Background

Mosquitoes are among the insect vectors that transmit deleterious human diseases, which pose major public health challenges especially in the poorest countries of the world (Awad and Shimaila, 2003). Their medical importance as vectors for the transmission of important diseases such as malaria, filariasis and viral diseases like yellow fever, dengue fever, rift valley fever that cause morbidity, mortality, economic loss and social disruption are documented (Becker et al., 2003).

Culex pipiens group are the principal vector of Bancroftian filariasis in areas where Wuchereria bancrofti has nocturnal periodicity, but, in other places, other species of mosquitoes may serve as vector of the parasite (White, 1987).

Culex quinquefasciatus which is the principal vector of Bancroftian filariasis in the tropical cities of the world has continued to multiply and increase in many towns due to increasing urbanization and resultant proliferation of unsanitary collections of water and this attitude stabilizes and perpetuates the disease transmission by the mosquito (Service, 1989; Nwoke andEbo, 1993).

The popular control methods that have been used to manage mosquito population include: source reduction, biological control using natural enemies of mosquitoes and chemical method using toxic pesticides. Although ranging degrees of success have been recorded with these various strategies, they equally have various levels of limitations. Apart from cost of application, slowness in operation and the need for skilled manpower for effective implementation, there is also a growing concern about the health and environmental risks associated with most control strategies. As a result of the various limitations using chemical insecticides, plant extracts are been investigated as an insecticides for controlling larval and adult mosquitoes as they are considered to be more environmentally friendly and safer than synthetic insecticides.

Hyptis suaveolens(L) Poit is a dicotyledonous, annual plant, which is a member of Laminaceae (mints family); and genus Hyptis is distributed evenly in the tropic of West Africa. The plant is a well known pseudo cereal plant having branches and long piliferous stem. Based on its ethnobotanical evidence, people believed it is a strong mosquito repellent (Peerzada, 1997).

Senna hirsutais a large genus of flowering plant in the family Fabaceae and this group is native throughout the tropics with a small number of species reaching the temperate region (Navie et al., 2002).Senna species therefore contains compounds that are of good value in the development of environmentally safe pest- management agents.

The study therefore seeks to evaluate the lethal concentration and efficacy of the leaves extract of Hyptis suaveolens and Senna hirsuta as a larvicide and as repellent against the larvae and adult of Culex quinquefasciatus mosquitoes in the laboratory and also to determine the synergistic potential of the two plant extracts as both larvicides and repellents against the mosquito.

1 Materials and Methods

1.1 Study area

Onitsha (6⁰/0’N 6⁰ 47⁰ E) is a commercial city characterized with incessant refuse disposal which ranges from water sachet to solid waste such as tins, plastic containers which serves as a breeding sites for mosquitoes.

1.1.1 Collection of eggs and larvae of Culex quinquefasciatus

The larvae for the study were collected at Onitsha (Odoakpu and Fegge), Anambra State using ladles, bowels, sieves of 0.25mm and 0.5mm mesh sizes and 1000ml specimen bottles. The eggs were collected from ovitraps at the fermentation pots of cassava (Maniohot esculenta) and bread fruit (Artocarpus altilis) washing sites at the popular Ose market in Onitsha. After the collection, proper identification of the eggs and the larvae was done at Insectary of National Arbovirus and Vector Research Centre Enugu.

1.1.2 Hatching of eggs and rearing of larvae to adult stage

The eggs collected were introduced into plastic bowls containing 500 mls of distilled water. The eggs hatched to 1^st instar larval stage and were reared to pupae and adult at temperature 28±3°C and relative humidity of 80±5% measured with thermo-hygrometer. Care was taken to separate the male from the female mosquito using the Identification keys of (Reuben et al., 1994).

The leaves collected were washed and dried for 6 days under room temperature. The dried samples were pulverized into fine powder using an electric grinder. Oil from the pulverized samples of Hyptissuaveolens and Senna hirsuta were extracted with hexane as the solvent using Soxhlet apparatus. The extracts of the leaves of Hypti ssuaveolens and Senna hirsuta were then concentrated using water bath, which removed the hexane component leaving behind greenish viscous oil for Hyptis suaveolens and brownish green viscous oil for Senna hirsuta (both of which are essential oils). The concentrated extract was collected and stored in a refrigerator at 4°C as the stock solution for further use.

