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Larvicidal Activity of Medicinal Plant Extracts Against Aedes aegypti, Ochlerotatus togoi, and Culex pipiens pallens (Diptera: Culicidae)
J. Asia-Pacific Entomol. 7(2): 227 -232 (2004) www.entomology.or.kr
Larvicidal Activity of Medicinal Plant Extracts Against Aedes aegypti, Ochlerotatus togoi, and Culex pipiens pal/ens (Diptera: Culicidae) 2
2
Young-Cheol Yang, I1-Kwon Park', Eun-Hee Kim , Hoi-Seon Lee3 and Young-Joon Ahn * Department of Advanced Organic Materials Engineering, Chonbuk National University, Chonju 561-756, Korea IPorestry Research Institute, Seoul 151-742, Korea 2School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea 3Paculty of Biotechnology, College of Agriculture, Chonbuk National University, Chonju 561-756, Korea
Abstract The toxicities of methanol extracts from 28 medicinal plant species to early 4th instar larvae of Aedes aegypti, Ochlerotatus togoi (Aedes togoi), and Culex pipiens pal/ens were determined in the laboratory. Responses varied according to plant and mosquito species. At a concentration of 100 ppm, >90% mortality of the three species was obtained with the extracts of Cinnamomum cassia bark, Illicium verum fruit, Piper nigrum fruit, Zanthoxylum piperitum fruit, and Kaempferia galanga rhizome. P. nigrum fruit extract gave 100% mortality of larvae of Ae. aegypti and 0. togoi at 5 ppm and 96% mortality of larvae of C. pipiens pal/ens at 2.5 ppm. Z. piperitum fruit extract gave 85, 100, and 48% mortality in larvae of Ae. aegypti, 0. togoi, and Cx. pipiens pal/ens at 10 ppm, respectively. The plants described merit further study as potential mosquito larval control agents. Key words Aedes aegypti, biopesticides, Culex pipiens pal/ens, medicinal plants, mosquito larvicides, natural insecticides, Ochlerotatus togoi
Introduct ion The yellow fever mosquitoes, Aedes aegypti (L.) and Ochlerotatus togoi (Theobald), and the northern house mosquito, Culex pipiens pal/ens (Coq.), are widespread and serious disease vectoring insect pests. Mosquito abatement is primarily dependent on continued applications of organophosphates like temephos and fenthion, insect growth regulators like diflubenzuron and methoprene, and bacterial larvicides like Bacillus thuringiensis H14 and Bacillus sphaericus, which are still the most effective larvi cides (Rozendaal, 1997). Their repeated use has *Corresponding author. E-mail: [email protected] Tel: +82-2-880-4702; Fax: +82-2-873-2319 (Received January 26, 2004; Accepted April 13, 2004)
disrupted natural biological control systems and led to outbreaks of mosquitoes (DeBach and Rosen, 1991), often resulted in the widespread development of resistance (Brown, 1983), has undesirable effects on nontarget organisms, and fostered environmental and human health concerns (Hayes and Laws, 1991). These problems have highlighted the need for the development of new strategies for selective mosquito larval control. Plants may be an alternative source of materials for mosquito larval control because they constitute a rich source of bioactive chemicals. Because of this, much effort has been focused on plant extracts or phytochemicals as potential sources of commercial mosquito control agents (Amason et al., 1989a; Miyakado et al., 1989; Sukumar et al., 1991; Hostettmann and Potterat, 1997). Little work has been done in relation to the control of mosquito larvae, despite the excellent pharmacological actions of medicinal plants (Tang and Eisenbrand, 1992; Namba, 1993). This paper describes a laboratory study in which we examined the insecticidal activity of methanol extracts from 28 medicinal plant species against early 4th instar larvae of Ae. aegypti, 0. togoi, and Cx. pipiens pal/ens.
Materials and Methods Mosquito species Cultures of Ae. aegypti and Cx. pipiens pal/ens were maintained in the laboratory for seven years without exposure to any insecticide. 0. togoi larvae were collected from the abandoned salina at Inchon city in August 2001. Adult mosquitoes were maintained on a 10% sucrose solution and blood from a live mouse, while larvae were reared in a plastic tray (24 X 35 X 5 em) containing sterilized diet (40 mesh chick chow powder:yeast, 4:1 by weight). They were kept
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at 28 ± 2 °C, 70 ± 5% relative humidity (RR), and a photoperiod of 16:8 (L:D) h.
evaporation at 40°C. The yield of each methanolic extraction is given in Table 1.
Plants and sample preparation
Bioassays
A total of 28 medicinal plant species were purchased from the Boeun medicinal herb shop at Kyungdong market, Seoul (Table 1). They were dried in an oven at 40°C for 2 days and finely powdered. Each 50 g sample was extracted with 300 ml methanol twice at room temperature for 2 days and filtered. The combined filtrate was concentrated to dryness by rotary
Concentrations of test plant extracts were prepared by serial dilution of a stock solution of the plant extracts in ethanol. Each plant extract dissolved in 1 ml ethanol was suspended in distilled water with Triton X-100 added at the rate of 10 mg/liter. Batches of 25 early 4th instar larvae of Ae. aegypti, 0. togoi, and ex. pipiens pallens then were separately put into
Table 1. Medicinal plants tested for mosquito larvicidal activity Tissue used'
Yield (%t
Angerica dahurica Bentham et Hooker
Ro
17.7
Cnidium officinale Makino
Rh
10.0
Family Apiaceae
Species
Foeniculum vulgare Miller
Fr
4.9
Acorus calamus var. angustatus Besser
Rh
10.1
Acorus gramineus Solander
Rh
9.5
Compositae
Artemisia princeps var. orientalis Hara
Wp
6.6
Dioscoreaceae
Dioscorea batatas Decaisne
Rh
2.4
Fabaceae
Gleditsia horrida Makino
Fr
17.3
Glycyrrhiza glabra L.
