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Larvicidal effects of cymbopogon citratus (lemon grass) extract against Culex quinquefasciatus qularvae (Diptera, culicidae).

Introduction

The use of traditional insect repellents/killers is widespread among the different cultures and communities of Africa and beyond. Plants have been used since ancient times to repel/kill blood-sucking insects in the human history (1). However, their use against pests decreased when chemical products became developed.

Larviciding is a successful way of reducing mosquito densities in their breeding places before they emerge into adults. Larviciding largely depends on the use of synthetic chemical insecticides--organophosphates (e.g. temephos, fenthion), insect growth regulators (e.g. diflubenzuron, methoprene), among others. Although effective, their repeated use has disrupted natural biological control systems and sometimes resulting in the widespread development of resistance (2, 3). These problems have warranted the need for developing alternative strategies using ecofriendly products. Plants offer an alternative source of insect-control agents because they contain a range of bioactive chemicals (4), many of which are selective and have little or no harmful effect on non-target organisms and the environment (5).

Much effort has, therefore, been focused on plant extracts or phytochemicals as potential sources of mosquito control agents or as lead compounds (6). In this context, essential oils have received much attention as potentially useful bioactive compounds against insects (2) showing a broad spectrum of activity against insects, low mammalian toxicity and degrading rapidly in the environment. Studies of essential oils obtained from the plants, Cymbopogan citratus (6), Hemidesmus indicus (7), Zanthoxylum armatum (3), and many other plants (2, 4) have demonstrated promising larvicidal activities against mosquito vectors.

The search for new strategies or natural products to control destructive insects and vectors of diseases is desirable due to the prevalent occurrence of vector resistance to synthetic insecticides (8); and the problem of toxic non biodegradable residues contaminating the environment and undesirable effects on non target organisms (9).

Anopheles Anopheles: see mosquito. Click the link for more information. Stephensi, Anopheles aegypti and Culex. quinquefasciatus (Diptera : Culicidae) are the major urban vectors of malaria, dengue and lymphatic filariasis, respectively (3). The approach to combat these diseases largely relied on interruption of the disease transmission cycle by either targeting the mosquito larvae through spraying of stagnant water breeding sites or by killing the adult mosquitoes using insecticides (10). The essential oil from Cymbopogon citratus exhibited 70.3% repellency against Aedes aegyptii and 100% repellency against Anopheles stephensi and Culex quinquefasciatus (11). However, the oil exhibited no effect on Aedes aegyptii larvae at one, two and twenty four hour's exposure (1). In the present paper we report the larvicidal activity of Cymbopogon citratus (lemon grass) extract against Culex quinquefasciatus. The results of the present study would be useful in promoting research aiming at the development of new agent for mosquito control based on bioactive chemical compounds from indigenous plant source.

Materials and Methods

Collection of Culex quinquefasciatus larvae

The larvae were sourced from slow moving gutters within the main campus of Ahmadu Bello University, Samaru (11[degrees]10'N, 07[degrees] 39'E). The larvae were immediately transferred to the laboratory of the of Biological Sciences Department and kept in rearing cages (30 x 45 x 60 cm) and were fed on yeast (baker's yeast). Emergent adult, female mosquitoes were fed with blood from immobilized live pigeon with skinned chest hung over and tied to the cage to facilitate egg laying (12). Eggs were collected on ovi-positioning pads placed angularly onto water surface and transferred into rectangular perspex containers (15 x 10 x 20 cm). Emergent larvae were indentified as 3rd instar stage using published Keys. Late 3rd instars were isolated for toxicity test and fed with baker's yeast and dog biscuits, until ready for test.

Collection and preparation of plant extract

The leaves of C. citratus were collected from staff residential quarters of Ahmadu Bello University, Samaru-Zaria, Nigeria, and brought to the laboratory. The leaves were dried under shade at room temperature (29 [+ or -] 1[degrees]C) for about 20 days. The completely dried leaves were powdered and sieved to get fine powder of leaf. The methanol-leaf extracts from the sieved fine leaf powder was obtained by using Soxhlet apparatus. Two hundred and fifty grams of leaf powder was dissolved in 200 ml of methanol (as a solvent) and extracted in the Soxhlet apparatus (13), for 8 h over a mantle heater at 55[degrees]C. The methanol extracts were concentrated using a vacuum evaporator at 45[degrees]C under low pressure. After complete evaporation of the solvents, the concentrated extracts were collected and stored in a refrigerator for later use.