1.2 Formulation of the essential oil

The extracts were taken to be 100% concentration and was diluted to 30%, 20%, 15%, 10% and 5% by adding 6mls, 4 mls, 3 mls, 2 mls and 1 ml of the oil to 14 mls, 16 mls, 17 mls, 18 mls and 19 mls of acetone respectively using 20 mls syringe. The control was prepared by the addition of 1 ml of acetone to 19 mls of water.

1.3 Larval bioassay

Standard procedures used were those of (Mulla et al., 2003) and a little modification of (WHO, 1981) procedures for larval bioassay. The modifications are in the number of larvae used (from twenty- five to twenty) and the number of replications (from four to three).

Aliquots of 1ml in ml/ml of the plant extracts were added in 250 ml beakers containing 200 ml of distilled water for both Hyptis suaveolens and Senna hirsutaoil extracts. Each beaker was challenged with batches of twenty fourth instar larvae of Culex quinquefasciatus respectively.

Larval mortalities were counted after 3 hours of treatment. Mortality served as the end point for the test. Larvae were considered either alive (clearly moving normally) or dead (no movement and no response to gentle probing with a fine glass rod).

1.4 Repellency test (Using the two oil extracts separately)

Arm in cage testing method of WHO (1996) and Faradin et al. (2002) were used to test the repellency effect of Hyptis suaveolens and Senna hirsuta oil against Culex quinquefasciatus mosquitoes. The various concentrations of the extract evaluated were 30%, 20%, 15%, 10% and 5%. Ten different cages measuring 50×50×50 cm were used and each cage containing twenty female mosquitoes. However five different cages containing one hundred mosquitoes (twenty mosquitoes /cage) were used for each plant oil extract. Five adult human volunteers who did not apply any lotion, perfume, oil or soap on the fore arm participated in the test. The fore arm of each volunteer from the elbow to the tips of the fingers was washed with water and left to dry.

The right arms (used for the treatment) were rubbed with 1ml of each concentration of each plant extract while the left arm (which was not treated) served as control. Before each test, the readiness of the mosquitoes to bite was confirmed by having the volunteers put their untreated arms in the cage for 20 minutes.

The repellent index was calculated using the formular:

Where, Ta=Number of mosquitoes in the control; Tb=Number of mosquitoes tested/treated (Schreck et al., 1997).

1.5 Synergistic test (Larvae Bioassay using the combination of the two oil extracts)

Aliquots of 0.5 ml of Hyptis suaveolens oil extract were added to 0.5 ml of Senna hirsuta oil extract thereby making it up to 1ml dosage of each of the concentrations. The mixture were added into a plastic cups containing 200 ml of distilled water each and subsequently twenty fourth instar larvae were introduced into each cup. Each treatment was replicated three times. Counts of dead and moribund larvae were taken at 1 hour interval for 3 hours. Mortality served as the end point for the test and the result was used to determine the LC₅₀ value for the extract.

1.6 Repellency test (combining the two oil extracts)

An aliquot of 0.5 ml of each concentration of Hyptis suaveolens extract were added to 0.5 ml of each concentration of Senna hirsuta thereby making up 1ml of each concentration and was used to test for repellency action against the adult Culex quinquefasciatus mosquito. The mixtures used were rubbed from the elbow to the finger tip and were then exposed to the mosquitoes in the cage. The left untreated arm was used as control.

1.7 Quantitative phytochemical analysis

Chemical tests for the screening and identification of bioactive chemical constituents in the medicinal plants under study were carried out on the extracts using the standard procedures as described by Obadoni andOchuko (2001).

1.7.1 Determination of saponin

Exactly 5 g of the samples was put into 20% acetic acid in ethanol and allowed to stand in a water bath at 50°C for 24 hours. This was filtered and the extracts were concentrated using water bath to one quarter of the original volume. Concentrated NH₄OH was added drop- wise to the extract until the precipitate was complete.

The whole solution was allowed to settle and the precipitate was collected by filtration and weighed. The saponin content was weighed and calculated in percentage (Obadoni andOchuko, 2001).