Ro
21.9
Labiatae
Agastache rugosa O. Kuntze
Wp
9.5
Schizonepeta tenuifolia Briquet
Wp
8.1
Araceae
Thymus mandschuricus Ronniger
Wp
28.0
Lauraceae
Cinnamomum cassia Presl
Ba
5.1
Magnoliaceae
Illicium verum Hooker
Fr
26,1
Magnolia obovata Thunberg
Ba
5.8
Myrtaceae
Eugenia caryophyllata Thunberg
Fb
37.8
Paeoniaceae
Paeonia suffruticosa Andrews
Rb
18.6
Piperaceae
Piper nigrum L.
Fr
10.1
Polygonaceae
Rheum coreanum Nakai
Rh
41.6
Prmulaceae
Lysimachia davurica Ledebour
Wp
9.0
Rutaceae
Evodia rutaecarpa Thoms
Fr
9.5
Zanthoxylum coreanum Nakai
Fr
17.4
Zanthoxylum piperitum de Candolle
Fr
20.7
Zanthoxylum schinifolium Siebold et Zuccarini
Fr
16.2
Stemona japonica Miquel
Ro
15.2
Stemonaceae Thymelaeaceae
Aquillaria agallocha Roxburgh
Hw
6.6
Valerianaceae
Nardostachys chinensis Batalin
Rh
12.9
Zingiberaceae
Kaempferia galanga L.
Rh
7.1
• Ba, bark; Fb, flower bud; Fr, fruit; Hw, heart wood; Rb, root bark; Rh, rhizome; Ro, root; and Wp, whole plant b (Weight of crude methanol extract/weight of dried test material) x 100
Mosquito larvicidal activity of medicinal plants
the paper cups (270 ml) each containing 250 ml of the test solution. The toxicity of each plant extract was determined with 4 to 7 concentrations ranging from 0.63 to 200 ppm. If a plant extract exhibited less than 40% mortality at a given concentration, further bioassay was not conducted. Controls received ethanol-Triton X-lOO solution. Treated and control larvae were held under the same conditions used for colony maintenance. Evaluation of larvicidal activity was made 24 h after treatment. Larvae were considered dead if they did not move when prodded with a wooden dowel. All treatments were replicated three times. Statistical analyses
The percentage mortality was determined and transformed to arcsine square-root values for analysis of variance (ANOVA). Treatment means were compared and separated by Scheffe test at P = 0.05 (SAS Institute, 1996). Means ± SE of untransformed data are reported.
Results When the methanol extracts from 28 medicinal plant species were bioassayed, significant differences were observed in toxicity to early 4th instar larvae of Ae.
229
aegypti (Table 2). At a concentration of 5 ppm, 100% mortality was observed with Piper nigrum fruit extract. Zanthoxylum piperitum fruit extract gave 100, 85, and
19% mortality at 25, 10, and 5 ppm, respectively. The extracts from Illicium verum fruit and Kaemfera galanga rhizome caused 100% mortality at 100 ppm, whereas the larvicidal activity decreased significantly at 50 ppm. No mortality was observed in the controls. The toxicity of test plant extracts to early 4th instar larvae of 0. togoi larvae was examined (Table 3). P. nigrum fruit extract as above gave 100% mortality at 5 ppm. Z. piperitum fruit extract produced 100 and 48% mortality at 10 and 5 ppm, respectively. K. galanga rhizome extract showed 100, 78, and 36% mortality at 100, 50, and 25 ppm, respectively. Cinnamomum cassia bark extract was highly effective at 100 ppm but the larvicidal activity decreased significantly at 50 ppm. No mortality was observed in the controls. Table 4 shows the toxic effects of the test plant extracts on early 4th instar larvae of Cx. pipiens pallens larvae. At 5 ppm, 100% mortality was obtained with P. nigrum fruit extract. Z. piperitum fruit extract gave 100 and 48% mortality at 25 and 10 ppm, respectively. 1. verum fruit extract gave 100 and 35% mortality at 50 and 25 ppm, respectively. K. galanga rhizome extract showed 100 and 43% mortality at 100 and 50 ppm, respectively. There was no mortality in the controls. Toxic effects of P. nigrum fruit extract on larvae of the three mosquito species were investigated at 2.5,
Table 2. Mortality of early 4th instar larvae of Ae. aegypti by methanol extracts of medicinal plants Mortality (mean ± SE), % Plant species'
Concentration (ppm) 200
A. dahurica C. officinale
100
50
25
95±2.7abc
57±3.5b
96±2.3ab
61±2.7b
16±2.3c
67±1.3b
45±3.5b
F. vulgare
100±O.Oa
A. calamus
39±2.7d
A. gramineus
51±1.3d
A. rugosa
43± 1.3d
10
5
21 ±3.5c
9±1.3b
9± 1.3d
C. cassia
81 ± 1.3c
I. verum
IOO±O.Oa
IOO±O.Oa
51 ±3.5b
15±3.5b
P. nigrum
100±O.Oa
100±O.Oa
IOO±O.Oa
lOO±O.Oa
100±O.Oa
100±O.Oa
z. z. z.
100±O.Oa
IOO±O.Oa
100±O.Oa
100±O.Oa
85±1.3b
19±3.5b
100±O.Oa
37± 1.3b
coreanum piperitum schinifolium
K. galanga
84±2.3bc
60±2.3b
21±1.3c
40±2.3c
37±3.5d 100±O.Oa
Means within a column followed by the same letter are not significantly different (P before ANOV A. Means ± SE of untransformed data are reported. a Plants showing less than 20% mortality at 200 ppm are not presented.
