Plant extracts as larvicide

Larvicidal activities of aquous extracts of nineteen Indian plants against two mosquito species, Aedes(s) albopictus and Culex quinquefasciatus.
D. M. Badruddoza and S. M. K. Rahman
Crop Protection and Toxicology Laboratory, Department of Zoology, Rajshahi University, Bangladesh. e-mail: [e-mail].

ABSTRACT
Methanol extracts of 19 indigenous plants were evaluated as mosquito larvicide. Among these, pericarp of Zanthoxylum limonella was found to have the most promising larvicidal properties against Aedes(s) albopictus and Culex quinquefasciatus with LD90 values at 0.47 ppm and 0.73 ppm, respectively. The extract of Piper nigrum was also found very effective (LD90) on the larvae of both the species at 6.8 ppm and 8.4 ppm, respectively. The extracts of the remaining plant parts showed LD90 values at above 100 ppm concentration. Extract of Calotropis gigantea was found to be the least effective ( LD90 values at 962.8 ppm and 1091.8 ppm) against the larvae of both the species. However, plant extracts were found more effective against Aedes(s) albopictus larvae than against Culex quinquefasciatus larvae.
Keywords: Mosquito larvicide, plant extract, larvicidal activity, Zanthoxylum limonella, Piper nigrum, Calotropis gigantea, Aedes(s) albopictus, Culex quinquefasciatus.

INTRODUCTION
The geo-climatic condition of Bangladesh supports fast proliferation and growth of natural fauna and flora. Many of the herbs and shrubs are found to have promising medicinal properties, mosquito larvicidal and mosquito repellent properties. Owing to the fact that application of synthetic larvicide has envenomed the surroundings as well as nontarget organisms, natural products of plant origin with insecticidal properties have been tried as an indigenous method for the control of a variety of insect pests and vectors in the recent past1-5. Phytotoxicological activity of Tagetes erecta was demonstrated by Sharma and Saxena6 against second and third instar larvae of Anopheles stephansi. Azadiractin, the active ingredient of Azadiracta indica has long been recognised for its mosquito larvicidal capability. Long before the advent of synthetic insecticides, plants and their derivatives were being used to kill the pests of agriculture, veterinary and public health importance. Insecticidal activities of plant-derived compounds have been evaluated and few of these exploited commercially7. Laboratory and field trials of plant extracts and purified chemicals showed mosquito larvicidal activity. Larvicidal activity of certain extracts of plant parts such as Acorus calamus, Adhatoda vesica, Croton tiglium, Mentha arvensis, Ocimum basilicum, and Vitex negundo against Culex larvae was reported by Deshmukh8. Also, the alcoholic extract of Artemisia annua was reported to be more toxic than the hexane extract and acetone extract of Artemisia vulgaris against Culex larvae. Similarly, the extract of Cuscuta reflexa was found more effective than the extract of Cannabis sativa, against Culex larvae9. According to Sharma and Shrivastava10, the extract of Cuscuta reflexa was the most toxic to Culex larvae, followed by extracts of Artemisia annua, Carica papaya, and Lantana indica. Latha11 reported Piper longum and Zingiber wightianum extracts at 80 mg/l causing complete mortality on Culex quinquefasciatus and 60 mg/l for Culex sitiens. Fruit and seed extracts of Abrus precatorious were found toxic to adult mosquitoes12 and crude extract of Withospermum arvense were found toxic to Aedes aegypti larvae13. Several such reports related to the toxic properties of plant products against mosquito larvae like peel oils of three citrus fruits against Culex pipiens14 and larvicidal properties of leaf extracts of Solanum nigrum15 are available.
Insecticides of plant origin have been extensively used on agricultural pests, and to a vary limited extent, against insects vectors of public health importance, which deserve careful and thorough screening. The use of plant extracts for insect control has several appealing features, as these are generally more biodegradable, less hazardous, and rich storehouse of chemicals of diverse biological activity. Moreover, herbal sources give a lead for discovering new insecticides. Therefore, biologically active plant materials have attracted considerable interest in mosquito control programmes in the recent times. The present study deals with the screening of locally available herbs and shrubs for mosquito larvicidal properties.