1.7.2 Cardiac glycosides determination

To 1 ml of the extract was added 1 ml of 2% solution of 3, 5-DNS (Dinitro Salicylic acid) in methanol and 1 ml of 5% aqueous NAOH. It was boiled for 2 minutes (until brick- red precipitate was observed) and the boiled sample was filtered.The weight of the filter paper was weighed before filtration. The filter paper with the absorbed residue was dried in an oven at 50°C till dryness and the weight of the filter paper with residue was noted.

1.7.3 Tannin determination by titration

The Follins Dennis titrating method as described by (Pearson, 1974) was used .To 20 g of the crushed sample in a conical flask was added 100 mls of petroleum ether and covered for 24 hours. The sample was then filtered and allowed to stand for 15 minutes allowing petroleum ether to evaporate. It was then re- extracted by soaking in 100 ml of 10% acetic acid in ethanol for 4 hours.The sample was then filtered and the filtrate was collected. 25 ml of NH₄OH were added to the filtrate to precipitate the alkaloids. The alkaloids were heated with electric hot plate to remove some of the NH₄OH still in solution. The remaining volume was measured to be 33 ml. 5 ml of this was taken and 20 ml of ethanol was added to it. It was titrated with 0.1M NaOH using phenlphthalyne as indicator until a pink end point is reached. Tannin content was then calculated in %.

1.7.4 Phytate determination

0.2 g of the sample was weighed into 250 ml conical flask and soaked in 100 ml of 2% concentrated Hcl for 3 hours. The sample was then filtered.50 ml of the filtrate was placed in 250 ml beaker and 100 ml distilled water was added. 10 ml of 0.3% ammonium thiocyanate solution was added as indicator and titrated with standard iron (III) chloride solution.

1.7.5 Oxalate precipitation

The extraction was done by boiling 2 g of the sample in 40 ml of water for 30 minutes in a reflux condenser. 10ml of 2% NaCO₃ was added and boiled for another 30 minutes. The liquid extract was filtered and washed with hot water till wash water showed no alkaline reaction. The combine water wash and filtrate was concentrated to a small volume and allowed to cool. Hcl (1:1) was added drop wise with constant stirring until the final acid concentration after neutralization was about 1% at which stage a heavy precipitate appeared, which was allowed to flocculate. Extract was carefully filtered into 250 ml flask, made up to mark and kept overnight. Supernatant liquid was filtered through a dry filter paper in a dry beaker. An aliquot of the filtrate in a 400 ml beaker was diluted with water to 200 ml and re-acidified with acetic acid.10 ml of a 10% CaCl₂ solution was added to the medium and stirred very well to induce CaO₃ precipitate to appear and left to settle overnight.The clear supernatant liquid was carefully decanted off through what-man filter paper NH₄OH solution. The content was boiled and allowed to settle overnight. Oxalic acid was determined by titrating against 0.05N KMnO₄.

1.7.6 Alkaloid determination

Five grams (5 g) of the sample was weighed into a 250 ml beaker and 200 ml of 20% acetic acid in ethanol was added and covered and allowed to stand for 4 hours at 25°C. This was filtered with filter paper No. 42 and the filtrate was concentrated using a waterbath to one quarter of the original volume. Concentrated NH₄OH was added drop wise to the extract until the precipitate was complete. The whole solution was allowed to settle and the precipitate was collected and washed with dilute NH4OH (1% ammonia solution). Then, filter with pre weighed filter paper. The residue on the filter paper is the alkaloid, which is dried in the oven (precision electrothermal model BNP9052 England) at 80°C. The alkaloid content was calculated and expressed as a percentage of the weight of the sample analyzed.

1.8 Data analysis

Mortality data obtained were subjected to Log-probit analysis using (Finney, 1971) for determining LC₅₀. Two-way Analysis of Variance (ANOVA) was also performed on the mortality data in order to determine the level of significance in the effect of both concentration and time. P-values were calculated to check whether the mortality observed from different concentrations were significantly different. Phytochemical analysis were carried out to determine the active compounds in the plants.

