EVALUATION OF INSECTICIDAL EFFECT OF VEGETABLE OILS EXTRACTED FROM THE RIPE SEEDS OF AZADIRACHTA INDICA AND KHAYA SENEGALENSIS IN ADULT ANOPHELES GAMBIAE S.L. IN MAROUA, FAR NORTH REGION CAMEROON

Abstract

The insecticidal effect of vegetable Oils of two plants in the Far North Region Cameroon was evaluated on the adult population of Anopheles gambiae s.l. in Maroua, through of net bed in the laboratory. The plant oils of Azadirachta indica and Khaya senegalensis were extracted from mature seeds, then diluted with petroleum ether. Plots of net bed were impregnated with these solutions. After 24 hours of exposure, the plant oil extracts showed a repellent effect on insecticide in adult A. gambiae. A. indica provided an LC^sub 50^ of 2.77g/m^sup 2^, and K. senegalensis an LC^sub 50^ of 2.79g/m^sub 2^. For lethal 50 hours (HL^sub 50^), A. indica provided 11 hours 56 mins 42 seconds of HL^sub 50^ and K. senegalensis gave 14 h51mn19s. As cons, the Mosquito Impregnated Net has an LC^sub 50^ of 2.30 x 10^sub -4^g/m^sub 2^ and an HL^sub 50^ of 2 hours 52 min 28seconds. Vegetable oil of A. indica presented quite high toxicity compared to that of K. senegalensis in A. gambiae.

Headnote

The insecticidal effect of vegetable Oils of two plants in the Far North Region Cameroon was evaluated on the adult population of Anopheles gambiae s.l. in Maroua through of net bed in the laboratory. The plant oils of Azadirachta indica and Khaya senegalensis were extracted from mature seeds, then diluted with petroleum ether. Plots of net bed were impregnated with these solutions. After 24 hours of exposure, the plant oil extracts showed a repellent effect on insecticide in adult A. gambiae. A. indica provided an LC^sub 50^ of 2.77g/m^sup 2^, and K. senegalensis an LC^sub 50^ of 2.79g/m^sup 2^. For lethal 50 hours (HL^sub 50^), A. indica provided 11 hours 56 mins 42 seconds of HL^sub 50^ and K. senegalensis gave 14 h51mn19s. As cons, the Mosquito Impregnated Net has an LC^sub 50^ of 2.30 × 10^sup -4^g/m^sup 2^ and an HL^sub 50^ of 2 hours 52 min 28seconds. Vegetable oil of A. indica presented quite high toxicity compared to that of K. senegalensis in A. gambiae. However, these two vegetable oils have a lower toxicity than the deltamethrin usually used for to the concentration of 5.5 × 10^sup -4^g/m^sup 2^ insecticide effect.

KEYWORDS: Evaluation, insecticidal effect, Vegetable Oils, bed net, Anopheles gambiae, Cameroon.

INTRODUCTION

In Africa, Malaria is the main cause of medical consultation.Nearly 50% of mortality and morbidity among children under 6 years are caused by malaria (PNLP, 2006). According to the World Malaria Report" of 2005, the World Health Organization (WHO) reported that 107 countries and territories have areas which are concerned by the transmission of malaria and some 3.2 billion people live in endemic areas. The fight against the malaria vector must be adapted to target vectors. Preventive control is based on long lasting impregnated nets, on the indoor residual spraying of insecticides and, to a lesser extent, on the destruction of breeding area. Active materials of insecticides which are mostly used, belong to organophosphates, pyrethroids and carbamates synthesis (Djogbénou, 2009). Plants sampling based on ethnobotanical survey are known to show greater percentage yield of bioactive useful compounds than other methods (Usman et al., 2014). Vector resistance to insecticides synthesis constitutes the main limits to their usage. Resistance to organophosphates and carbamates has been recently discovered in West Africa in the Anopheles gambiae the main vector's malaria (WHO, 2006). The World Health Organization recognizes that the most practical to control resistance, in alternate effect spraying consists in using alternatively insecticide of different types following a pre-established program or according to the results of resistance. Very few studies have been conducted on the present effect interest of the insecticides extracts of local plants that could be prevented. Azadirachta indica and Khaya senegalensis are very used in Far North Region Cameroon and they have insect repellents and insecticides effects (Dansou et al., 2007, Fawrou et al., 2006). Some farmers function on the basis of solution reward leaves of these plants as insecticide so as to destroy and drive away pests (Mamadou, 1997). Unlikely, the information provided by the population reveals that extracts have a repellent effect on mosquitoes, once they ambolmed themselves with. On the social plan, a new efficient method to fight against some malaria vector agents must significantly reduce the mortality rate of this disease. On the economic plan, oils extracted from the ripe seeds of local plants are accessible to all at low cost. This could enormously reduce budgets, funding for the prevention and fight against malaria and all the diseases transmitted by mosquitoes. The overall objective of this work is done to evaluate the effect of insecticide vegetable oils extracted from the ripe seeds of Azadirachta indica and Khaya senegalensis in adult Anopheles gambiae sl in the city of Maroua.