MATERIALS AND METHODS
Plant materials collected from the foothill forests of Chittagong Hilltracs, Rajshahi and some other parts of the country were segregated as leaf, twig, flower, tuber, root, etc and were air dried in a shady place to retain their active ingredients intact. Dried materials were powdered in a table model grinder for extraction and each of the powdered plant material (500 g) was soaked in methanol in a airtight wide mouth bottle and kept for 7 days. After that, the cold extracts from the bottles along with methanol were filtered and kept in petridishes for drying at room temperature. The average yield of crude extract (500 g) of powder was 3.5 g. Dried extracts were used for larvicidal bioassay as per WHO standards. Stock solutions were prepared by dissolving plant extract (1 g) in water (1 l) to make its strength 1000 ppm. Teepol (1 ppm) was also added as emulsifying agent. Different concentrations were prepared by adding required doses of stock solution in beakers containing water (250 ml).
Laboratory-reared Aedes albopictus and Culex quinquefasciatus were used for larvicidal bioassay under laboratory conditions (27o ± 2 °C and 75 ± 5 % RH). Twenty five mosquito larvae of third or early fourth instar were released in each beaker for 24 h with a concurrent control for every set of experiment. Three replicates were kept for each concentration. No food was added in the beaker as per WHO norms. Mortality was recorded after 24 h of treatment and mortality corrected by Abbot‘s formula. Data were analysed by probit analysis16.

         Pr = Po-Pc100-Pc  100
where,
         Pr = corrected mortality %
         Po = observed mortality % and
         Pc = control mortality %

RESULTS AND DISCUSSION
Out of 19 plants, pericarp extract of Zenthoxylum limonella was found to possess the most effective larvicidal activity against Aedes(s) albopictus and Culex quinquefasciatus with LD90 values at 0.47 ppm and 0.73 ppm, respectively, followed by seed extract of Piper nigrum, Piper longum, and leaf extract of Spilanthes acmella (Table 1). Among the others, peel extract of Citrus aurantifolia, leaf extract of Mentha arvensis, and flower extract of Tagetas patula were also found to have larvicidal properties. Leaf extracts, of Lippia nodiflora, Chrysanthemum cinerarifolium, Leucus linifolia, Lantana camara, fruit extract of Capsicum annuum, root extract of Moringa oleifera, and rhizome extract of Zingiber officinalis also showed larvicidal (LD90) activity against both the species with a higher dose.
As compared to the other herbal extracts, concentration of Zenthoxulum limonella extract for LD90 value was very low for both the species. Results of Piper nigrum were also encouraging considering other plant extracts tested against Aedes albopictus and Culex quinquefasciatus. LD50 values of Zenthoxulum limonella showed at 0.01 ppm and 0.02 ppm and for Piper nigrum, at 0.56 ppm and 0.65 ppm accordingly. It may be the plant alkaloid that have toxic effect on mosquito larvae. According to Saxena5, et al. plant alkaloids resulted in a significant loss in fecundity Percentage of test mortality  and fertility in the adult species of mosquitoes. Sharma and Saxena6 also found that the petroleum ether extract of Tagetes erectes had toxic effect on larvae of Anopheles stephensi and on its significant growth index. In the present study, post-treatment data analysis on growth and fertility could not be carried out. Mwangi and Mukiama17 observed that one fraction of Melia volkensi fruit karnel extract had growth inhibition activity at low concentration, whereas two other fractions had acute toxic effects on

Table 1. Mosquito lervicidal efficacy of some plant extracts
plant species with common name          part used          Culex quinquefasciatus
         Aedes(s) albopictus