2 Results

2.1 Determination of the effective concentration of Hyptis suaveolens and Senna hirsuta oil separately as larvicide on the fourth instar larvae of Culex quinquefasciatus

The mean percentage mortality of the different concentration of Hyptis suaveolenson the 4^th instar larvae were shown below (Table 1; Table 2). Larval mortality observed after the first, second and third hour gave the percentage mortality of 18%, 32% and 61% for the three hours. The concentration from the highest (30%) to the least (5%) gave the mortality of 80%, 70%, 60%, 55% and 40% respectively. It was observed that the mortality was concentration and time dependent. Percentage mortality increased with increasing exposure time. No mortality was observed in the control which was acetone. Statistical analysis using ANOVA revealed that the mortality due to the concentration was significant (P<0.05).

After 3 hours of exposing the larvae to the extract, larval mortality was observed to be highest at the 30% concentration of the extract. The least larval mortality of 45% was observed when the larvae were exposed to the 5% concentration for 3 hours. Statistical analysis using ANOVA showed that there was a significant difference (P< 0.05), for concentration at P-value is 0.001 which implies the existence of enough evidence to reject the null hypothesis and accept the alternative and conclude that the effect of concentration is significantly different. An increase in mortality as the exposure time increased was observed across the concentrations (Table 3).

Probit (Hyptis suaveolens) = 4.11 + 1.10 Log-dose (Hyptis suaveolens)

LC₅₀= 0.8091 (6.4430)

LC₅₀ = 6.4%

Probit (Senna hirsuta) = 3.42 + 1.81 Log-dose (Senna hirsuta)

LC₅₀= 0.8729 (7.4633)

LC₅₀ =7.5%

From the probit graph below (Figure 1) it was deduced that 6.4% concentration of the extract of Hyptis suaveolens killed 50% of the larvae and 7.5% of Senna hirsuta  killed 50% of the larvae.

2.2 Determining the repellency action of the two oil extracts separately on the adult female Culex quinquefasciatus

The maximum repellency effect as shown in the Table 4 observed at 30% concentration which gave a percentage repellency of 70%. The least concentration 5% exhibited only 40% repellency of the adult mosquitoes after 3 hours. Repellency decreased with decreasing concentration and increased as the exposure hours increased. After one hour of exposure, 26% were repelled from the imbibing blood. After the second and the third hours of exposing the arm, 41% and 56% of the test mosquitoes were repelled from the arm respectively. Statistical analysis using ANOVA revealed that the repellency due to concentration was significant (P<0.05).

The repellent effect of the leaf extract of Senna hirsutaat different concentrations was presented in the Table 5. The highest repellency effect was observed at 30% concentration which repelled 50% of the mosquitoes used. At the first hour of the test, 5 out of the 20 mosquitoes used for the test were repelled from imbibing blood from the volunteer, 7 and 10 mosquitoes were repelled as well in the second and third hour respectively. The least concentration 5% exhibited only 25% repellency of the adult mosquitoes after 3 hours. Repellency increased with increasing concentration and time. Using the statistical analysis ANOVA, it was shown that there was a significant difference in the use of the toxicant. As the exposure time increases from one hour to the third hour, percentage repellency increased from 18%, 29% to 38 % respectively across the five concentrations and there was also a significant difference (P< 0.05) in the effect of time on the rate of repellency.

2.3 To determine the synergistic potential of the two plant extracts as both larvicide and repellent

The mixture of Hyptis suaveolens and Senna hirsuta extract oil at the ratio of 0.5 ml each showed a promising larvicidal effect against the larvae of Culex quinquefasciatus (Table 6). Mortality observed after the first, second and third hour were 20%, 44% and 73%. It was observed that the mortality was dose and time dependent. No mortality was observed in the control which was acetone. The least concentration of 5% had a mortality of 50% after 3 hours while the highest concentration of 30% had percentage mortality of 95. Using statistical analysis of variance, the synergistic effect of concentration was significant (P<0.05).

Regression analysis: Probit versus Log-dose of the combined extract (Figure 2)

The regression equation is

Probit = 3.422 + 2.0309 Log-dose of the combined plant extracts

LC₅₀= 0.7769 (5.98)

LC₅₀ = 6%

2.4 The Synergistic effect of Hyptis suaveolens oil and Senna hirsuta oil as a repellent against the adult female Culexquinquefasciatus

The mixture of the two oil extracts exhibited an enhanced repellency than the individual oil extract. The highest repellent effect was observed in the 30% concentration in which a total of 15 out of 20 mosquitoes were repelled after 3 hours and the lowest repellent effect was observed at 5% concentration which had a percentage repellence of 35% after 3 hours repelling only 7 mosquitoes as seen in Table 7. The repellent effect of the mixture was observed to be dose and time dependent and using the statistical analysis it showed the synergistic effect of concentration as a repellent on the adult mosquito is significantly different. It was observed that as the exposure time increases the percentage repellency increased (Table 8).