MATERIALS AND METHODS

Presentation and study of the site

Maroua, the Capital of Diamaré Division and the Far North Region of Cameroon, is located between 10° 31 ' - 10° 39' North latitude and 14° 13 ' - 14° 24' East longitude. It has an in altitude of 400 m in the Savannah (Casalprime, 2008). Maroua town is divided into three sub-divisions. Maroua town stretches along the shores of the Mayo - Kaliao which passes longitudinally from West to East. On a global view, the relief of this Region is made up of all the plains combined with the Mandara Mountains and mountains on the border with Nigeria. With an altitude of 1000 m of mountains and 300 m of plain, Maroua is a transitional zone between the desert areas of North and the wet climate of the South Sudanese (Yann, 2000). The Region is of the Sudano - Sahelian climate with an average temperature of 35°C, with the highest in March and April which goes up to 45°C and with a minimum of 25°C (Yann, 2000). A very short rainy season alternates with a dry season relatively long, depending on the years. The annual interest rainfall of about 800-900 mm is distributed over 50 to 60 days of precipitation (Mechia, 2005). The harmattan is the most dominant wind of the Region and blows almost all the year (Méchia 2005). The vegetation consists of thorny steppes and Sahelian interest dominated by some species such as Azadirachta indica, Khaya senegalensis, Balanites aegyptiaca, Anogeissus leiocarpus and Boswellia dalzieli which grow on the rocky soils (Boutrais, 1984). The population of the Maroua town has gone from 201, 372 inhabitants (GPC, 1987) to about 400, 000 inhabitants in 2013 according to the Maroua I council. More than forty different ethnic groups live in the Region (Yengue et Yann, 2002).

METHODOLOGY

Sampling larvae

The harvesting of the Anopheles larvae was conducted during the months of March, April, May and June 2013. Samples were made using ladles enamel handle. The larvae were collected by using a pipette bulb and placed in a metal tray of 50 cm of diameter in the laboratory of the Institute of Agricultural Research for Development (IRAD) of Maroua.

Rearing larvae of Anopheles gambiae

The larvae were maintained in breeding conditions with different water deposits. The identification and recognition of Anopheles larvae were made from their parallel position with respect to the water surface (Fig. 1). The study took into consideration the only Anopheles genus. Containers for mosquito larvae bore the characteristics of the sampling sites. In each tray, a maximum of 100 larvae were introduced. Nutritional powder made from prowns and biscuits were served as food to the larvae. The water from the trays were from time to time renewed to avoid water pollution resulting from the presence of the nutritional powder. Two to three days after the introduction of the larvae in large trays, the nymphs appeared. The nymphs were in turn removed from the plates and transferred to transparent glasses. The glasses containing water house were placed inside cubic cages made of tulle net with a volume of 8000 cm3 (Fig.2). In each cage, a petridish containing a sugar juice based on 10 % sucrose was placed, to serve as an emerging food to adult mosquitoes. With their emergence in cages, adult mosquitoes were fed with blood of Rabbits before being submitted to the various toxicity tests to avoid biased results that would be linked to poor diet to the Anopheles. The determination of adult Anopheles species was made using the identification keys of Gillies & De Meillon (1968) and Gillies & Coetzee (1987). Meanwhile, seed collection Azadirachta indica and Khaya senegalensis continued.

Collection of fruits and seeds

The seeds were collected from ripe fruits. In fact, these fruits were harvested in Pitioaré quarter (Camp IRAD) and at the Camp SIC neighborhood. Azadirachta indica (Fig.3) produces berries that fall when ripe. Seeds are collected within a shell in fruit lying below a thin layer of chair. Khaya senegalensis (Fig.4) produces fruit capsular, dehiscent, 4 or 5 valves, 5 to 10 cm of diameter, and light gray at maturity. Dried fruits open and release seeds.

