J. Asia-Pacific Entomol. 9(4): 389-395 (2006)www.entomology.or.kr

PEST MANAGEMENT

Growth Inhibitory Nature of Artemisia annua Extract againstCulex autnauetesctetus (Say)

Preeti Sharma, Lalit Mohan and CN. Srivastava*

Applied Entomology & Vector Control Laboratory, Department of Zoology, Faculty of Science, Dayalbagh Educational Institute(Deemed University), Dayalbagh, Agra-282 005 (India)

Abstract Petroleum ether (Pee), carbon tetrachloride(Cte) and methanol extract (Mee) of Artemisia annua,Chenopodium album and Sonchus oleraceus werescreened for their efficacy against Culex quinque­fasciatus larvae. Pee of A. annua, Mee of A. annuaand Ch. album, Cte of A. Annua were found effectivein descending order after 24 and 48 hrs of treatment.Pee of A. annua, the most potent extract with LCso78.2 ppm was selected to study its influence on thedevelopment and metamorphosis of the culicine mos­quito. The extract significantly affected the hatching,larval development, pupal transformation and alsolengthened the larval and pupal periods. Growth index'was remarkably reduced. Treated culicine eggs, larvaeand pupae showed deformities including disruptionof the body wall, distorted alimentary canal, damagedtracheal network and arrested histogenesis. The ex­tract has remarkable effect on the metamorphosis andhigh larvicidal potential, hence, can be used as aneffective alternative to the existing synthetic pesticidesfor the control of Cx. quinquefasciatus.

Key words Phytoextract, culicine larvae, develop­ment, metamorphosis

Introduction

Vector borne diseases constitute the major cause ofmorbidity in most of the tropical and subtropicalcountries and have always been a challenge to themedical professionals struggling for the welfare ofhumanity. Mosquitoes are the most deadly vector forseveral of these disease causing organisms. Thenuisance value of the Culex mosquito can be visu­alized by the increasing incidence of Dengue feverin India and other countries of the world and also

*Corresponding author.Email: chandnarayan_dei@rediffmail.comTel: +91-9319103817(R); Fax: +91-5622801226

(Received April 24, 2006; Accepted December 14, 2006)

by the alarming cases of filariasis, encephalitis andyellow fever. For the suppression of these diseases,pathogen control and management of the vector, Culexmosquitoes are concurrently urgent task. DDT andother conventional synthetic pesticides previouslyconsidered a panacea to overcome mosquito densityand longevity are handicapped under the presentscenario of mosquito control as the frequent andextensive usage of these insecticides has inducedresistance in many species of mosquitoes (WHO,1998; Mehrotra, 1992). Further, this has also con­tributed to gradual environmental deterioration, bioac­cumulation, non-target action and other health relatedhazards (Tyagi, 2003). These deleterious effects causedby the continuous application of chemical insecticideshave encouraged the scientists to solve this globalproblem by evolving an alternative technology, whichis the application of eco-friendly and biodegradablephytoproducts. The rich Indian tropical flora, a sourceof medicinal and insecticidal bioactive metabolites,is as an effective option. These phytochemicals areeasily available, economical, biodegradable and envi­ronmentally non-hazardous with least biomagnifica­tions. These have successfully been tested for variousbiocidal activities against different stages of mosquitolarvae (Evans and Kaleysa, 1988; Zaurrough et al.,1988; Sharma et al., 2004; Mohan et al., 2005; Sharmaet al., 2005), adults (Saxena and Sumithra, 1985) andfor their insect growth regulating activity (Deshmukhand Renapurkar, 1987; Sharma et al., 2006 a & b).The sincere efforts are going on to further investigatethe pesticides of botanical origin. The present inves­tigation is a step forward in this direction. The in­secticidal flora of Agra is exploited for the manage­ment of Culex quinquefasciatus and the extracts ofArtemisia annua, Chenopodium album and Sonchusoleraceus were screened for their larvicidal activity.The growth regulating effect of the most effectiveextract on the life cycle of the vector is further studied.This may prove useful in better and timely applicationof extract against the right stage of the mosquito.