3 Discussion

This study has shown the potency of Hyptis suaveolens and Senna hirsuta against the larvae of Culex quinquefasciatus and as a repellent to adult of Culex quinquefasciatus. The plant extracts have the LC₅₀ values of 6.4% and 7.5% respectively and this shows that 6.4% of Hyptis suaveolens extract will be required to kill 50% of the test samples and also 7.5% of Senna hirsuta extract will be needed to kill 50% of the mosquito larvae used for the bioassay. Larval mortality was high in the 3^rd hour for Hyptis suaveolens and for Senna hirsuta by killing 80% and 90% of the test sample at the highest concentration. This result is comparable with earlier findings of (Arivoliand Samuel, 2011).

The biological activities of the leaf extracts of Hyptis suaveolens and Senna hirsuta might be due to the presence of various phytochemical compounds which includes saponin, alkaloid, tannins, phythic acid, cardia glycosides   and oxalic acid found in the plants. These compounds jointly or independently contribute to produce larvicidal and repellent effects against the mosquitoes. Most authors have reported that active compounds, such as saponin are soluble in both organic solvent and water, and they work by interacting with the cuticle membrane of the mosquito larvae ultimately disarranging the membrane which is the most probable reason for larval death (Macedo et al., 2007). (Weisman and Chapgain, 2003) reported that saponin extracted from the plant Balanites aegyptica exhibited 100% mortality against the larvae of Aedes aegypti.Alkaloid, glycosides and steriodisolated from plant caused a 100% larval mortality of Culex quinquefasciatus (Jayashree et al., 2003).

In comparing the relative toxicity of both extracts Senna hirsuta and Hyptis suaveolens on the larvae of Culex quinquefasciatus, it was observed from the result that Senna hirsuta leaf extract caused higher mortality of 90% at LC₅₀ 7.5% than Hyptis suaveolensextract which caused 80% mortality at LC₅₀ 6.4% concentration and this shows that 100% mortality is achievable if the time is increased above 3 hours and the concentration above 30%. From the phytochemical analysis carried out, Senna hirsuta has 15% saponin while Hyptis suaveolens has 10% saponin. The findings of Weisman and Chapgain (2003) reported that plants with more saponin component are mostly insecticidal. It was also reported by (Jang et al., 2002) that a species of the same family Senna tora leaves and seed extracts showed a good larvicidal and repellent effect against the filarial vector Culex quinquefasciatus.

Generally, individual botanical insecticides are slow in action and active only at high concentration which makes them impractical and uneconomical for field application (George and Vincent, 2005).Mixture of more than one extract from plants with insecticidal activity and with different modes of action against the vectors proves to be effective and is recommended for integrated resistance management in insect pests (Ru et al., 1998).

In this study, evaluation of the larvicidal efficacy of the combined extract of Hyptis suaveolens and Senna hirsuta on the 4^th instar larvae of Culex quinquefasciatus was carried out and the result showed that there was 95% mortality at LC₅₀ 6% which showed an increase in mortality from the individual plant extract.

The result of this study is comparable with that of (Tanprasit, 2005) which revealed that mixture of Hyptis suaveolens and Lantana camara possessed significantly higher larvicidal activity against Aedes aegypti than those of the individual substances.

The leaf extractof Hyptis suaveolens and Senna hirsuta was observed in this study as repellent. Essential oil from Hyptis suaveolens at 30% concentration repelled 70% of the mosquito while the essential oil from Senna hirsuta at 30% concentration repelled 50% of the mosquito. Hyptis suaveolens repellence was highest in the first hour for 30% and 20% concentration. This is in agreement with the study done by (Abagvi and Alavo, 2011) that repellence was high in the first 15 minutes to 1 hour of study and that study. Also according to them, it was shown that low concentration of Hyptis suaveolens essential oil induced maximal repellency rate against Anopheles gambiae.