Treatment of fruits and seeds

The ripe fruits of Azadirachta indica were having very high water content. They were dried before removing the seeds. This allows the seed to separate itself easily from the shell. Dried fruits of Khaya senegalensis do not need to be dried first. For cones, the fruit should be picked because they fall after having released their seeds. After drying the fruits, the seeds were then subjected to roasting and grinding, and then pulped between the millstones. The molding thus obtained is steamed until oil oozes. The following steps were followed to obtain vegetable oils.

Pressing by traditional method

Pressing is optimized with 5 % to 8 % with an impurity content of 2 % of humidity. A manual pressing was used, which is actually home made equipment but which provides good extraction rates.

Sedimentation and filtration

It consists of letting in rest the extract obtained by pressing. The heaviest substances settle at the bottom of the container can be recovered with floating oil above. The operation was repeated several times until the obtention of clear oil. The oil obtained was then filtered to remove the impurities.

Impregnating of bed nets with extracts

Nets industrially impregnated with delthamétrin, pyrethroids, DDT were done with the concentrations of 55 mg/m2; 0.2 g/m2 and 8 mg/m2 respectively. The obtention of effective concentration was made using several ranges of concentration tests: 0.1 g/m2; 0.2 g/m2; 0.3 g/m2; 0.4 g/m2; 0.5 g/m2; 1.5 g/m2; 2.5 g/m2; 3.5 g/m2; 5 g/m2, with the surfaces of the cones being very low compared to the areas (m2) used industrially. A similarity was used to determine the masses of extracts on the surface of a cone. The following formula was obtained: X = 0.0169 × M (M is the mass per square meter, X is the mass value brought to the surface of 0.0169 m2). Knowing the different masses needed for testing, oil volumes corresponding to each mass were determined. It was enough indeed to find the density of oils and deduct the volumes. To determine the density, a known volume of oil was weighed on an electronic balance and the density of vegetable oils has been obtained by applying the formula d = m/v (where m is the mass and v the volume), and each volume was obtained using the formula m = v/d. After calculation, we obtained: (d1 = density of the Azadirachta indica oil, d1 = 0.82) and (d2 = oil Khaya senegalensis density, d2 = 0.80). Cones used for the tests were made thanks to the model cones of WHO for this purpose. The end caps serving as bottle packaging of mineral water were used. These nozzles, like WHO-cones have the characteristics of being made of polyethylene, a material on which the mosquito scarcely clings on. The mosquito will have no other choice but to stay in flight or landing on the substrate (impregnated mosquito net). In order not to lose in mass quantities, it has been established that for a surface mosquito net of 0.0169 m2, you need a volume of 2 ml of organic solvent. The suitable organic solvent used herein to diluate the oil is the petroleum ether. With the help of a pipette, 2 ml of ether are removed and poured into a petri dish containing the mass of the corresponding active substance. After a complete dissolution of the oil in the ether, the plots of the nets were soaked. Then the nets were left to dry under the sun for a period of one hour so as to allow the plant extract to fully adhere on the and to completely let evaporate the ether. Meanwhile, the negative control was based solely on ether which is used to assess its influence on mosquitoes.

Tests of insecticidal effect of oils on adults Anopheles gambiae

Impregnated nets were hanged to the base of the cone using a transparent tape. A total of 13 cones were used including 10 reserved for the different concentrations of each type of extracted plants and 3 others like cones' witnesses. One is impregnated with deltamethrin 55 mg/m2, actually used by the population and being distributed by the government to the population. This net was considered as a positive control to assess the effectiveness of the extracted oils from ripe seeds of Azadirachta indica and Khaya senegalensis. Another cone was equipped with mosquito nets soaked in ether only; this was to ensure the neutrality of ether on mosquitos during the test. After the distribution of cones and the fixing of nets, 30 unsexed adults anopheles were introduced into each cone with vacuum insects. The cone containing mosquitoes are closed by a piece of cotton wool and these cones were suspended separately in the laboratory using a string to allow ventilation. After the introduction of the insects in the cones, the insecticide efficiency is measured by their Knock-down effect and they died after 24 hours of observation. In order to determine the Knock-down effect, three minutes after the reaction were observed on the mosquitoes. The number of mosquitoes shocked or stunned or dead were recorded in a table. Then every hour up to twelve hours, the last observation was made after the expiration of the last 12 hours. The test of each concentration was repeated three times to minimize errors. The averages of the three replicates were calculated.