Bioassays

The acute toxicity bioassay tests to determine the 24-hour lethal concentration that will result to 50% mortality of the test organisms (24h-[LC.sup.50]) was conducted by static renewable method (14). Each treatment was in triplicate with serial dilutions made from the stock solution. After a series of pilot bioassays, the definitive concentrations of toxicant used were 2.0, 5.0, 10.0, 13.0 and 15.0g/L. Each treatment bioassay was carried out in triplicate and replicated. The bioassays were conducted in 100ml capacity glass beakers. Each treatment concentration and the control were inoculated with ten larvae using a 1ml syringe. The larvae were viewed with the aid of a stereoscopic microscope and assumed dead when heartbeat ceased. All dead larvae were removed from the media by sucking out with the syringe.

The water quality of the bioassays was determined at the beginning and at the end of the experiments. The dissolved oxygen (DO) and total hardness were determined by the methods of APHA (15). The pH values were determined by using a Philips pH meter model PW9418. The water temperatures were taken with a mercury-in-glass thermometer. SPSS 17.0 statistical software was used to compute the [LC.sup.50] value.

Results

The mean water quality parameters are 25.00 [+ or -] 0.70 [degrees]C for temperature, 7.75 [+ or -] 0. 35mg/L for dissolved oxygen, 76.0 [+ or -] 1.41 mg/LCaC[O.sub.3] for total hardness and 7.6 [+ or -] 08 for pH. There was no significant difference (p > 0.05) in the values of water quality physicochemical during the course of experimentation. The leaf extract of C. citratus exhibited various degrees of larvicidal efficacy against Culex quinquefasciatus. On introduction to C. citratus extract toxicant; the larvae become hyperactive and showed erratic swimming and loss of balance. The results of mortality rate in relation to concentration of C. citratus leaf extract are given in Table 1. The extract's computed [LC.sup.50] value of 3.495 g/L with 2.852g/L and 4.575 g/L as lower and upper limits respectively, was obtained by using log-probit regression of SPSS 17.0 for Windows/Microsoft Excel programme. Toxic behavioral response and onset of mortality was dose dependant.

Discussion

The anti-mosquito properties of C. citrates extracts observed during this investigation is similar to other findings on Cymbopogon species. Our study shows that C. citrtus crude extract has tremendous larvicidal potential. The main chemical constituents of three different fractions (methanol, 80% aqueous ethanol and water) obtained from an essential oil-free infusion of extracts of Cymbopogon citratus leaves includes: Tannins, phenolic acids (caffeic and p-coumaric acid derivatives) and flavone glycosides (apigenin and luteolin derivatives) (16). However, the main chemical constituents of the crude extract and oil from Cymbopogon spp are alpha- cubebene, camphene, citronellal, geraniol, limonene, palmitic acid and sabinene (4).

Thus the behavioral responses and mortality of Culex quinquefasciatus observed in the present investigation could be attributed to phytochemicals present in the leaf extracts of Cymbopogon citrates. Earlier studies observed that phytochemicals have a major role in mosquito control programme (5, 17, 18). Gopieshkhanna and Kannabiran (7) have observed the presence of carbohydrates, saponins, phytosterols, phenols, flavonoids and tannins in 3 different plant extracts as having mosquito larvicidal activity. Pelah et al. (19) reported the use of commercial saponin from Quillaja saponaria bark as a natural larvicidal against Aedes aegypti and Culex pipens. Isolated triterpenoids from Lantana camara were found to have an antibacterial activity (20). Also Cardiac glycoside was found to have a acaricidal effect against larva and adult stages of the camel tick (21).

The 24-[LC.sup.50] of Cymbopogon citratus in this investigation was estimated to be 3.495g/L. However, some studies showed that sensitivity of Culex spp was lesser towards many plant extracts and essential oils (1, 3, 4, and 17). The variations in the toxicity of crude extract and essential oil against different mosquito species are not uncommon (6), due to qualitative and quantitative variations of constituents like monoterpenes, saponins, phytosterols, phenols, flavonoids and tannins in the crude extract and essential oil composition. The findings of the present studies, therefore, suggest the use of Cymbopogon citrates leaf extract as a local resource in controlling mosquito larvae. The source constraint may not allow their practical utility in larger breeding habitat; however, the plant source may be utilized by local people for controlling mosquito larvae in small breeding places like water coolers, tree holes, abandoned wells, drums and containers in and around the rural/suburban dwellings. Such practice would not only reduce the chemical burden on the environment but also promote sustainable utilisation of locally available bioresource by rural communities. Further studies may be directed towards enhancing the efficacy of such extarcts with the use of potentiating/synergistic agents, developing suitable formulations and their bioefficacy evaluation in real field conditions.