Senna hirsuta gave repellence of 50% for 3 hours and repellence was highest in the third hour for all the concentrations. Though not much has been done using Senna hirsuta as a repellent against mosquito, however, other species of Senna has been reported to be a good repellent. From the findings of (Govindarajan et al., 2011), it was reported that methanol extract Senna fistula exhibited a repellence of 50.39% against Aedes aegypti for 10% concentration and 69% repellency in the 50% concentration.

Synergistic action was seen in the study when the extracts of Hyptis suaveolens and Senna hirsuta were used to repel the adult female of Culex quinquefasciatus. The result of the study showed a repellency of 75% on the test mosquito used.

This study is comparable to that of (Takawiraand Samuel, 2012) that plant extract combination of Colophospermum mopane, Dicomaanomala and Lippia javanicaagainst the malaria vector Anopheles gambia gave a higher repellency effect than the individual plant extracts. The result of the study showed that at 30% concentration, the leaf extracts of both plants was evidently larvicidal and repellent to the larvae and the adult Culex quinquefasciatus.

From the result, plant extracts of Hyptis suaveolens and Senna hirsuta passed the benchmark of eliminating more than 50% of the larvae and also repelling more than 50% adult of Culex quinquefasciatus from biting the volunteers. It is also important to note that throughout the use of the essential oil in the study, no skin reaction or dermatological effect was reported. It is therefore encouraged to use these plant extracts especially in their combined formulation to control mosquitoes.

The findings of the present study revealed that hexane leaf extract of Hyptis suaveolensand Senna hirsuta exhibited larvicidal and repellent action against Culex quinquefasciatus and this can effectively be used to control the filarial vector which has placed our country on the third most endemic countries to the disease. In this perspective, the combination of plant extracts like the extract from Hyptis suaveolens and Senna hirsuta should be integrated as a potential bio-pesticide to be tried in the field.

References

Awad O.M., and Shimaila A, 2003, Operational use of neem oil as an alternative to anophelinelarvicide, Part A: laboratory and field efficacy, Journal of Eastern Mediterranean Health, 9:637-645

Abagvi A.Z., and Alavo T.B.C., 2011, Essential Oil from Bush Mint, Hyptissuaveolens, is as Effective as DEET for Personal Protection against Mosquito Bites,Open Entomology Journal, 5: 45-48

https://doi.org/10.2174/1874407901105010045

Arivoli S., and Samuel T., 2011,Bioefficacy of Citrulluscolocynthis(L.),Schrad (Cucurbitaceae) whole plantextracts against Anopheles stephensi, Aedesaegypti and Culexquinquefasciatus(Diptera: Culicidae),International Journal of Current Research, 3(4): 296-304

Becker N., Petriae D., Zgomba M., Boase C., Dahl C., Lane J., and Kaiser A., 2003,Mosquitoes & their control,New York: Kluwer Academic Plenum Publisher,pp.1-16

https://doi.org/10.1007/978-1-4757-5897-9

PMCid:PMC1796847

Faradin M.S., and Day J.F., 2002, Comparative efficacy of insect repellents against mosquito bites, New England Journal of Medicine,347: 13-18

https://doi.org/10.1056/NEJMoa011699

PMid:12097535

Finney D.J., 1971,Probit analysis, 3^rd edition, Cambridge University press, Cambridge pp.36

Govindarajan M., Sivakumar R., Rajeswari M., and Yogalakshmi K., 2011, “Chemical composition and larvicidal activity of essential oil from Menthaspicata (Linn.)against three mosquito species,” Parasitology Research110,(5): 2023-2032

https://doi.org/10.1007/s00436-011-2731-7

PMid:22139403

George S., and Vincent S.,2005, “Comparative efficacy of Annona squamosaLinn and Pongamiaglabra Vent to Azadirachtaindica A. Juss against mosquitoes,” Journal of Vector Borne Diseases,42(4): 159-163

Jayashree G., Kurup M., Sudarslal S., Jacob V.B., 2003, Anti-oxidant activity of Centellaasiatica on lymphoma-bearing mice,Fitoterapia 74:431-434

https://doi.org/10.1016/S0367-326X(03)00121-7

Jang Y.S., Baek B.R., Yang Y.C., Kim M.K., and Lee H.S., 2002,Larvicidal activity of leguminous seed and grains against Aedesaegypti and Culexpipiennspallens, American Journal on Mosquito Control Association,18(3): 210-213