Data analysis

According to Finney DJ (1971), a lethal concentration of 50 (LC50) and a lethal time of 50 (HL50) were obtained from the following formulas: LC50 = log10-1 (y-b / a); HL50 = log10-1 (y-b / a).

The concentration-response graph for each exposure time is plotted against the logarithm of the concentrations by means of software.The right probit = f (log concentration) is plotted using the Microsoft Office Excel 2007 software; the equation of the line known as the correlation coefficient is also obtained with this software.

Concentrations and times that kill 50% of mosquitoes (LC50 and HL50) were determined graphically.

The regression lines have yielded correlation coefficients, which in turn helped to highlight the different correlations compared to the Long Lasting Insecticide treated nets.

RESULTS

Profit yield of the extraction of vegetable oils

Mature seeds of A. indica and K. senegalensis showed relatively high yields of vegetable oils as weighing 1500g which gave of seeds 600g and 450g of vegetable oil, resulting 40% and 30% respectively for A. indica and K. senegalensis. Vegetable oils of A. indica and K. senegalensis are the most popular products in the Far North Cameroon, given their wide range of applications in the treatment of diseases such as malaria, helminthes infections, constipation, itching. Many people confirm that anointing of these two products provides temporary protection against the biting of insects especially mosquitoes. Depending on the selling period, the price for a liter of these plants is between 6000F and 13000F in Maroua town, Cameroon. Sellers are more and more opting for retail sale to allow easy access to all.

Mortality rate of vegetable oils A .indica and K. senegalensis on Anopheles gambiae

The data obtained helped to draw graphs on mortality rate by using time for each concentration of the vegetable oils and for each batch of mosquitoes. The graph on mortality rate versus time and concentration of the extract from Azadirachta indica on vegetable oil in anopheles is represented by figure 5. According to figure 5, the mortality rate increases with the concentration of the extract of Azadirachta indica oil. In fact, the correlation coefficients are positive (r = 0.9593). This study showed a dose- mortality of all oils tested, with an increase in larval mortality, when the dose increases. The activity of the oils could be due to the active substances contained in the plant material. Figure 6 below shows the evolution of the mortality rate depending on the time and the concentrations of Khaya senegalensis in Anopheles gambiae sl. The observation of figure 6 shows that mortality increases with the concentration of extracted oil of Khaya senegalensis. This observation is confirmed by the correlation coefficient r = 0.9572. It was found a very high mortality rate of mosquitoes in the active control of Insecticide treated nets, 100% after about 1 hour 30 min of exposure. This confirms the powerful effect of the insecticide Deltamethrin. Deltamethrin is effective at a very low dosage (55mg/m2) compared to lethal oils of Azadirachta indica and Khaya senegalensis.

Determination of lc50 of A. indica and K. senegalensis

The determination of the extract of vegetable oil concentration that causes a mortality rate of 50% among adult anopheles was made from regression equation of mortality rates, expressed as probit value, and depending on the concentrations of extracts of vegetable oil in decimal logarithm value (Fig.7 and Fig.8).

Apparently, the LC50 of A. indica and K.senegalensis are almost equal (table 1). However, they are very high in A. indica (2.77g/m2) and in K. senegalensis (2.79 g/m2) compared to that of the Insecticide treated nets (MILDA) with an LC50 of 2.30 × 10-4g/m2. There is no significant difference at α = 5 % between the LC50 of A. indica and K. senegalensis. Taken at this dosage, deltamethrin is 4279 times more effective than A. indica and 4488 faster than K. senegalensis. LC50 obtained from anopheles adults which showed that two oil extracts are interesting in terms of toxicity. In reality, the lethal concentrations vary depending on the method of extraction and of the extract of the plant or plant part in question.

Determination of hl50 A. indica, K. senegalensis and long acting insecticide treated nets (milda)

The determination of hours from which the plant extract concentration induced 50 % mortality of Anopheles gambiae was performed. This parameter was obtained from the equation of regression mortality expressed in probit value based on exposure times in decimal logarithm (table 2). The HL50 obtained revealed that Azadirachta indica provided HL50 smaller values about 11h56min42s than Khaya senegalensis (14H51min19s). A significant difference at the 5% level between HL50 A. indica and K. senegalensis was observed. This means that the vegetable oil from Azadirachta indica is higher than K. senegalensi toxicity. The values of correlation coefficient obtained show that there is a close correlation between the duration of exposure and the mortality rate (0.9 ≤ r ≤ 1).