=
0.05, Scheffe test). Mortalities were transformed to arcsine square root
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Table 3. Mortality of early 4th instar larvae of 0. togoi by methanol extracts of medicinal plants
Mortality (mean ± SE), % Plant species'
Concentration (ppm) 200
100
A. dahurica
95±l.3b
56±2.3b
C. officinale
100±0.Oa
17±l.3d
F. vulgare
49± l.3e
9± l.3e
A. gramineus
87± l.3c
20±2.3d
C. cassia
100±0.Oa
100±0.Oa
l. verum
60±2.3e
21±l.3d
100±0.Oa
100±0.Oa
coreanum
77± 1.3d
35±2.7c
Z. piperitum
100±0.Oa
P. nigrum
z.
Z. schinifolium K. galanga
50
25
10
5
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
48±2.3b
100±0.Oa
78±2.0b
36±2.3b
9±l.3d
29±I.3c
32±2.3f 100±0.Oa
Means within a column followed by the same letter are not significantly different (P = 0.05, Scheffe test). Mortalities were transformed to arcsine square root before ANOVA. Means ± SE of untransfonned data are reported . • Plants showing less than 20% mortality at 200 ppm are not presented.
Table 4. Mortality of early 4th instar larvae of Cx. pipiens pal/ens by methanol extracts of medicinal plants
Mortality (mean ± SE), % Plant species' A. dahurica
c.
officinale
F. vulgare
Concentration (ppm) 200
100
100±0.Oa
65±2.7c
20±2.3cd
87±1.3b
31 ± l.3bc
95± 1.3abc 100±0.Oa
50
25
10
5
29± l.3e
A. calamus
81±3.5cd
49±3.5cd
II ±2.7e
A. gramineus
95±2.7ab
65±3.5c
13±1.3de
C. cassia
93±1.3bc
44±2.3de
ll±1.3e
l. verum
100±0.Oa
100±0.Oa
100±0.Oa
35±2.7b
P. nigrum
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±O.Oa
100±0.Oa
coreanum
63±1.3d
8±2.3f
piperitum
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
48±2.3b
1l±2.7b
lOO±O.Oa
43±1.3b
19± l.3c
z. z. z.
schinifolium
K. galanga
31±2.7e lOO±O.Oa
Means within a column followed by the same letter are not significantly different (P = 0.05, Scheffe test). Mortalities were transformed to arcsine square root before ANOV A. Means ± SE of untransfonned data are reported. • Plants showing less than 20% mortality at 200 ppm are not presented.
1.25, and 0.63 ppm (Table 5). At 2.5 ppm, the fruit extract gave 64, 56, and 100% mortality in larvae of Ae. aegypti, 0. togoi, and Cx. pipiens pal/ens, respectively. The larvicidal activity of the fruit extract
against Cx. pipiens pal/ens decreased significantly at 0.63 ppm.
Mosquito larvicidal activity of medicinal plants
231
Table 5. Mortality of early 4th instar larvae of three mosquito species by methanol extract of P. nigrum fruit Mortality (mean ± SE), % Mosquito species
Concentration (ppm) 2.5
1.25
0.63
Ae. aegypti
64±2.3a
13±3.5b
O±O.Oc
0. togoi
56±2.3a
11± 1.3b
O±O.Oc
Cx. pipiens pallens
96±2.3a
79± I.3b
27± 1.3c
Means within a row followed by the same letter are not significantly different (P before ANOV A. Means ± SE of untransformed data are reported.
Discussion Plant extracts and phytochemicals have potential as products for mosquito control because many of them are selective, may biodegrade to nontoxic products, and may be applied to mosquito breeding places in the same way as conventional insecticides (Sukumar et al., 1991; Hostettmann and Potterat, 1997). Many plant extracts and essential oils possess larvicidal activity against various mosquito species (Jacobson, 1989; Miyakado et al., 1989; Berenbaum, 1989; Sukumar et al., 1991; Hostettmann and Potterat, 1997). Additionally, some plant-derived materials are found to be highly effective against insecticideresistant insect pests (Amason et al., 1989b; Ahn et al., 1997). Sukumar et al. (1991) has pointed out that the most promising botanical mosquito control agents are in the families Asteraceae, Cladophoraceae, Lamiaceae (formerly Labiatae),Meliaceae, Oocystaceae, and Rutaceae. In the present study, potent larvicidal activity against Ae. aegypti, 0. togoi, and Cx. pipiens pallens was observed with plants in the families Magnoliaceae, Piperaceae, Rutaceae, and Zingiberaceae. The differential responses of various mosquito species are influenced by extrinsic and intrinsic factors such as the plant species, the parts of the plant, the solvents used for extraction, the geographical location where the plants were grown, and the application methods (Sukumar et al., 1991). Minijas and Sarda (1986) reported that crude extract of the fruit pods from Swartzia madagascariensis Desvaux produced higher mortality in larvae of Anopheles gambie (Giles) than larvae of Ae. aegypti but was ineffective against larvae of Culex quinquefasciatus (Say). Sujatha et al. (1988) observed differential susceptibilities of larvae of three mosquito species to petroleum ether extracts of Acarus calamus L., Ageratum conyzoides L., Annona squamosa L., Bambusa arundanasia, Madhuca Iongifolia L., and Citrus medica L.: extracts of A. calamus and B. arundanasia were most effective against Cx. quinquefasciatus and Anopheles stephensi Liston, respectively, while C. medica extract affected
=
0.05, Scheffe test). Mortalities were transformed to arcsine square root
only An. stephensi larvae and M longifolia extract was ineffective against this species. In this study, larvicidal responses varied according to mosquito and plant species. Methanol extracts of C. cassia bark, /. verum fruit, P. nigrum fruit, Z. piperitum fruit, and K. galanga rhizome exhibited potent larvicidal activity against Ae. aegypti, 0. togoi, and Cx. pipiens pallens. It has been reported that P. nigrum fruit extract has potent larvicidal activity against ex. pipiens pallens (Miyakado et al. 1989). Variation in mosquito response to the extracts related to plant species has been studied. Differences in the larvicidal effects on Ae. aegypti among the steam distilled oils from the whole plant of Tagetes erecta L., Tagetes minuta L., and Tagetes patula L. have been reported (Green et al., 1991; Perich et al., 1994): T. minuta had the most potent larvicidal activity. In the present study, the larvicidal activity against Ae. aegypti, 0. togoi, and Cx. pipiens pallens was more pronounced with the fruit extract of Z. piperitum than with those of Zanthoxylum coreanum and Zanthoxylum schinifolium. These results suggest that chemical composition between the two plant species may be different. In conclusion, some plant species described appear to have potentials as mosquito larval control agents. Further research is required on these plant-derived constituents active against mosquito larvae for developing into effective formulations for control of mosquito larvae in field ecosystem. Acknowledgment This work was supported by the Korea Science and Engineering Foundation (ROI20010000880) and the Ministry of Education & Human Resources Development for Brain Korea 21 Project of the Korean Government to YJA.