390 1. Asia-Pacific Entomol. Vol. 9 (2006)

Materials and Methods

Extract preparation

The leaves of the plants selected i.e. A. annua, Ch.album and S. oleraceus collected from the Institutecampus, after thorough washing, were dried in shadeand crushed manually. These were subjected toSoxhlation with petroleum ether, carbon tetra chlorideand methanol subsequently. After complete extractioni.e. for 72 hrs, the solvents were evaporated from eachextract by vacuum rotary evaporator to obtain crudepetroleum ether (Pee), carbon tetrachloride (Cte) andmethanol extract (Mee).

Larvicidal assay

The stocks of the desired concentrations were pre­pared in appropriate solvents (ethyl alcohol or ace­tone) for each of these extracts. The stock solutionwas further diluted to prepare a range of test con­centrations. One ml of the test concentrations wasdiluted in 249 ml of water in 500 ml capacity beakers.Twenty 3rd instar culicine larvae were exposed to thesetest concentrations according to Standard W.H.O. Pro­cedure (1975). All the experiments were set in tri­plicate along with the controls at 25±2°C and 85%relative humidity. Larval mortality data due to thetoxic effect of the extracts were recorded after 24and 48 hrs of treatment, were further proceeded ac­cording to Probit Analysis (Finney, 1971) to calculateLC so.

Morphometric and developmentalexperi ments

Twenty un-hatched culicine eggs were exposed todifferent concentrations of the most potent extractscreened for the morphometric studies. The percenthatching, larval mortality, pupal transformation, pupalmortality and adult emergence were noted along withthe duration of larval and pupal stages. The sameexperiments were conducted with I ml alcohol andde-chlorinated water for examination under the controland untreated conditions. The deformities producedin the treated eggs, emerging larvae and pupae werealso noticed under zoom stereo binocular microscope.The changes in size of the eggs, larvae and pupaewere also noted. Growth index was calculated bydividing the percentage of adult emergence from theeggs treated by the total developmental period (Saxenaand Saxena, 1992).

Results

Larvicidal activity of the extracts

The extracts tested revealed appreciable results againstCx. quinquefasciatus larvae (Table I). Pee of A. annuaexhibited minimum LCso, value, 78.2 and 74.2 ppmafter 24 and 48 hrs of exposure, respectively. It wasfollowed by Mee of A. annlla and Ch. album andCte of A. annua with LCso values 360.1, 403.7 and415.9 ppm after 24 hrs and 159, 366.1 and 412.6ppm after 48 hrs of treatment, respectively. The Peeand Cte of Ch. album and Pee, Cte and Mee of S.oleraceus with LCso 2529, 5009.6, 6273, 2228.4 and5438 ppm after 24 hrs and 2011, 2254, 5439, 1792.3and 3741.1 ppm after 48 hrs of exposure, exhibitedthe least bioactivity. All the LCso values were wellwithin the 95% confidence limits. Pee of A. annua,being the most potent larvicide, was selected forfurther development and growth related studies.

Effect of the extract on hatching andlarval development

The observations based on the morphometric anddevelopmental studies (Table 2 and 3) reveal that ontreatment of the eggs with the extract, they becamehighly de-shaped and swollen, increasing in size froman average of 0.604 mm to 0.610 mm indicatingodema (Fig. 1). The extract reduced the hatching from100% at control to 85% at 7.5 ppm, 80% at 15 ppm,45% at 30 ppm, 40% at 60 ppm, 35% at 120 and240 ppm. Successfully hatched larvae developed de­formities as the larval stage progressed. The averagebody size of the 4th instar larvae reduced from 4.117mm to 3.479 mm. In most of the treated larvae, therewas constricted body wall, partly damaged alimentarycanal and distorted tracheal network (Fig. 2). Thelarval period prolonged as compared to control beingunaltered at 7.5 ppm, 9 days at 15 and 30 ppm andII days at 60, 120 and 240 ppm.