DISCUSSION

We have noticed that mature seeds of A. indica and K. senegalensis showed relatively high yields of vegetable oils, 40% and 30% respectively. Meanwhile, Chougourou et al., (1997) have found rather a higher oil yield of 43.4 % with A. indica seeds. In fact, Azevodo et al., (2002) justifed this through the extraction efficiency of a plant which depends on several factors, namely the nature of the soil, climate and the harvest season. The repellency of the extracts of A. Indica and K.senegalensis on insects is increasingly confirmed by several researches. Fawrou et al., (2006) conducted a study on A. indica oil in pre-imaginal stages of Culex quinquefasciatus and confirmed that A. Indica oil was made effective at both larvae and nymph levels. Our results corroborate those of Tchoumbougnang et al., (2009) on the larvicidal activity of four plants cultivated in Cameroon (Cymbopogon citratus (DC.) Stapf, Ocimum canum Sims, Ocimum gratissimum L and Thymus vulgaris L) on Anopheles gambiae Giles 1902. Hence, the results of the tests performed on larvicidal activities on essential oils show a direct relationship between the percentage of the mortality of the larvae L4 and the concentration of essential oils. In addition, Chougourou et al., (2012) revealed that the bioactive substances such as azadirachtin, curcin, Ricin and thevetin are respectively present in A. indica seeds. Furthermore, Sharma et al., (1993) also showed that a mixture of oil Azadirachta indica in coconut oil has provided a 100% protection for 12 hours against anopheles bites and those against sandfly vectors of leishmaniasis, 100% overnight against Phlebotonus argentipes and 100% for 7 hours against Phlebotonus papatasis. These results show that the vegetable oil from Azadirachta indica has a residual effect. In addition, Dua et al., (1994) showed that with cream Azadirachta indica, two grams per person provides protections of 80-90 % against Aedes, Culex and Anopheles. Sharma et al., (1994) in their work showed that Azadirachta indica oil combustion of 1% in kerosene in the usual lamps provided 100% protection against anopheles' bites. The chemical compositions of several plants give their insecticidal properties. Several other effects were observed on insects; and the study of the chemical composition of Khaya senegalensis revealed the presence of alkaloids, terpenoids, phenolics and flavonoids that variously affect the existence or the development cycle of pests (Boeke et al., 2004). Essential oils further demonstrate their multiple activities, anti-oxidant properties that come from the multitude and diversity of their chemical constituents (Saotoing, 2005). Similar studies conducted by Njan Nlôga et al., (2007) showed the insecticidal effect of six plants in the Northern part of Cameroon. These plants were: Ocimium canum, Plectrancthus glandulosus, Laggera pterodonta, Eucalyptus camaldulensis, Hyptis spicigera, Pittosporum viridiflorum. Their LC50 ranged from 11.95 to 71 mg/m2. Moreover, previous work Traboulsi (2002) had demonstrated the insecticidal activity of four medicinal plants harvested in Lebanon (Myrtus communis L., Lavandula stoechas L., Origanum L. and Mentha microphylla syriacum K.Koch) on larval Culex pipiens molestus Forskal. LC50 obtained were between 16 and 89mg/l. The studies conducted by Gauvin et al., (2002) on the composition of the extracts from the seeds of Azadirachta indica showed that this oil contains a mixture of more than 100 compounds. Among these, the azadirachtin (C35H14O16) is one of the most important bio-insecticides (Mordue and Blackwell 1993; Schmutterer, 1990; Zongo et al., 1993). Furthermore, these same authors have demonstrated that azadirachtin is not the only active and responsible insect for mortality tested substance, but it acts synergically with a mixture of several substances in the extract.

CONCLUSION

The results of this study show that vegetable oils extracted from Azadiratchta indica and Khaya senegalensis have an insecticidal effect on mature Anopheles of Maroua. LC50 obtained are 2.77g/m2 for Azadirachta indica and 2.79 g/m2 for Khaya senegalensis. The HL50 obtained are 11 hours 56 min 42 seconds for Azadirachta indica and 14 heures 51 minutes 19 seconds for Khaya senegalensis. However, these two vegetable oils have much lower insecticidal effect than that of deltamethrin (2.30 x10-4g/m2) used for Long Lasting Insecticide Treated Nets. Azadiratchta indica is the plant which is widely spread in the Northern region Cameroon. People know that this plant has an insecticidal effet but do not know the lethal dosage of toxicity. In the future work, we will tray to characterise the bio-active substances contained in the oil in order to formulate it.

