ISSN:
Journal of Biology and Medicine
Research Article       Open jbmcess      Peer-Reviewed

Bioefficacy 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
*Corresponding author: Adesina Femi Peter, Department of Biology Federal University of Technology Akure, Ondo State, Nigeria, Tel: +2347033028164; E-mail: femi.adesina@outlook.com
DOI: 10.17352/jbm.000004
Received: 19 November, 2018 | Accepted: 27 November, 2018 | Published: 28 November, 2018
Keywords: Clerodendrum; Anopheles gambiae; Plant extract; Larvae; Pupae

Cite this as

Ileke KD, Adesina FP (2018) Bioefficacy of Larvicidal and Pupicidal properties of Clerodendrum capitatum and Bridelia machrantha leaves extracts against Malaria Vector, Anopheles gambiae giles [Diptera: Culicidae]. J Biol Med 2(1): 007-011. DOI: 10.17352/jbm.000004

Studies were conducted under ambient condition to assess the bio-efficacy 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, Twenty- two 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 significant 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.

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, filariasis, 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 identified to transmit malaria in Nigeria and Anopheles gambiae was reported as a primary vector of malaria among other species in the southern Nigeria [4-10].

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 [5,9-13].

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 [8,10,15-18]. Therefore, the present study investigates the toxicity of Clerodendrum capitatum and Bridelia machranth extract on the larvae and pupae of Anopheles gambiae.

Materials and Methods

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 field. The clean transparent white bucket was filled 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 first instar larva. These larvae were taken into the laboratory. In the laboratory, the larvae were identified 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 fine 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 1mm2 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 filter papers and solvent evaporated using a rotary evaporator at 30 to 40oC 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.

Preparation of standard stock solution

Standard stock solutions were prepared by dissolving 4g of the crude extracts in 1Litre of water. From these stock solutions, different concentrations of 25 mg/L, 50 mg/L, 100 mg/L, 150 mg/L and 200 mg/L were prepared and these aqueous solutions were used for the various experiments.

Bioassy

Toxicity of Extracts on Larvae of Mosquito: 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 obtained from the culture were introduced separately into the various plant extracts along with a set of control containing rain water without any test solution and all tested concentrations were replicated four times. The number of dead larvae were counted and recorded accordingly after 24 hours of treatment.

Dead larvae were those incapable of rising to the surface or without the characteristic diving reaction when the water was disturbed [21].

% Larval Mortality=  Number of dead larvae Number of larvae introduced  x  100 1 MathType@MTEF@5@5@+=feaaguart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbnvMCYL2DLfgDOvMCaeXatLxBI9gBaerbd9wDYLwzYbItLDharuavP1wzZbItLDhis9wBH5garqqtubsr4rNCHbGeaGakY=3j0xXdbba91rFfpec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqaq=JfrVkFHe9pgea0dXdar=Jb9hs0dXdbPYxe9vr0=vr0=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@83F5@

The LC50 and LC90 were carried out by Probit analysis.

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.

% Pupal Mortality=  Number of dead pupae Number of pupae introduced  x  100 1 MathType@MTEF@5@5@+=feaaguart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbnvMCYL2DLfgDOvMCaeXatLxBI9gBaerbd9wDYLwzYbItLDharuavP1wzZbItLDhis9wBH5garqqtubsr4rNCHbGeaGakY=3j0xXdbba91rFfpec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqaq=JfrVkFHe9pgea0dXdar=Jb9hs0dXdbPYxe9vr0=vr0=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@80CD@

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 (LC50), the 90% lethal concentration (LC90) and their 95% confidence limits.

Results

Contact toxicity of C. capitatum and B. machrantha Extracts on An. gambiae Larvae

Effects of C. capitatum and B. machrantha extracts on percentage mortality of An. gambiae larvae is presented in table 1. Larvae mortality of An. gambiae occurred in a dosage-dependent manner. There was no significant 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. Generally, An. gambiae larvae mortality increased with increase in hours of exposure.

Table 1: Effect of C. capitatum and B. machrantha Extracts on % mortality of Larvae An.gambiae after 24 hours of exposure.
Plant Extracts Concentration (mg/L)
25 50 100 150 200
C. capitatum 30.00±2.22b 47.50±2.50b 60.00±2.22b 90.00±2.22b 100.00±0.0b
B. machrantha 22.50±2.50b 35.00±2.89b 50.00±2.22b 77.50±2.50b 92.50±2.50b
Control 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a
Mean ± Standard error followed by the same letters, superscript at the end of each value, down the column are not significantly different (p > 0.05) from one another using New Duncan’s Multiple Range Test.

Contact toxicity of C. capitatum and B. machrantha Extracts on An. gambiae Pupae

Table 2 shows the effects of contact toxicity of C. capitatum and B. machrantha extracts on percentage mortality of An. gambiae larvae after 24 hours of exposure. Mortality of An. gambiae pupae occurred in a dosage-dependent manner. There was no significant differences (p>0.05) in toxicity level of the C. capitatum and B. machrantha extracts on An. gambiae pupae at concentrations tested (62.5mg/L, 125mg/L, 250mg/L, 500mg/L and 1000mg/L). Clerodendrum capitatum caused 30%, 35%, 57.5%, 72.5% and 97.5% mortality of An. gambiae larvae after 24 hours of exposure at concentrations 62.5mg/L, 125mg/L, 250mg/L, 500mg/L and 1000mg/L respectively. Bridelia machrantha extract at concentrations 62.5mg/L, 125mg/L, 250mg/L, 500mg/L and 1000mg/L caused 15%, 30%, 47.5%, 65% and 90% mortality of An. gambiae larvae after 24 hours of exposure, respectively. Generally, An. gambiae pupae mortality increased with increase in hours of exposure.