Effect of the extract on pupation

Larval deformities hampered the further transformationof larvae into pupae and only 76% of larvae couldsuccessfully pupate at 7.5 ppm. This was furtherreduced to 66% at 15ppm, 63% at 30 ppm, 61% at60 ppm, 47% at 120 ppm and 42.8% at 240 ppm.

In most of the transformed pupae, slight reductionin body size was noticed from an average of 3.990mm to 3.906 mm. The body wall was highly damagedand histogenesis was arrested in contrast to active

Growth Inhibitory Response of Artemisia Against Culex Mosquito 391

Table 1. LCso of the extracts tested against the larvae of Culex quinquefasciatus.

PlantExtraction Exposure period Chi-square Regression LCso Fiducial

solvent (Hours) (X2

) equation (ppm) limits

24 0.39 0.94X+2.53 415.9429.95

Carbon 401.85tetrachloride

48 0.72 0.97X+2A6 412.6426.83398.37

24 0.92 1.26X+1.78 360.1369.98

ArtemisiaMethanol

350.12annua

48 1.86 0.96X+2.87 159163.72154.18

24 0.92 1.16X+2.79 78.283.59

Petroleum 72.80ether

48 30.72 0.80X+3A1 74.278.9269.38

24 7.52 0.23X+4.14 5009.65170.80

Carbon 4848.30tetrachloride

48 0.17 0.64X+3A8 22542305.802202.20

24 2048 9.90X-20.74 403.7416.56

ChenopodiumMethanol

390.74album

48 1.73 8.28X-16.20 366.1380041351.79

24 1.21 OAOX+3.64 25292645.30

Petroleum 2522.60ether

48 0.72 1.23X+1.23 20112241.201778.90

24 2.08 2.58X-3.64 2228042356.50

Carbon 2100.20tetrachloride

48 0.36 3.15X-5.24 1792.31895.301689.20

24 3.85 1.l0X+0.89 54385562.60

Sonchus Methanol5312.50

oleraceus48 1.98 1.10X+1.07 3741.1

3835.103377.10

24 1.75 1.72X-1.53 62736633.60

Petroleum 5912.30ether

48 1.78 1.59X-0.96 54395751.605126.10

Table 2. Effect of the petroleum ether extract of Artemisia annua on development and growth index of Culex quinquefasciatus.

S. Concentration Eggs % Average % % Average % % % Average GrowthNo. (ppm) Treated transfor- larval Larval transfor- pupal pupal transfor- transfor- develop- Index*

mation period Mortality mation period mortality mation mation mental (alb)of eggs (days) of larvae (days) of pupae of eggs period

into larvae into pupae into adults into (days)Adults (b)

(a)

l. 7.5 20 85 8 24 76 2 23 76 50 10 5

2. IS 20 80 9 34 66 2 31 69 36 II 3.27

3. 30 20 45 9 37 63 2 34 66 19 II 1.73

4. 60 20 40 II 49 61 3 40 60 14.4 14 1.02

5. 120 20 35 11 53 47 3 50 50 10 14 0.71

6. 240 20 35 II 57.2 43 60 40 06 14 0.42

7. CONTROL 20 100 8 30 70 2 IS 85 60 10 6

8. UNTREATED 20 100 8 4 96 2 2 98 94 10 9.4

* Statistical evaluation carried out by using student 't' test (p<O.OOI) compared to the control and untreated.

392 1. Asia-Pacific Entomol. Vol. 9 (2006)

Table 3. Average length of eggs, larvae and pupae of Culex quinquefasciatus with their body division under control andafter exposure: to Pee of A. annua for 48 hours.