ACKNOWLEDGEMENT

We are grateful to the authorities of the University of Maroua from whom we received funding for research and the publication of this article.

Sidebar

Cite this article:

Saotoing P, Tchuenguem Fohouo F.N, Febal H.A, Amba A.H.B, Amadou H, Nawe T.U.S (2014), EVALUATION OF INSECTICIDAL EFFECT OF VEGETABLE OILS EXTRACTED FROM THE RIPE SEEDS OF AZADIRACHTA INDICA AND KHAYA SENEGALENSIS IN ADULT ANOPHELES GAMBIAE S.L. IN MAROUA, FAR NORTH REGION CAMEROON, Global J Res. Med. Plants & Indigen. Med., Volume 3(8): 312-322

References

REFERENCES

Baldet T., (1995). Étude comparative de deux stratégies de lutte contre Culex quinquefasciatus Say, 1823 par Bacillus sphaericus Neide, 1904 dans la ville de Maroua (Nord-Canleroun). Thèse de Doctorat Universite de Montpellier II. 376p.

Boeke, S. J., Baumgart, I. R., van Huis, A., VanLoon, J. J. A., Dicke, M. et Kossou, D. K., (2004). Toxicity and repellence of african plants traditionally used for the protection of stored cowpea against Callosobruchus maculatus. Journal of Stored Products Research. 40 : 423- 438.

Brahim Aouinty, Saadia Oufara, Fouad Mellouki et Saadia Mahari., (2006). Évaluation préliminaire de l'activité larvicide des extraits aqueux des feuilles du ricin (Ricinus communis L.) et du bois de thuya (Tetraclinis articulata) sur les larves de quatre moustiques culicidés : Culex pipiens (Linné), Aedes caspius (Pallas), Culiseta longiareolata(Aitken) et Anopheles maculipennis (Meigen), Biotechnol. Agron. Soc. Environ. 10 (2) : 67-71.

Casalprime D., (2008). Rapport général de la Société AZIMUT-GEOCART de la prise de vue aérienne, numérisation, appuis photo gramétrique, aérotriangulation, restriction et ortho- photo. Ville de Maroua. p388.

Chougourou C. Danie, Dellouh P. Lesline , Agbaka Alphons, N'guessan K. Raphael et Gbenou Joachim D., (2012). Toxicité et effets répulsifs de certaines huiles extraites des plantes locales Béninoises sur la mouche domestique Musca domestica L. (Diptera Muscidae). Journal of Applied Biosciences 55: 3953-3961.

Dansou K. Kossou, P., Atachi, Tohouédé E. Zannou et Soulémane B., (2007). Évaluation de l'Activité Insecticide de deux Plantes Hyptis suaveolens (Linn) et Khaya senegalensis (A. Juss) sur les insectes ravageurs du niébé (Vigna unguiculata L. Walp.) Sciences & Nature Vol. 4 N°1 : 17-26.

Djogbénou L., (2009). Lutte antivectorielle contre le paludisme et résistance des vecteurs aux insecticides en Afrique. Institut Régional de Santé Publique (IRSP), Ouidah, Bénin. Med Trop 2009 ; 69 : 160-164.

Dual V.K., Nagpal B.N. et Sharma V. P., (1995). Repellent action of neem cream against mosquitoes. Indian J. Malariol. 32, 47-53.

Fawrou S, Demba N.R. et Mady N., (2006). Étude comparative de deux produits de neem (huile et poudre) sur les stades préimaginaux du moustique Culex quinquefasciatus (Diptera : Culicidae) Afrique Science 02(2) : (212-225).

Gauvin, Belanger, Nebie et Guy Boivin., (2003). Azadirachta indica: l'azadirachtine est-elle le seul ingrédient actif Phytoprotection, société de protection des plantes du Québec, 84 : 115-119.

Gillies M.T. et Coetzee, (1987). The Anopheline of Africa South Sahara (Ethiopian) Zoogeographical region, Johannesburg: South Afr inst Med.Second.editon: Research.343p.