Literature Cited Ahn, Y.J., M. Kwon, H.M. Park and C.G. Han. 1997. Potent insecticidal activity of Ginkgo bilaba-derived trilactone terpenes against Nilaparvata lugens. pp. 90105, in Phytochemicals for pest control, Eds. P.A. Hedin, R.M.
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Hollingworth, E.P. Masler, J. Miyamoto and D.G. Thompson. Am. Chem. Symp. Ser. 658, American Chemical Society, Washington DC. Amason, J.T., B.J.R. Philogene and P. Morand. 1989a. Insecticides of plant origin. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Amason, J.T., B.J.R. Philogene, P. Morand, K. Imire, S. Iyengar, F. Duval, C. Soucy-breau, J.C. Scaiano, N.H. Werstiuk, B. Hasspieler and A.E.R. Downe. 1989b. Naturally occurring and synthetic thiophenes as photoactivated insecticides. pp. 164172, in Insecticides of plant origin, Eds. J.T. Amason, BJ.R. Philogene and P. Morand. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Berenbaum, M.R. 1989. North american ethnobotanicals as sources of novel plant-based insecticides, pp. 1124, in Insecticides of plant origin, Eds. J.T. Amason, B.J.R. Philogne and P. Morand. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Brown, AW.A 1983. Insecticide resistance as a factor in the integrated control of Culicidae. pp. 161235, in Integrated mosquito control methodologies, Eds. M. Laird and J.W. Miles. Academic Press, New York. DeBach, P. and D. Rosen. 1991. Biological control by natural enemies. 2nd ed. Cambridge University Press, Cambridge, United Kingdom. Green, M.M., J.M. Singer, DJ. Sutherland and C.R. Hibben. 1991. Larvicidal activity of Tagetes minuta (marigold) toward Aedes aegypti. J. Am. Mosq. Control Assoc. 7: 282286. Hayes, J.B., Jr and E.R. Laws, Jr. 1991. Handbook of pesticide toxicology, Vol. I. Academic Press, San Diego. Hostettmann, K. and O. Potterat. 1997. Strategy for the isolation and analysis of antifungal, molluscicidal, and larvicidal agents from tropical plants. pp. 1426, in Phytochemicals for pest control, Eds. P.A Hedin, R.M. Hollingworth, E.P. Masler, J. Miyamoto and D.G.
Thompson. Am. Chern. Symp. Ser. 658, American Chemical Society, Washington DC. Jacobson, M. 1989. Botanical pesticides: past, present, and future. pp. 110, in Insecticides of plant origin, Eds. J.T. Amason, BJ.R. Philogne and P. Morand. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Minijas, J.N. and R.K. Sarda. 1986. Laboratory observations on the toxicity of Swartzia madagascariensis (Leguminosae) extract to mosquito larvae. Trans. R. Soc. Trop. Med. Hyg. 80: 460-461. Miyakado, M., 1. Nakayama and N. Ohno. 1989. Insecticidal unsaturated isobutylamides from natural products to agrochemical leads. pp. 173187, in Insecticides of plant origin, Eds. J.T. Amason, B.J.R. Philogsne and P. Morand. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Namba, T. 1993. The encyclopedia ofwakan-yaku (traditional Sino-Japanese medicines) with color pictures. Hoikusha, Osaka, Japan. Perich, M.J., C. Wells, W. Bertsch and K.E. Tredway. 1994. Toxicity of extracts from three Tagetes against adults and larvae of yellow fever mosquito and Anopheles stephensi (Diptera: Culicidae). J. Med. Entomol. 31: 833837. Rozendaal, J.A. 1997. Vector control Geneva, Switzerland: World Health Organization. SAS Institute. 1996. SAS/STAT user's guide, Version 6. SAS Institute, Cary, North Carolina. Sujatha, C.H., V. Vasuki, T. Mariappan, M. Kalyanasundaran and P.K. Das. 1988. Evalulation of plant extracts for biological activity against mosquitoes. IntI. Pest Control 30: 122-124. Sukumar, K., Perich, MJ. and L.R. Boobar. 1991. Botanical derivatives in mosquito control: a review. J. Am. Mosq. Contr. Assoc. 7: 210-237. Tang, W. and G. Eisenbrand. 1992. Chinese drugs of plant origin. Springer, New York.
