Bioefficacy of Larvicidal and Pupicidal properties of Clerodendrum capitatum and Bridelia machrantha leaves extracts against Malaria Vector, Anopheles gambiae giles [Diptera: Culicidae]

Studies were conducted under ambient condition to assess the bio-effi cacy of Clerodendrum capitatum and Bridelia machrantha leaves against developmental stages (larva and pupa) of malaria vector, Anopheles gambiae. One hundred millimeter (100mm) of aqueous solutions of the various plant extracts at various concentrations of 25mg/L, 50mg/L, 100mg/L, 150mg/L and 200mg/L were each put in a labelled transparent bowl. Twenty (20) 3rd/4th larval instar were introduced separately into the C. capitatum and B. machrantha extracts along with a set of control containing rain water without any test solution and all tested concentrations were replicated four times. Similarly, Twentytwo days old pupae of Anopheles mosquito were introduced separately into the various plant extracts. The number of dead larvae and pupae were counted and recorded accordingly after 24 hours of treatment. The results showed that Larvae and pupae mortality of An. gambiae occurred in a dosage-dependent manner. There was no signifi cant differences (p>0.05) in toxicity level of the two plant extracts on An. gambiae larvae at concentrations 25mg/L, 50mg/L, 100mg/L, 150mg/L and 200mg/L. Clerodendrum capitatum at concentrations 25mg/L, 50mg/L, 100mg/L, 150mg/L and 200mg/L caused 30%, 47.5%, 60%, 90% and 100% mortality of An. gambiae larvae after 24 hours of exposure, respectively. Bridelia machrantha extract caused 22.5%, 35%, 50%, 77.5% and 92.5% mortality of An. gambiae larvae after 24 hours of exposure at rates 25mg/L, 50mg/L, 100mg/L, 150mg/L and 200mg/L, respectively. Similar trend of results were recorded on the pupae. C. capitatum had lower LC50 and LC90 recorded when compared to B. machrantha that had the higher value on both larvae and pupae. The use of natural insecticide in the implementation of national policy for the control of malaria vectors should be promoted as they are easily degradable, medicinal and eco-friendly. Research Article Bioeffi cacy of Larvicidal and Pupicidal properties of Clerodendrum capitatum and Bridelia machrantha leaves extracts against Malaria Vector, Anopheles gambiae giles [Diptera: Culicidae] Ileke Kayode David and Adesina Femi Peter* Department of Biology, School of Science, Federal University of Technology, P.M.B. 704, Akure, Ondo State, Nigeria Received: 19 November, 2018 Accepted: 27 November, 2018 Published: 28 November, 2018 *Corresponding author: Adesina Femi Peter, Department of Biology Federal University of Technology Akure, Ondo State, Nigeria, Tel: +2347033028164; E-mail:


Introduction
One of the insects that are of medical importance is mosquito, it is more predominant in tropical countries, thereby putting up to 3.2 billion people at risk of malaria [1].
It abhors parasite (Plasmodium spp.) that can cause diseases like malaria, fi lariasis, yellow fever and denque on human [2] and it has resulted to 44500 deaths out of 216 million malaria cases reported in the world [1]. This disease is transmitted only by the Anopheline mosquitoes from the order Diptera. Out of 400 species of Anopheles described, 40 species are important vectors of human malaria worldwide [3]. Many species has been identifi ed to transmit malaria in Nigeria and Anopheles gambiae was reported as a primary vector of malaria among other species in the southern Nigeria [4][5][6][7][8][9][10].
Mosquito net, repellant cream and paint, insecticide and mosquito coil among others are well known as mosquito control methods in Nigeria. Over the decades, synthetic insecticide has been the most used because of its effectiveness. Nevertheless, its negative effects on human health, non target organisms (including aquatic animals) and environment are a major concern. It has also make mosquito population develop resistance against its application [5,[9][10][11][12][13].
Searching for new compounds that are highly effective against mosquito and at the same time that are eco-friendly, degradable, not toxic to humans and non target organisms, and inexpensive has being a current trend in research. Several researchers have suggested aromatic plants and their essential oils as natural insecticides for pest control because they have few harmful effects on ecosystem structure and function [14].
Eggs, larvae, pupae and adults of different species of mosquito had been subjected to different plant extracts and volatile oils to assess the insecticide ability of botanicals [8,10,[15][16][17][18]. Therefore, the present study investigates the toxicity of Clerodendrum capitatum and Bridelia machranth extract on the larvae and pupae of Anopheles gambiae.

Collection and rearing of larva and pupa mosquito
Mosquito baits, consisting of shallow containers with a large surface area were established under a partial shade in an open fi eld. The clean transparent white bucket was fi lled with rain water to mimic mosquito natural breeding environment and to attract adult female for oviposition. Ten grams (10g) of yeast (Bakers' yeast) were sprinkled on the surface of the water and allowed to decompose slowly to nourish the developing larvae. Wild mosquitoes were allowed to freely visit the baits and to lay eggs. The water was monitored for 3-5 days for the development of the egg and fi rst instar larva. These larvae were taken into the laboratory. In the laboratory, the larvae were identifi ed into species level using the morphological keys [19]. The Anopheles larvae were separated from the mixed culture and transferred into another plastic container containing rain water. Some of the larvae were used for the larvicial tests. The Anopheles larvae were further nurtured to pupae for 3-5 days for pupicidal tests.