Gillies M.T. et De Meillon B., (1968). The Anopheline of Africa South Sahara, Johannesburg: South Afr inst Med.Second.editon: Research. 214- 223p.

Mamadou S., (1997). Effet de l'extrait aqueux des feuilles de neem (Azadrachta indica A. juss) sur la population de Thrips et le rendement du nièbe (Vigna unguiculata). Rapport de stage, ISRAKNRAI Bambey. Sénégal. P.29.

Mechia V., (2005). Une analyse socio- économique des activités rurales à Maroua.

Mordue, A.J. et A. Blackwell. (1993). Azadirachtin: an update. J. Insect Physiol.

Njan Nlôga A.M, Saotoing P, Tchouankeu J. C. and Messi J., (2007). Effect of Essential Oils of Six Local Plants Used Insecticide on Adults of Anopheles gambiae, Giles 1902. Journal of Entomology 4(6) : 444-450.

Olivry j.C., (1986). Fleuves et rivières du Cameroun. Collection Monographies hydrodrologiques de l'ORSTOM/MESIRES n° 9, 733 pp.

OMS, (2005). Roll Back Malaria Project: Resources Support Network for Prevention and control of Malaria Epidemics. Document WHO/CDS/RBM/RSN/EPI/2005. Organisation Mondiale de la Santé et Ministère de la Santé Publique (Programme Nationale de Lutte contre le paludisme), 88 pages.

OMS, (2006). Paludisme : lutte antivectorielle et protection individuelle.

Programme Nationale de Lutte contre le Paludisme., (2006). Rapport d'évaluation de la faisabilité et de l'acceptabilité de l'utilisation des Combinaisons Thérapeutiques à base d'Arthémisinine pour le traitement du paludisme à domicile au Cameroun 112 p

RGPH, (1987). Recensement Général de la Population et de l'Habitat

Saotoing P., (2005). Diversités culicidienne dans la partie septentrionale du Cameroun et effet des huiles essentielles sur les adultes d'Anopheles gambiae. Université de Yaoundé I Cameroun. Thèse de Doctorat.

Schmutterer, H. (1990). Properties and potential of natural pesticides from the neem

Sharma V.P. et Ansari M.A., (1994). Personal protection from mosquitoes (Diptera: Culicidae) by burning neem oil in kerosene. Entomol. Soc. Am.31, 505- 507.

Sharma V.P., Ansari M.A. et Razdan R.K., (1999). Mosquitoes repellent action of neem (Azadirachta indica). All. J.Am. Mosq. Control. Assoc., 9, 359-360.

Tchoumbougnang F., Dongmo P.M., Sameza M.L., Nkouaya E.G., Fotso G.B., Zollo P.H et Menut C., (2009). Activité larvicide sur Anopheles gambiae Giles et composition chimique des huiles essentielles extraites de quatre plantes cultivées au Cameroun. Biotechnol. Agron. Soc. Environ. 13(1): 77-84.

Traboulsi A.F., (2002). Insecticidal properties of essential plant oils against the mosquito Culex pipiens molestus (Diptera: Culicidae). Pest Manage. Sci., 58: 491-495. Tree, Azadirachta indica. Annu. Rev. En-tomol. 35 : 271-297.

Usman Idris Nda-Umar, Mohammed Gbate, Abdulkadir Nda Umar, Abdullahi Mann. (2014). Ethnobotanical study of medicinal plants used for the treatment of malaria in nupeland, north central Nigeria. Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 4 | April 2014 | 112-126. ISSN 2277-4289 | www.gjrmi.com | International.

Yann L'hote., (2000). Climatologie. In : Atlas de la province de l'Extrême-Nord Cameroun. Eds. Seignobes C. et Iyebi- Mandjek O., IRD. MINREST, Paris, 27-33.

Yengue J.L et Yann C., (2002). L'arbre et la ville dans la région de Maroua (Extrême-Nord Cameroun), sciences et changements planétaires/sécheresse, 13(3): 155-163.

Zongo, J.O., C. Vincent et R.K. Stewart. (1993). Effects of neem seed kernel extracts on egg and larval survival of the sorghum shoot fly, Atherigona soccata Rondani (Dipt.: Muscidae). J. Appl. Entomol. 115 : 363-369.

Source of Support: University of Maroua, Cameroon

Conflict of Interest: None Declared