Larvicidal Activity of Medicinal Plant Extracts Against Aedes aegypti, Ochlerotatus togoi, and Culex pipiens pal/ens (Diptera: Culicidae) 2
2
Young-Cheol Yang, I1-Kwon Park', Eun-Hee Kim , Hoi-Seon Lee3 and Young-Joon Ahn * Department of Advanced Organic Materials Engineering, Chonbuk National University, Chonju 561-756, Korea IPorestry Research Institute, Seoul 151-742, Korea 2School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea 3Paculty of Biotechnology, College of Agriculture, Chonbuk National University, Chonju 561-756, Korea
Abstract The toxicities of methanol extracts from 28 medicinal plant species to early 4th instar larvae of Aedes aegypti, Ochlerotatus togoi (Aedes togoi), and Culex pipiens pal/ens were determined in the laboratory. Responses varied according to plant and mosquito species. At a concentration of 100 ppm, >90% mortality of the three species was obtained with the extracts of Cinnamomum cassia bark, Illicium verum fruit, Piper nigrum fruit, Zanthoxylum piperitum fruit, and Kaempferia galanga rhizome. P. nigrum fruit extract gave 100% mortality of larvae of Ae. aegypti and 0. togoi at 5 ppm and 96% mortality of larvae of C. pipiens pal/ens at 2.5 ppm. Z. piperitum fruit extract gave 85, 100, and 48% mortality in larvae of Ae. aegypti, 0. togoi, and Cx. pipiens pal/ens at 10 ppm, respectively. The plants described merit further study as potential mosquito larval control agents. Key words Aedes aegypti, biopesticides, Culex pipiens pal/ens, medicinal plants, mosquito larvicides, natural insecticides, Ochlerotatus togoi
Introduct ion The yellow fever mosquitoes, Aedes aegypti (L.) and Ochlerotatus togoi (Theobald), and the northern house mosquito, Culex pipiens pal/ens (Coq.), are widespread and serious disease vectoring insect pests. Mosquito abatement is primarily dependent on continued applications of organophosphates like temephos and fenthion, insect growth regulators like diflubenzuron and methoprene, and bacterial larvicides like Bacillus thuringiensis H14 and Bacillus sphaericus, which are still the most effective larvi cides (Rozendaal, 1997). Their repeated use has *Corresponding author. E-mail: [email protected] Tel: +82-2-880-4702; Fax: +82-2-873-2319 (Received January 26, 2004; Accepted April 13, 2004)
disrupted natural biological control systems and led to outbreaks of mosquitoes (DeBach and Rosen, 1991), often resulted in the widespread development of resistance (Brown, 1983), has undesirable effects on nontarget organisms, and fostered environmental and human health concerns (Hayes and Laws, 1991). These problems have highlighted the need for the development of new strategies for selective mosquito larval control. Plants may be an alternative source of materials for mosquito larval control because they constitute a rich source of bioactive chemicals. Because of this, much effort has been focused on plant extracts or phytochemicals as potential sources of commercial mosquito control agents (Amason et al., 1989a; Miyakado et al., 1989; Sukumar et al., 1991; Hostettmann and Potterat, 1997). Little work has been done in relation to the control of mosquito larvae, despite the excellent pharmacological actions of medicinal plants (Tang and Eisenbrand, 1992; Namba, 1993). This paper describes a laboratory study in which we examined the insecticidal activity of methanol extracts from 28 medicinal plant species against early 4th instar larvae of Ae. aegypti, 0. togoi, and Cx. pipiens pal/ens.
Materials and Methods Mosquito species Cultures of Ae. aegypti and Cx. pipiens pal/ens were maintained in the laboratory for seven years without exposure to any insecticide. 0. togoi larvae were collected from the abandoned salina at Inchon city in August 2001. Adult mosquitoes were maintained on a 10% sucrose solution and blood from a live mouse, while larvae were reared in a plastic tray (24 X 35 X 5 em) containing sterilized diet (40 mesh chick chow powder:yeast, 4:1 by weight). They were kept
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J. Asia-Pacific Entomol. Vol. 7 (2004)
at 28 ± 2 °C, 70 ± 5% relative humidity (RR), and a photoperiod of 16:8 (L:D) h.
evaporation at 40°C. The yield of each methanolic extraction is given in Table 1.
Plants and sample preparation
Bioassays
A total of 28 medicinal plant species were purchased from the Boeun medicinal herb shop at Kyungdong market, Seoul (Table 1). They were dried in an oven at 40°C for 2 days and finely powdered. Each 50 g sample was extracted with 300 ml methanol twice at room temperature for 2 days and filtered. The combined filtrate was concentrated to dryness by rotary
Concentrations of test plant extracts were prepared by serial dilution of a stock solution of the plant extracts in ethanol. Each plant extract dissolved in 1 ml ethanol was suspended in distilled water with Triton X-100 added at the rate of 10 mg/liter. Batches of 25 early 4th instar larvae of Ae. aegypti, 0. togoi, and ex. pipiens pallens then were separately put into
Table 1. Medicinal plants tested for mosquito larvicidal activity Tissue used'
Yield (%t
Angerica dahurica Bentham et Hooker
Ro
17.7
Cnidium officinale Makino
Rh
10.0
Family Apiaceae
Species
Foeniculum vulgare Miller
Fr
4.9
Acorus calamus var. angustatus Besser
Rh
10.1
Acorus gramineus Solander
Rh
9.5
Compositae
Artemisia princeps var. orientalis Hara
Wp
6.6
Dioscoreaceae
Dioscorea batatas Decaisne
Rh
2.4
Fabaceae
Gleditsia horrida Makino
Fr
17.3
Glycyrrhiza glabra L.