Conclusion

In this present study the leaf extract of Cymbopogon citratus (lemon grass) obtained using methanol was found to have larvicidal activity against Culex quinquefasciatus, thus proposing the use this leaves as a mosquito control agent. Also the results of this investigation indicate that the grass could be studied further in detail and its beneficial effect to the control of vector borne diseases could be utilized for healthy environments.

References

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[2] Cheng SS, Chang HT, Chang ST, Tsai KH, Chen W.J., 2003. Bioactivity of selected plant essential oils against the yellow fever mosquito Aedes aegypti larvae. Biores Technol 89: 99-102.

[3] Tiwary, M., Naik, S.N., Tewary, D.K., Mittal, P.K.and Yadav, S., 2007. Chemical composition and larvicidal activities of the essential oil of Zanthoxylum armatum DC (Rutaceae) against three mosquito vectors. Journal of Vector Borne Diseases.1:1-11.

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[11] Joseph, C.C., Ndoile, M.M., Malima, R.C. and Nkuniya, M.H.M., 2004. Larvicidal and mosquitocidal extracts, a coumrin, isoflavonoids and pterocarpans from Neorautanenia mitis. Trans R Soc Trop Med Hyg. 98: 451-5.

[12] Amer, A. and Mehlhorn, H., 2006. Repellency effect of forty-one essential oils against Aedes, Anopheles, and Culex mosquitoes. Parasitol Res 99(4):478-490

[13] Poopathi, S., 1999. Effects of Bacillus sphaericus and Bacillus thuringensis Var isrealiensis on the ultrastructural changes in the midgut of Culex quinquefasciatus. J. Entomol. Res. 23: 347-357.

[14] Vogel, A.I., 1978. Text book of practical organic chemistry. The English Language Book Society and Longman, London, p 1368

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[16] APHA., 1998. Standard Methods for the Examination of Water and Wastewater (Greenberg AE, Clesceri LS & Eaton AD eds), 20th edn. American Public Health Association (APHA) Inc, Washington DC.

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[18] Hag, E.L., Nadi, E.A., El, A.H. and Zaitoon, A.A., 1999. Toxic and growth retarding effects of 3plant extracts on Culex pipiens larvae (Diptera : Culicidae). Phytother. Res., 13: 388-392.

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[20] Pelah, D., Abramovich, Z., Markus, A. and Wiesman, Z., 2002. The use of commercial saponin from Quillaja saponaria bark as a natural larvicidal agent against Aedes aegypti and Culexpipiens. J. Ethnopharmacol., 81(3): 407-409.

[21] Mahmoud, S., Alaa, K., Xiaoyang, L. and James, S., 1999. Antibacterial triterpenoids isolated from Lantana camara. Pharmaceut. Biol., 37(1): 63-66.

[22] Al-Rajhy, D.H., Alahmed, A.M., Hussein, H.I. and Kheir, S.M., 2003. Acaricidal effects of cardiac glycosides, azadirachtin and neem oil against the camel tick, Hyalomma dromedaril (Acari: Ixodidae) . Pest Management Sci., 59(11): 1250-1254.

Adakole J.A. and Adeyemi A.F.F.

Department of Biological Sciences, Ahmadu Bello University, Zaria. Nigeria E-mail: drjaadakole@yahoo.com
Table 1: Probit table for 24-h of larvicidal effects of Cymbopogon
citratus extract against Culex quinquefasciatus

Conc. Log of 24-h mortality Total
tanks Conc. Replicate Replicate Replicate observed
(g/L) A B C mortality

Control 0.00 0 0 0 0
1 0.000 1 0 2 3
2 0.301 2 2 3 7
3 0.477 4 3 5 12
4 0.602 5 4 6 15
5 0.699 6 6 6 18

Conc. Total Residual Probability
tanks expected
(g/L) mortality

Control 0 0 0
1 2.088 -0.088 0.070
2 6.987 0.013 0.233
3 11.567 0.433 0.386
4 15.242 -0.242 0.508
5 18.095 -0.095 0.603

N = total number of organisms exposed = 30
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Author:Adakole, J.A.; Adeyemi, A.F.F.
Publication:International Journal of Applied Environmental Sciences
Date:May 1, 2012
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