Mulla M.S., Thavara U., Tawatsin A., Chompoosri J., Zaim M., and Su T.Y., 2003, Laboratory and field evaluation of novaluron, a new insect growth regulator (IGR), against Aedesaegypti, Journal of Vector Ecology,28:241-254

Macedo M.L.R., Freire M.G.M., Silva M.B.R., and Coelho L.C.B.B, 2007, Insecticidal action of Bauhinia monandraleaf lectin (BmoLL) against Anagastakuehniella(Lepidoptera:Pyralidae), Zabrotessubfasciatus and Callosobruchusmaculatus (Coleoptera:Bruchidae), Comparative Biochemistry and Physiology Part A, Molecular and Integrative Physiology, 146: 486-498

https://doi.org/10.1016/j.cbpa.2006.01.020

PMid:16488638

Nwoke B.E.B., and Ebo. J.C., 1993, Human activities in south-eastern Nigeria and their potential danger to the breeding of mosquito vectors of human diseases, Annals of Medical Sciences,24:180-182

Navie S.C, Markwell B, Playford J., and Adkins S.W., 2002,Suburban and Environmental Weeds: an interactive identification and information system, The University of Queensland, St. Lucia, Queensland

Obadoni B.O., and Ochuko P.O., 2001, Phytochemical studies and comparative efficacy of the crude extracts of some homeostatic plants in Edo and Delta States of Nigeria, Global Journal of Pure Applied Science, 86: 203-208

Pearson D., 1974,The chemical analysis of foods. 6th edition, Churchill Livingstone, Edinburgh pp.451

Peerzada N., 1997, Chemical composition of the essential oil of Hyptissuaveolens, Molecule 2: 165-168

https://doi.org/10.3390/21100165

Ru L.J., Rui C.H., Fan X.L., ZhaoT.E., and WeiC., 1998, “Realized heritability analysis of resistance to single and multiple insecticides in Lipaphiserysimi (Kaltenbach) and Helicoverpaarmigera (Hübner),” ActaEntomologicaSinica,41(3): 243-249

Reuben R., Tewaris S.C., Hiriyan J., and Akiyama J., 1994, Illustrated keys to species of Culex associated with Japanese encephalitis in Southeast Asia (Dipteria: Culicidae),Mosquito System,26:75-96

Schreck C.E., and McGover T.T., 1977, Repellents and other personal protection strategies against Aedesalbopictus, American Journal of Mosquito Control Association,5:247-252

Service M.W., 1989, Lymphatic filariasis, In: Demography and Water-Borne Diseases,Service, M.W. (Ed),CRC Press, Boca pp.69-72

Tanprasit P., 2005, Biological Control of Dengue Fever Mosquitoes (Aedesaegypti Linn.) Using Leaf Extracts of Chan (Hyptissuaveolens (L.)Poit.)and Hedge Flower (Lantana camara Linn.), Thesis Suranaree University of Technology, Nakhon Ratchasima, Thailand

Takawira K, and Samuel J., 2012, “Malarial control with mosquito repellent plants: Colophospermum mopane,Dicomaanomala and Lippiajavanica”, World J Life Sci. and Medical Research, Vol.2, No.4, pp.141-149

White G.B., 1987, Medical entomology: mosquitoes,pp.1404-1435 In:Mason-Bahr, P.E.C. &Apted, F.I.C (ed) Mason’s Tropical Diseases, 19^th Edition. London, Baillere Tindall, 1557pp

WHO, 1996, Report of the WHO informal consultation on the evaluation and testing of insecticides, CTD/WHOPES/IC/96.1. Control of Tropical Diseases Division,Geneva.WHO

WHO, 1981, WHO Expert Committee on Insecticide,Instructions for determining the susceptibility or resistance of mosquito larvaeto insecticides, WHO/VBC/8.807

Weisman Z., and Chapgain B.P., 2003, Dengue Bulletin 27: 168-173. World Journal of Life Sci. and Medical Research, 2(4):141-143