Ro
21.9
Labiatae
Agastache rugosa O. Kuntze
Wp
9.5
Schizonepeta tenuifolia Briquet
Wp
8.1
Araceae
Thymus mandschuricus Ronniger
Wp
28.0
Lauraceae
Cinnamomum cassia Presl
Ba
5.1
Magnoliaceae
Illicium verum Hooker
Fr
26,1
Magnolia obovata Thunberg
Ba
5.8
Myrtaceae
Eugenia caryophyllata Thunberg
Fb
37.8
Paeoniaceae
Paeonia suffruticosa Andrews
Rb
18.6
Piperaceae
Piper nigrum L.
Fr
10.1
Polygonaceae
Rheum coreanum Nakai
Rh
41.6
Prmulaceae
Lysimachia davurica Ledebour
Wp
9.0
Rutaceae
Evodia rutaecarpa Thoms
Fr
9.5
Zanthoxylum coreanum Nakai
Fr
17.4
Zanthoxylum piperitum de Candolle
Fr
20.7
Zanthoxylum schinifolium Siebold et Zuccarini
Fr
16.2
Stemona japonica Miquel
Ro
15.2
Stemonaceae Thymelaeaceae
Aquillaria agallocha Roxburgh
Hw
6.6
Valerianaceae
Nardostachys chinensis Batalin
Rh
12.9
Zingiberaceae
Kaempferia galanga L.
Rh
7.1
• Ba, bark; Fb, flower bud; Fr, fruit; Hw, heart wood; Rb, root bark; Rh, rhizome; Ro, root; and Wp, whole plant b (Weight of crude methanol extract/weight of dried test material) x 100
Mosquito larvicidal activity of medicinal plants
the paper cups (270 ml) each containing 250 ml of the test solution. The toxicity of each plant extract was determined with 4 to 7 concentrations ranging from 0.63 to 200 ppm. If a plant extract exhibited less than 40% mortality at a given concentration, further bioassay was not conducted. Controls received ethanol-Triton X-lOO solution. Treated and control larvae were held under the same conditions used for colony maintenance. Evaluation of larvicidal activity was made 24 h after treatment. Larvae were considered dead if they did not move when prodded with a wooden dowel. All treatments were replicated three times. Statistical analyses
The percentage mortality was determined and transformed to arcsine square-root values for analysis of variance (ANOVA). Treatment means were compared and separated by Scheffe test at P = 0.05 (SAS Institute, 1996). Means ± SE of untransformed data are reported.
Results When the methanol extracts from 28 medicinal plant species were bioassayed, significant differences were observed in toxicity to early 4th instar larvae of Ae.
229
aegypti (Table 2). At a concentration of 5 ppm, 100% mortality was observed with Piper nigrum fruit extract. Zanthoxylum piperitum fruit extract gave 100, 85, and
19% mortality at 25, 10, and 5 ppm, respectively. The extracts from Illicium verum fruit and Kaemfera galanga rhizome caused 100% mortality at 100 ppm, whereas the larvicidal activity decreased significantly at 50 ppm. No mortality was observed in the controls. The toxicity of test plant extracts to early 4th instar larvae of 0. togoi larvae was examined (Table 3). P. nigrum fruit extract as above gave 100% mortality at 5 ppm. Z. piperitum fruit extract produced 100 and 48% mortality at 10 and 5 ppm, respectively. K. galanga rhizome extract showed 100, 78, and 36% mortality at 100, 50, and 25 ppm, respectively. Cinnamomum cassia bark extract was highly effective at 100 ppm but the larvicidal activity decreased significantly at 50 ppm. No mortality was observed in the controls. Table 4 shows the toxic effects of the test plant extracts on early 4th instar larvae of Cx. pipiens pallens larvae. At 5 ppm, 100% mortality was obtained with P. nigrum fruit extract. Z. piperitum fruit extract gave 100 and 48% mortality at 25 and 10 ppm, respectively. 1. verum fruit extract gave 100 and 35% mortality at 50 and 25 ppm, respectively. K. galanga rhizome extract showed 100 and 43% mortality at 100 and 50 ppm, respectively. There was no mortality in the controls. Toxic effects of P. nigrum fruit extract on larvae of the three mosquito species were investigated at 2.5,
Table 2. Mortality of early 4th instar larvae of Ae. aegypti by methanol extracts of medicinal plants Mortality (mean ± SE), % Plant species'
Concentration (ppm) 200
A. dahurica C. officinale
100
50
25
95±2.7abc
57±3.5b
96±2.3ab
61±2.7b
16±2.3c
67±1.3b
45±3.5b
F. vulgare
100±O.Oa
A. calamus
39±2.7d
A. gramineus
51±1.3d
A. rugosa
43± 1.3d
10
5
21 ±3.5c
9±1.3b
9± 1.3d
C. cassia
81 ± 1.3c
I. verum
IOO±O.Oa
IOO±O.Oa
51 ±3.5b
15±3.5b
P. nigrum
100±O.Oa
100±O.Oa
IOO±O.Oa
lOO±O.Oa
100±O.Oa
100±O.Oa
z. z. z.
100±O.Oa
IOO±O.Oa
100±O.Oa
100±O.Oa
85±1.3b
19±3.5b
100±O.Oa
37± 1.3b
coreanum piperitum schinifolium
K. galanga
84±2.3bc
60±2.3b
21±1.3c
40±2.3c
37±3.5d 100±O.Oa
Means within a column followed by the same letter are not significantly different (P before ANOV A. Means ± SE of untransformed data are reported. a Plants showing less than 20% mortality at 200 ppm are not presented.