                   LD50
ppm          LD90 ppm          Regression equation          LD50          LD90 ppm          Regression equation
Zanthoxylum limonella
(Bezarmoni)          
Pericarp          
0.02          
0.73          
Y =0.775x+4.0ô2          0.01          
0.47          
Y =0.749x+4.270
Piper nigrum
(jaluk)          
Seed          
0.65          
8.42          
Y = I.0053x+4,758          0.56          
6.S0          
Y= 1.1225x4-4.1220
Spilanthes acmella          
Leaf          
57.40          
108.30          
Y = 4.l627x - 2.35111          54.10          
I13.90          
Y = 3.8490x + 1.6784
Piper longum
(Pipoli)          
Seed          
70.10          
113.90          
Y = 5.669x±5.4902          54.50          
2.10          
Y=5.4766x-4-4.5187
Citrus aurantifolia          
Peel          
96.40          
128.40          
Y = 10.20lx + 15.274          79.50          
116.70          
Y 7.6293x + 9.53 IS
Mentha arvensis
(Pudina)          
Leaf          
98.90          
l93.O0          
Y = 4.3440x - 3.6756          90.20          
111.00          
Y = 4.3528x + 3.5360
Tagetespatula
(Nargee)          
Floer          
118.10          
261.070          
Y=3.5l26x+2.30l4          84.79          
15.10          
Y 3.5780x+l.9266
Lippia nodiflora
(Nemu sak)          
Leaf          
190.00          
301.60          
Y =6.9745x I 1.0557          190.10          
267.50          
Y=7.9629x- 13.147
Chrysanthemum cinerariifolium
(Indrmalati)          
Leaf          
210.00          
398.60          
Y= 4.72 13x - 6.0653          227.00          
323.90          
Y 4.4089x - 5.4081
Capsicum annuum
(Jalakia)          
Fruit          
2411.70          
314.70          
1 = 10.1775x- 19.261          
208.20          
320.80          
1 =6.I373x-9.279I
Leucas linifolia
(Doron)          
Leaf          
250.00          
315.00          
1 12.8550x—25.874          
233.20          
305.50          
1 10.8577-20.766
Lantana camara
(Guti phul)          
Leaf          
273.50          
374.70          
Y=9.2798x- 17.6632          
268.30          
364.20          
Y=Q.I651x- 17.3114
Ocimum sanctum
(Tulshi)          
Leaf          
288.20          
528.30          
1 4.51 62x - 6.2776          
176.90          
271.00          
Y = 7. 1467x-11.067
Moringa oleifera
(Sajina)          
Root          
317.90          
486.60          
1 = 6.791 7x -12.0074          
283.40          
498.20          
y 4.9387x - 7.1333
Hibiscus rosa sinensis
(Joba)          
Leaf          
380.50          
613.30          
1 =6.3069x— 11.3451          
294.90          
501.90          
1 5.3382x-8.1973
Zingiber officinalis
(Ada)          
Rhizome          
390.10          
628.30          
1 = 5.9706x - 10,4801          
521.10          
524.20          
1 = 5.5004x - 0.5389
Curcuma amada
(Amda)          
Rhizome          
395.10          
606.90          
Y =6.4218x - 11.7044          
181.20          
783.10          
Y = 5.7232x-10.377
Adnenthera pavonia
(Lalchandan)          
Seed          
474.90          
834.90          
Y=5.0250x - 8.4563          
42w. 10          
743.70          
Y = 4.7796x - 7.5467
Calotropis gigantean
(Akan)          
Leaf          
628.90          
1091.80          
Y = 5.1569x 9.4424          
467.00          
962.80          
Y = 3.8702x 5.3486

the mosquito larvae. As per Pushpalatha and Muthukrishnan18, leaf extracts of Vitex negundo, Nerium oleander, and seed of extract of Syzygium jambolanum at very low concentration had larvicidal activity against Culex quinquefasciatus and Anopheles stephensi, and also extended the duration of larval instars pupation. Mudrigal,13 et. al. reported that methanol extract of Lithospernum arvense were toxic to Aedes aegypti larvae. Fractionation of Zenthoxylum limonella extract may give more active compound for larval control of mosquitoes. It is evident from the present study that herbal extracts might have promising larvicidal efficacy. Plants are rich source of bioactive organic chemicals and offer an advantage over synthetic pesticides as these are less toxic, less prone to development of resistance, and easily biodegradable. Screening and identification of effective compounds available in Bangladesh will certainly bring more success towards the control of mosquitoes.

ACKNOWLEDGEMENTS
The authors are grateful to Professor Dr. Md. Nurul Islam and Shah Hosain Ahmad Mahdi for the help providing the laboratory facilities and to Professor M. Khalequzzaman in the statistical analysis of the data.

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