S. Plant extract Eggs Fourth Instar Larvae (mm) Pupae (mm)No. (mm) Head Thorax Abdomen Total Cephalothorax Abdomen Total

(i) Artemisia annua 0.610 0.744 0.882 2.252 3.479 1.890 2.100 3.906Pee (±0.044) (±0.022) (±0.058) (±0.206) (±0.330) (±0.172) (±0.325) (±0.245)

(ii) CONTROL* 0.604 0.655 0.755 2.688 4.095 1.480 2.520 3.990(±0.011) (±0.041) (±0.096) (±0.191) (±0.595) (±0.112) (±O.l59) (±0.195)

(iii) UNTREATED 0.605 0.653 0.762 2.640 4.117 1.487 2.506 3.993(±0.002) (±0.004) (±0.003) (±0.006) (±0.024) (±0.005) (±0.006) (±0.008)

* The acetone being a common solvent for Pee of Artemisia annua, same control experimental data are considered in both the cases.

(A) (B)

Fig. 1. Culicine egg under control (A) and treated (B) with Artemisia annua (Pee). SH, Shell. Scale line: 1 mm x 100

(A) (B)

Fig. 2. Culicine larva under control (A) and treated (B) with Artemisia annua (Pee). FBR, feeding brush; ANT, antenna;HE, head; CE, compound eye; TH, thorax; BR, bristles; AB, abdomen; HLT, haemolymphatic tissue; ALC, alimentary canal;RSI, respiratory siphon. Scale line: 1 Hun x 20

Growth Inhibitory Response of Artemisia Against Culex Mosquito 393

histolysis (Fig. 3). The pupal period was not effectedat 7.5, 15 and 30 ppm but was lengthened to 3 daysat 60, 120 and 240 ppm. The percentage relationshipbetween different developmental stages with extractconcentrations is shown in Fig. 5.

at 240 ppm. Growth index was descended from 7.5at control to 6.5, 6, 2.5, 1.3, 1.15 and 0.36 at 7.5,15, 30, 60, 120 and 240 ppm, respectively (Fig 4).

DiscussionEffect of the extract on adultemergence and growth index

Pupal deformities further influenced the adult emer­gence which was 76% at 7.5 ppm, 69% at 15 ppm,66% at 30 ppm, 60% at 60 ppm, 50% at 120 ppmand 40% at 240 ppm. The percentage of adult emer­gence from the eggs exposed was also reduced from50 at 7.5 ppm to 6.0% at 240 ppm. The developmentalperiod of 10 days was also prolonged up to 14 days

Significant larvicidal activity was found in some ofthe extracts tested. The petroleum ether extract of A.annua was found more toxic as compared to thedifferent extracts of Artemisia annua, Chenopodiumalbum and Sonchus oleraceus against culicine larvaewith LCso of 78.2 and 74.2 ppm after the exposureof 24 and 48 hrs, respectively.

The most potent extract, i.e. Pee of A. annuaexhibited considerable ovicidal activity and the hatch-

(A) (B)

Fig. 3. Anopheline pupa under control (A) and treated (B) with Artemisia annua (Pee). CT, cephalothorax; CE, compoundeye; RUA, rudiments of appendages; AB, abdomen; HLT, haemolymphatic tissue; TG, tracheal gill; RT, respiratory trumpet.Scale line: I mm x 20

8%LARoIAL...mTALfTY

240.00CCNTR:l.. 7.50 15.00

J11 Jt ::=::.mill ---,ooo,-..."-"'."""-'''--'''IIIIL.......-........ -.JIIUlj. -,. '

20,00·

60.00<

4000'

00.00'

120,00,

100,00.

1__Growth Index IGrowth Index10

9

8

7

6

5

4

3

2

1

o J-~~-~r~~-.~~-,--,~_··_·~~ .r~~~·~-'\ '? ...':> '!? <§> ...<f' rI' of'~(} "rJ>

.;:,~

Fig. 4. Effect of Pee of A. annua on growth index of Cx.quinquefasciatus.

Fig. 5. Effect of Pee of A. annua on development of Cx.quinquefasciatus.