=
0.05, Scheffe test). Mortalities were transformed to arcsine square root
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J. Asia-Pacific Entomol. Vol. 7 (2004)
Table 3. Mortality of early 4th instar larvae of 0. togoi by methanol extracts of medicinal plants
Mortality (mean ± SE), % Plant species'
Concentration (ppm) 200
100
A. dahurica
95±l.3b
56±2.3b
C. officinale
100±0.Oa
17±l.3d
F. vulgare
49± l.3e
9± l.3e
A. gramineus
87± l.3c
20±2.3d
C. cassia
100±0.Oa
100±0.Oa
l. verum
60±2.3e
21±l.3d
100±0.Oa
100±0.Oa
coreanum
77± 1.3d
35±2.7c
Z. piperitum
100±0.Oa
P. nigrum
z.
Z. schinifolium K. galanga
50
25
10
5
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
48±2.3b
100±0.Oa
78±2.0b
36±2.3b
9±l.3d
29±I.3c
32±2.3f 100±0.Oa
Means within a column followed by the same letter are not significantly different (P = 0.05, Scheffe test). Mortalities were transformed to arcsine square root before ANOVA. Means ± SE of untransfonned data are reported . • Plants showing less than 20% mortality at 200 ppm are not presented.
Table 4. Mortality of early 4th instar larvae of Cx. pipiens pal/ens by methanol extracts of medicinal plants
Mortality (mean ± SE), % Plant species' A. dahurica
c.
officinale
F. vulgare
Concentration (ppm) 200
100
100±0.Oa
65±2.7c
20±2.3cd
87±1.3b
31 ± l.3bc
95± 1.3abc 100±0.Oa
50
25
10
5
29± l.3e
A. calamus
81±3.5cd
49±3.5cd
II ±2.7e
A. gramineus
95±2.7ab
65±3.5c
13±1.3de
C. cassia
93±1.3bc
44±2.3de
ll±1.3e
l. verum
100±0.Oa
100±0.Oa
100±0.Oa
35±2.7b
P. nigrum
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
100±O.Oa
100±0.Oa
coreanum
63±1.3d
8±2.3f
piperitum
100±0.Oa
100±0.Oa
100±0.Oa
100±0.Oa
48±2.3b
1l±2.7b
lOO±O.Oa
43±1.3b
19± l.3c
z. z. z.
schinifolium
K. galanga
31±2.7e lOO±O.Oa
Means within a column followed by the same letter are not significantly different (P = 0.05, Scheffe test). Mortalities were transformed to arcsine square root before ANOV A. Means ± SE of untransfonned data are reported. • Plants showing less than 20% mortality at 200 ppm are not presented.
1.25, and 0.63 ppm (Table 5). At 2.5 ppm, the fruit extract gave 64, 56, and 100% mortality in larvae of Ae. aegypti, 0. togoi, and Cx. pipiens pal/ens, respectively. The larvicidal activity of the fruit extract
against Cx. pipiens pal/ens decreased significantly at 0.63 ppm.
Mosquito larvicidal activity of medicinal plants
231
Table 5. Mortality of early 4th instar larvae of three mosquito species by methanol extract of P. nigrum fruit Mortality (mean ± SE), % Mosquito species
Concentration (ppm) 2.5
1.25
0.63
Ae. aegypti
64±2.3a
13±3.5b
O±O.Oc
0. togoi
56±2.3a
11± 1.3b
O±O.Oc
Cx. pipiens pallens
96±2.3a
79± I.3b
27± 1.3c
Means within a row followed by the same letter are not significantly different (P before ANOV A. Means ± SE of untransformed data are reported.
Discussion Plant extracts and phytochemicals have potential as products for mosquito control because many of them are selective, may biodegrade to nontoxic products, and may be applied to mosquito breeding places in the same way as conventional insecticides (Sukumar et al., 1991; Hostettmann and Potterat, 1997). Many plant extracts and essential oils possess larvicidal activity against various mosquito species (Jacobson, 1989; Miyakado et al., 1989; Berenbaum, 1989; Sukumar et al., 1991; Hostettmann and Potterat, 1997). Additionally, some plant-derived materials are found to be highly effective against insecticideresistant insect pests (Amason et al., 1989b; Ahn et al., 1997). Sukumar et al. (1991) has pointed out that the most promising botanical mosquito control agents are in the families Asteraceae, Cladophoraceae, Lamiaceae (formerly Labiatae),Meliaceae, Oocystaceae, and Rutaceae. In the present study, potent larvicidal activity against Ae. aegypti, 0. togoi, and Cx. pipiens pallens was observed with plants in the families Magnoliaceae, Piperaceae, Rutaceae, and Zingiberaceae. The differential responses of various mosquito species are influenced by extrinsic and intrinsic factors such as the plant species, the parts of the plant, the solvents used for extraction, the geographical location where the plants were grown, and the application methods (Sukumar et al., 1991). Minijas and Sarda (1986) reported that crude extract of the fruit pods from Swartzia madagascariensis Desvaux produced higher mortality in larvae of Anopheles gambie (Giles) than larvae of Ae. aegypti but was ineffective against larvae of Culex quinquefasciatus (Say). Sujatha et al. (1988) observed differential susceptibilities of larvae of three mosquito species to petroleum ether extracts of Acarus calamus L., Ageratum conyzoides L., Annona squamosa L., Bambusa arundanasia, Madhuca Iongifolia L., and Citrus medica L.: extracts of A. calamus and B. arundanasia were most effective against Cx. quinquefasciatus and Anopheles stephensi Liston, respectively, while C. medica extract affected
=
0.05, Scheffe test). Mortalities were transformed to arcsine square root
only An. stephensi larvae and M longifolia extract was ineffective against this species. In this study, larvicidal responses varied according to mosquito and plant species. Methanol extracts of C. cassia bark, /. verum fruit, P. nigrum fruit, Z. piperitum fruit, and K. galanga rhizome exhibited potent larvicidal activity against Ae. aegypti, 0. togoi, and Cx. pipiens pallens. It has been reported that P. nigrum fruit extract has potent larvicidal activity against ex. pipiens pallens (Miyakado et al. 1989). Variation in mosquito response to the extracts related to plant species has been studied. Differences in the larvicidal effects on Ae. aegypti among the steam distilled oils from the whole plant of Tagetes erecta L., Tagetes minuta L., and Tagetes patula L. have been reported (Green et al., 1991; Perich et al., 1994): T. minuta had the most potent larvicidal activity. In the present study, the larvicidal activity against Ae. aegypti, 0. togoi, and Cx. pipiens pallens was more pronounced with the fruit extract of Z. piperitum than with those of Zanthoxylum coreanum and Zanthoxylum schinifolium. These results suggest that chemical composition between the two plant species may be different. In conclusion, some plant species described appear to have potentials as mosquito larval control agents. Further research is required on these plant-derived constituents active against mosquito larvae for developing into effective formulations for control of mosquito larvae in field ecosystem. Acknowledgment This work was supported by the Korea Science and Engineering Foundation (ROI20010000880) and the Ministry of Education & Human Resources Development for Brain Korea 21 Project of the Korean Government to YJA.