Table 2: Effect of C. capitatum and B. machrantha Extracts on % mortality of Pupae An. gambiae after 24 hours of exposure.
Plant Extracts Concentration (mg/L)
62.5 125.0 250.0 500.0 1000.0
C. capitatum 20.00 ±2.22c 35.00 ±2.25c 57.50 ±2.50c 72.50 ±2.50b 97.50 ±2.50c
B. machrantha 15.00 ±1.29b 30.00 ±2.22b 47.50 ±2.50b 65.00 ±2.25b 90.00 ±2.25b
Control 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00a 0.00 ±0.00a
Mean ± Standard error followed by the same letters, superscript at the end of each value, down the column are not significantly different (p > 0.05) from one another using New Duncan’s Multiple Range Test.

LC50 and LC90 of C. capitatum and B. machrantha Extracts on An. gambiae larvae and pupae

LC50 and LC90 of C. capitatum and B. machrantha Extracts on larvae and pupae of An. gambiae is shown on table 3, C. capitatum had lower LC50 and LC90 recorded when compared to B. machrantha that had the higher value on both larvae and pupae. LC50 and LC90 of C. capitatum on larvae were 50.14 mg/L and 172.30 mg/L respectively while that of B. machrantha was 69.27 mg/L and 268.94 mg/L respectively. On pupae, LC50 and LC90 of C. capitatum was 190.87 mg/L and 854.02 mg/L respectively while B. machrantha had 253.76 mg/L and 1305.47 mg/L respectively.

Table 3: LC50 and LC90 of C. capitatum and B. machrantha Extracts on An. gambiae larvae and pupae.
>Plant Extracts Concentration (mg/L)
Plant Extract LC50 (Lower – Upper Limit) LC90 (Lower – Upper Limit)
>Larvae >C. capitatum >50.14 (6.86 – 95.33) >172.30 (9.74 – 334.86)
>  >B. machrantha >69.27 (31.86 – 119.87) >268.94 (144.69 – 393.13)
>Pupae >C. capitatum >190.87 (120.81 – 283.49) >854.02 (505.09 – 1202.96)
>  >B. machrantha >253.76 (216.87 – 297.2 9) >1305.47 (983.16 – 1919.19)  

Phytochemicals screening of C. capitatum and B. machrantha Leaves

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 then-hexane, and aqueous extracts of C. capitatum and B. machrantha were identical. Alkaloids, saponins, tannins, phlobatannins, anthraquinones, flavonoids and cardiac glycosides were detected in both aqueous ant n-hexane extracts of C. capitatum and B. machrantha leaves.

Table 4: Phytochemicals in different extracts of C. capitatum and B. machrantha Leaves.
Phytochemicals n-hexane extract of C. capitatum Aqueous extract of C. capitatum n-hexane extract of B. machrantha Aqeous extract of B. machrantha
Alkaloids + + + +
Saponins + + + +
Tannins + + + +
Phlobatannins _ ­_ _­ _­
Anthraquinones _ ­_ _­ _­
Flavonoids + ­+ + +
Cardiac glycosides + + + +
Key: _Absent ; +  Present.

Discussion

This study revealed that botanicals extract can be alternatives to the use of synthetic insecticide in the control of insect vector. The two plants assessed had proven high insecticide ability against mosquito and this was in agreement with Ileke et al. [6], who reported the insecticidal potential of botanicals like Anacardium occidentale, Afromomum melegueta, Garcina kola and Citrus sinensis plants against malaria vector, mosquito, Anopheles gambiae. Akinkurolere et al. [15] found plant oils to be effective against mosquito larvae and pupae.

The high toxicity of these plants on mosquito larvae and pupae may be attributed to the active compounds present in these plants. The preliminary phytochemical analysis of extracts from the plants leaves was found to have presence of alkaloids, flavonoids, cardiac glycosides, tannins, anthtraquinones and sapronins. Flavonoid was present in the two plants tested which may be responsible for it high larvicidal and pupicidal potentials [5-7]. Also, the swimming ability of the mosquito’s larvae and pupae could have been affected, thereby hinder their swimming to the surface for oxygen [5,25].

Nevertheless, C. capitatum caused high mortality than B. machrantha leaves on Anopheles mosquito larvae and pupae. This result is in agreement with the findings of Morya et al., [26] who tested the powder leave of some plants including Clerodendrum against Corcyra cephalonica and among the plants, Clerodendrum sp was reported to have the highest biopesticide ability. Adesina et al. [27] on Bio-efficacy of Clerodendrum capitatum against Dermestes maculates larvae infestation on smoked catfish Claria gariepinus, found that the plant powder cause significantly (p < 0.05) larval mortality compared to the untreated fish at 24 h, 48 h and 72 h post infestation. Clerodendrum genus had being widely study for it bioactivities toward medicinal benefits as 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 [5,33,34] (Akinkurolere et al., 2006, Ileke and Oni, 2011; Ileke and Ogungbite, 2015).

Conclusion

Ultimately, due to the efficiency 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.

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© 2018 Ileke KD, et al. This is an open-jbmcess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 

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