394 J. Asia-Pacific Entomo\. vei. 9 (2006)

ing was reduced from 100% at control and 85% at7.5 ppm to 35% at 240 ppm. The eggs became highlyde-shaped and swollen indicating odema as a resultof endosmotic effect of the extract. In the remainingcases where the eggs hatched successfully shrinkagewas observed in average body size of 4th instar larvae,which reduced from 4.117 mm to 3.479 rom. More­over, body wall was constricted in most of the larvae,may be, due to insufficient availability of chitin. Also,alimentary canal and tracheae were damaged. Theseresults are supported by findings of Saxena et al.(1984) who had noticed similar morphological defor­mities, including darkening of the larval cuticle, duringmoulting and development of Cx. quinquefasciatusinduced by Ageratum conyzoides extract. Certainphysical abnormalities had also been noticed by Sherifet al. (1985) in Cx. quinquefasciatus on exposure toElodee nutallii and Spirogyra niiela extract. Further,Tabassum et al. (1993) has observed that plant extractsaffected larval morphology resulting in greater pig­mentation and alteration in shape head and abdomen.Larval period was prolonged with increase in con­centration of extract up to 11 days at 240 ppm. Thisdelay in pupation is in consonance with findings ofMehra (1998) where the culicine larval period wasextended by 7 to 9 days on treatment with Dedro­dendrum inerme extract. Percentage of deformed lar­vae increased with increase in concentration of theextract and only 43% larvae could pupate successfullyat 240 ppm. Some of the larvae were arrested as larvalpupal intermediates and the rest died due to larvicidalaction of the extract and larval mortality may beattributed to the rupturing of the new cuticle at thetime of moulting and the subsequent loss of hae­molymph.

Successful pupation followed deformed and dis­torted progress in most of the cases, with reductionin average body size and damaged body wall, whichmay again be attributed to interruption in chitinsynthesis. Histogenesis was destructed indicating theinterrupting role of the extract in normal proteinsynthesis during the process. Pupal period was ex­tended by 24 hrs at 240 ppm and this alteration inthe pupal period has also been observed by Perieraand Gurudatt (1990) where the larvae pupated lateror earlier than respective controls when treated withPee of Clerodendron inerma. Abnormalities producedin larvae and pupae reduced the adult emergence,which may be credited to interference in normal chitinsynthesis as interpreted by Saxena and Yadav (1983).Similar observations regarding the deformities pro­duced in Cx. pipiens larvae has also been noted byDoghhagiri and Elhag (2002) after treatment with theaqueous extract of Rhazya stricta and Calotropisprocera where larval development was significantlyreduced, consequently reducing the pupation and adult

emergence.The percentage of adult emergence in relation to

the eggs treated, was reduced from 60 at control to6 only at 240 ppm and developmental period wasprolonged up to 14 days at 240 ppm due to arrestedlarval-pupal and pupal-adult intermediates. Our resultsare supported by findings of Grant et al. (1982) wherethe extract of Artemisia cana has been reported notonly to cause larval mortality but also to produceabnormalities in the surviving pupae and adults likethe inability to emerge fully from the molt andexuviae. Moreover, comparable observations on thegrowth pattern and life cycle of Cx. quinquefasciatushas also been noticed by Jayaprakash et aZ. (1997).Phytoextract induced hampered development anddegenerative metamorphosis of other insects, as ob­served by Stark et al. (1990), Bhagwan et al. (1995),Suryakala et al. (1995), Agarwal (1993), further sup­port the present study on the growth inhibitory effectof the Pee of A. annua against Cr. quinquefasciatus.The extract possessing significant larvicidal action andgrowth inhibiting activity can be successfully employedfor effective control and field management of Ja­panese Encephalitis and Yellow Fever vector and canbe an effective biocontrol option for the existingchemical insecticide use for control of mosquitoes andother vectors.

Acknowledgements Authors are thankful to Prof. K. K. Dua,Head, Department of Zoology, for encouragement. We arealso grateful to Council of Scientific and Industrial Research,New Delhi for financial assistance.

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