Literature Cited Ahn, Y.J., M. Kwon, H.M. Park and C.G. Han. 1997. Potent insecticidal activity of Ginkgo bilaba-derived trilactone terpenes against Nilaparvata lugens. pp. 90105, in Phytochemicals for pest control, Eds. P.A. Hedin, R.M.
232
J. Asia-Pacific Entomol. Vol. 7 (2004)
Hollingworth, E.P. Masler, J. Miyamoto and D.G. Thompson. Am. Chem. Symp. Ser. 658, American Chemical Society, Washington DC. Amason, J.T., B.J.R. Philogene and P. Morand. 1989a. Insecticides of plant origin. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Amason, J.T., B.J.R. Philogene, P. Morand, K. Imire, S. Iyengar, F. Duval, C. Soucy-breau, J.C. Scaiano, N.H. Werstiuk, B. Hasspieler and A.E.R. Downe. 1989b. Naturally occurring and synthetic thiophenes as photoactivated insecticides. pp. 164172, in Insecticides of plant origin, Eds. J.T. Amason, BJ.R. Philogene and P. Morand. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Berenbaum, M.R. 1989. North american ethnobotanicals as sources of novel plant-based insecticides, pp. 1124, in Insecticides of plant origin, Eds. J.T. Amason, B.J.R. Philogne and P. Morand. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Brown, AW.A 1983. Insecticide resistance as a factor in the integrated control of Culicidae. pp. 161235, in Integrated mosquito control methodologies, Eds. M. Laird and J.W. Miles. Academic Press, New York. DeBach, P. and D. Rosen. 1991. Biological control by natural enemies. 2nd ed. Cambridge University Press, Cambridge, United Kingdom. Green, M.M., J.M. Singer, DJ. Sutherland and C.R. Hibben. 1991. Larvicidal activity of Tagetes minuta (marigold) toward Aedes aegypti. J. Am. Mosq. Control Assoc. 7: 282286. Hayes, J.B., Jr and E.R. Laws, Jr. 1991. Handbook of pesticide toxicology, Vol. I. Academic Press, San Diego. Hostettmann, K. and O. Potterat. 1997. Strategy for the isolation and analysis of antifungal, molluscicidal, and larvicidal agents from tropical plants. pp. 1426, in Phytochemicals for pest control, Eds. P.A Hedin, R.M. Hollingworth, E.P. Masler, J. Miyamoto and D.G.
Thompson. Am. Chern. Symp. Ser. 658, American Chemical Society, Washington DC. Jacobson, M. 1989. Botanical pesticides: past, present, and future. pp. 110, in Insecticides of plant origin, Eds. J.T. Amason, BJ.R. Philogne and P. Morand. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Minijas, J.N. and R.K. Sarda. 1986. Laboratory observations on the toxicity of Swartzia madagascariensis (Leguminosae) extract to mosquito larvae. Trans. R. Soc. Trop. Med. Hyg. 80: 460-461. Miyakado, M., 1. Nakayama and N. Ohno. 1989. Insecticidal unsaturated isobutylamides from natural products to agrochemical leads. pp. 173187, in Insecticides of plant origin, Eds. J.T. Amason, B.J.R. Philogsne and P. Morand. Am. Chem. Symp. Ser. 387, American Chemical Society, Washington DC. Namba, T. 1993. The encyclopedia ofwakan-yaku (traditional Sino-Japanese medicines) with color pictures. Hoikusha, Osaka, Japan. Perich, M.J., C. Wells, W. Bertsch and K.E. Tredway. 1994. Toxicity of extracts from three Tagetes against adults and larvae of yellow fever mosquito and Anopheles stephensi (Diptera: Culicidae). J. Med. Entomol. 31: 833837. Rozendaal, J.A. 1997. Vector control Geneva, Switzerland: World Health Organization. SAS Institute. 1996. SAS/STAT user's guide, Version 6. SAS Institute, Cary, North Carolina. Sujatha, C.H., V. Vasuki, T. Mariappan, M. Kalyanasundaran and P.K. Das. 1988. Evalulation of plant extracts for biological activity against mosquitoes. IntI. Pest Control 30: 122-124. Sukumar, K., Perich, MJ. and L.R. Boobar. 1991. Botanical derivatives in mosquito control: a review. J. Am. Mosq. Contr. Assoc. 7: 210-237. Tang, W. and G. Eisenbrand. 1992. Chinese drugs of plant origin. Springer, New York.