Collection of plant materials
The plants evaluated in this work were Clerodendrum capitatum and Bridelia machrantha leaves. The fully developed leaves of C. capitatum and B. machrantha, free of insecticides were obtained in fresh form a farm at Ibule Soro, Akure, Ondo State, Nigeria. They were taken to the Crop, Soil and Pest management Department, Federal University of Technology, Akure, Ondo State for authentication by Plant Taxonomist.

Extraction of plant materials
The leaves of C. capitatum and B. machrantha were washed with distilled water, shade dried, cut into small pieces and air dried for 14 days in the laboratory. The seeds of M. myristica were also air dried for 21 days before pulverized into fi ne powders using an industrial electric pulverizing machine at the Department of Animal Production and Health Laboratory of the Federal University of Technology Akure. The powders were further sieved to pass through 1mm 2 perforations and kept in air-tight plastic containers for storage before use at ambient temperature (28 ± 2) °C.
About 400g of C. capitatum and B. machrantha powders were soaked separately in an extraction bottle containing 500ml of absolute n-hexane for 3 days. The mixture was stirred occasionally with a glass rod and extraction was terminated after 3 days. Filtration was carried out using a double layer of Whatman No. 1 fi lter papers and solvent evaporated using a rotary evaporator at 30 to 40 o C with rotary speed of 3 to 6 rpm for 8 hours [20]. The resulting extracts were air dried in order to remove traces of solvent. The extracts were kept in labeled plastic bottles till when needed.

Toxicity of extracts on pupae of mosquito
Also, similar experiment as described above was carried out for pupae of Anopheles gambiae. One hundred millimeter (100mm) of aqueous solutions of the various plant extracts at various concentrations of 25mg/L, 50mg/L, 100mg/L, 150mg/L and 200mg/L were each put in a labelled transparent bowl. Twenty (20) two (2) days old pupae of Anopheles mosquito were introduced separately into the various plant extracts. They were replicated four times and rain water was used as control. The number of dead pupae were counted and recorded accordingly after 24 hours of treatment.
Number of dead pupae 100 % Pupal Mortality x Number of pupae introduced 1 

Phytochemical Screening of C. Capitatum and B. Machrantha Leaves
Chemical tests were carried out on the aqueous and methanol extracts of C. capitatum and B. machrantha leaves for the qualitative determination of phytochemical constituents using standard procedures as described by Harborne [22]. Trease and Evans [23], Sofowora [24].

Statistical analysis of data
Data were subjected to analysis of variance (ANOVA), and means were separated using the new Duncan's multiple Range test. The log-Probit model analysis was done to the data recorded in the larvicidal and pupicidal bioassay to assess the 50% lethal concentration (LC 50 ), the 90% lethal concentration (LC 90 ) and their 95% confi dence limits.  Table 2 shows the effects of contact toxicity of C. capitatum  Table 4 presented the result of the phytochemical screening of the methanol and aqueous extracts of C. capitatum and B. machrantha leaves. The phytochemicals present in thenhexane, and aqueous extracts of C. capitatum and B. machrantha were identical. Alkaloids, saponins, tannins, phlobatannins, anthraquinones, fl avonoids and cardiac glycosides were detected in both aqueous ant n-hexane extracts of C. capitatum and B. machrantha leaves.

Discussion
This study revealed that botanicals extract can be alternatives to the use of synthetic insecticide in the control    Control 0.00 ±0.00 a 0.00 ±0.00 a 0.00 ±0.00 a 0.00 ±0.00 a 0.00 ±0.00 a Mean ± Standard error followed by the same letters, superscript at the end of each value, down the column are not signifi cantly different (p > 0.05) from one another using New Duncan's Multiple Range Test.  well as its insecticidal ability [28].
It took more concentration for both plants to achieved higher mortality on pupae than on larvae. This means that larvae is more susceptible to these plants than pupae and this could be attributed to the active feeding stage of the larvae since pupae stages of the insect are not feeding [5,29,30].
Related observations were reported by Akinkurolere et al. [15], who obtained a 75.35% and 47% mortality of Anopheles gambiae larvae and pupae respectively. Also supported by Ileke and Ogungbite [5], who worked on the effectiveness of Altonia boonei on malaria vector. The strong choky odour of the plants may have also disrupted respiratory activity of the insect [31], blocking the spiracles thereby hinder the larvae breathe [32].
This could result into asphyxiation and death of the larvae

Conclusion
Ultimately, due to the effi ciency of C. capitatum and B.
machrantha leaves extracts against Anopheles mosquitoes in the study, natural insecticide proved to be an alternative approach to the control of mosquito and to the use of synthetic insecticides whose cost, phenomenon of resistance developed in mosquitoes treated and proven action on non-target organisms are challenges in malaria controls.