)60026-6

Govindarajan M., and Sivakumar R., 2012, Adulticidal and repellent properties of indigenous plant extracts against Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae), Parasitology Reserves, 110: 1607-1620

http://dx.doi.org/10.1007/s00436-011-2669-9

Govindarajan M., 2010, Larvicidal and repellent activities of Sida acuta Burm. F. (Family: Malvaceae) against three important vector mosquitoes, Asian Pacific Journal of Tropical Medicine, 3: 691-695

http://dx.doi.org/10.1016/S1995-7645(10)60167-8

Isman M.B., 2006, Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world, Annual Review of Entomology, 51: 45-66

http://dx.doi.org/10.1146/annurev.ento.51.110104.151146

Manimaran A., Cruz M.J., Muthu C., Vincent S., and Ignacimuthu S., 2012, Larvicidal and knockdown effects of some essential oils against Culex quinquefasciatus Say, Aedes aegypti (L.) and Anopheles stephensi (Liston), Advances in Bioscience and Biotechnology, 3: 855-862

http://dx.doi.org/10.4236/abb.2012.37106

Miresmailli S., Bradbury R., and Isman  M.B., 2006, Comparative toxicity of Rosmarinus officinalis L. essential oil and blends of its major constituents against Tetranychus urticae Koch (Acari: Tetranychidae) on two different host plants, Pest Management Science, 62: 366-371

http://dx.doi.org/10.1002/ps.1157

Mohan R., and Marimuthu G., 2014, Mosquito adulticidal properties of Delonix elata (Family: Fabaceae) against dengue vector, Aedes aegypti (Diptera: Culicidae), Journal of Coastal Life Medicine, 2(5): 389-393

Ogbuewu I.P., Odoemenam V.U., Obikaonu H.O., Opara M.N., Emenalom O.O., Uchegbu M.C., Okoli I.C., Esonu B.O., and Iloeje M.U., 2011, The growing importance of neem (Azadirachta indica A. Juss) in agriculture, industry, medicine and environment: a review, Resource Journal of Medicinal Plants, 5: 230-245

http://dx.doi.org/10.3923/rjmp.2011.230.245

Parekh J., Jadeja D., and Chanda S., 2005, Efficacy of aqueous and methanol extracts of some medicinal plants for potential antibacterial activity, Turkish Journal of Biology, 29: 203-210

Ranson H., N’Guessan R., Lines J., Moiroux N., Nkuni Z., and Corbel V., 2011, Pyrethroid resistance in African anopheline mosquitoes: what are the implications for malaria control? Trends in Parasitology, 27: 91-98

http://dx.doi.org/10.1016/j.pt.2010.08.004

Shaalan E.A., Canyon D., Younes M.W., Abdel-Wahab H., and Mansour A.H., 2005, A review of botanical phytochemicals with mosquitocidal potential, Environment International, 31: 1149-1166

http://dx.doi.org/10.1016/j.envint.2005.03.003

Tiwari S., Ghosh S.K., Ojha V.P., Dash A.P., and Raghavendra K., 2010, Reduced susceptibility to selected synthetic pyrethroids in urban malaria vector Anopheles stephensi: a case study in Mangalore city, South India, Malaria Journal, 9: 179

http://dx.doi.org/10.1186/1475-2875-9-179

Were P.K., 2008, Efficacy of Phytolacca dodecandra on Anopheles gambiae mosquito larvae. A PhD thesis submitted to the School of Environmental Studies, Moi University, Supervisor: Okeyo-Owuor, J.B., pp. 78-143

WHO, 2006, Guidelines for testing mosquito adulticides for indoor residual spraying and treatment of mosquito nets, WHO/CDS/NTD/WHOPES/GCDPP/2006.3

Yugi J.O., Okeyo-Owour J.B., Atieli F., Amito R., and Vulule J.M., 2014, Knockdown effect of crude ethanol extracts of Phytolacca dodecandra on Anopheles gambiae adults. Journal of Mosquito Research, 4(18): 1-7

http://dx.doi.org/10.5376/jmr.2014.04.0018