Bioresource Technology 97 (2006) 1599–1604

Phytoextract-induced developmental deformities in malaria vector

Preeti Sharma, Lalit Mohan, C.N. Srivastava ¤

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

Received 4 May 2005; received in revised form 27 July 2005; accepted 28 July 2005Available online 13 December 2005

Abstract

Larvicidal potential of petroleum ether (Pee), carbon tetrachloride (Cte) and methanol extract (Mee) of Artemisia annua, Cheno-podium album and Sonchus oleraceus was observed against malaria vector, Anopheles stephensi Liston. The Pee of A. annua with LC5016.85 ppm after 24 h and 11.45 ppm after 48 h of treatment was found most eVective, followed by Cte of A. annua and Ch. album, Peeof Ch. album and Mee of A. annua. However, no signiWcant larvicidal activity was observed in Mee of Ch. album and all the threeextracts of S. oleraceous. The Pee of A. annua was further investigated for its eVect on the metamorphosis and the development of themalaria vector. It inXuenced the early life cycle of An. stephensi by reducing the percentage of hatching, larval, pupal and adult emer-gence and also lengthening the larval and pupal periods. The growth index was also reduced signiWcantly. As the extract has remark-able eVect on the metamorphosis and high larvicidal potential, it could, therefore, be used as an eVective biocontrol agent against thehighly nuisant malaria vector.© 2005 Elsevier Ltd. All rights reserved.

Keywords: Anopheles stephensi; Artemisia annua; Chenopodium album; Sonchus oleraceus; Metamorphosis; Growth index

1. Introduction

Phytoproducts on account of minimal hazardouseVect on the environment and wide range of availabilityoVer promises in future mosquito control programmes.They have revolutionized the Welds of vector control asthey possess diVerent bioactive components and can beused as general toxicants against various larval stages ofthe mosquito (Sharma et al., 2004; Mohan et al., 2005)and other insect vectors of human diseases. Also, thesecan be used as repellants (Omolo et al., 2004), ovicides(Jarial, 2001; Thenmozhi and Kingsly, 2004), feedingdeterrents (Thioson et al., 2004) and adulticides (Yanget al., 2004). Though a lot of work has been done regard-ing the toxic eVect of phtyoproducts on a particular

* Corresponding author.E-mail address: chandnarayan_dei@rediVmail.com (C.N. Sri-

vastava).

0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2005.07.024

stage of the life cycle of a mosquito but the Wndings onthe eVect of the phytochemicals in relation to the devel-opmental aspects of mosquitoes are so far limited andhence require further attention. An attempt has, there-fore, been made in the present study to observe theimpact of most potent extract out of Artemisia annua,Chenopodium album and Sonchus oleraceus on the hatch-ing and post-hatching development of Anopheles step-hensi.

2. Methods

2.1. Preparation of the extracts and test concentrations

Leaves of the plants were collected during December–March from the adjoining area of our Institute. Leavesafter thorough washing and drying, were crushed andextracted in the Soxhlet apparatus with petroleum ether,

1600 P. Sharma et al. / Bioresource Technology 97 (2006) 1599–1604

carbon tetrachloride and methanol for 72 h each. Sol-vents were removed by vacuum rotary evaporator toobtain crude petroleum ether (Pee), carbon tetrachloride(Cte) and methanol extract (Mee). Stocks of desired con-centrations were prepared in ethyl alcohol or acetone foreach extract. A range of test concentrations was pre-pared for each extract from the stocks in 500 ml capacitybeakers containing 249 ml of tap water and 1 ml of testconcentration.

2.2. Treatment of larvae and mortality data collection

Twenty III instar anopheline larvae were exposed toeach extract according to the Standard WHO Procedure(1975). Experiments were set in triplicate along with con-trols at 27§ 2 °C and 85% relative humidity. The mortal-ity was recorded 24 and 48 h after treatment and wasproceeded further according to Probit analysis (Finney,1971) for the calculation of LC50 and other parameters.

2.3. Morphometric and developmental studies

For the morphometric studies, 20 unhatched anophe-line eggs were exposed to diVerent concentrations of themost potent extract screened. The percentage of larvalemergence, larval mortality and adult emergence wasnoted, along with the duration of larval and pupal peri-ods. Controls were conducted parallel to each experi-ment and related observations were noticed under zoomstereo binocular microscope.

3. Results

3.1. Larvicidal assay

The petroleum ether extract of A. annua was found themost eVective larvicide against anopheline mosquito, theLC50 being 16.85 and 11.45 ppm after 24 and 48 h of expo-

Table 1LC50 of the extracts tested against Anopheles stephensi larvae

Plant Extraction solvent Exposure period (h) Chi-square (�2) Regression equation LC50 (ppm) Fiducial limits

Artemisia annua Carbon tetrachloride 24 1.88 3.46 + 1.03X 30.80 34.3427.26

48 2.13 3.48 + 1.11X 21.18 24.5817.78

Methanol 24 3.83 21.12 + 9.93X 425.60 442.20409.00

48 0.68 25.57 + 11.68X 414.48 426.85402.10

Petroleum ether 24 5.59 2.99 + 1.63X 16.85 19.7213.98

48 11.18 3.62 + 1.29X 11.45 14.838.51

Chenopodium album Carbon tetrachloride 24 1.52 0.26X ¡ 2.26 212.15 215.41208.87

48 0.41 1.13 + 1.81X 140.10 143.30136.90

Methanol 24 0.38 3.88X ¡ 4.79 332.75 348.05317.45

48 8.24 0.94 + 1.68X 254.50 266.05242.99

Petroleum ether 24 1.17 1.45X ¡ 0.08 314.90 £ 10 333.85 £ 10300.40 £ 10

48 2.29 2.52 + 0.79X 126.25 £ 10 131.42 £ 10121.06 £ 10

Sonchus oleraceus Carbon tetrachloride 24 4.64 2.97X ¡ 7.92 221.98 £ 100 224.40 £ 100219.22 £ 100

48 1.97 4.41X ¡ 13.3 179.73 £ 100 181.06 £ 100178.39 £ 100

Methanol 24 4.39 2.04 + 0.76X 742.93 £ 10 760.00 £ 10725.85 £ 10

48 5.96 1.28 + 0.98X 591.90 £ 10 605.50 £ 10578.30 £ 10

Petroleum ether 24 14.99 0.66 + 1.16X 557.95 £ 10 572.62 £ 10543.32 £ 10

48 2.39 0.15 + 1.30X 537.03 £ 10 548.14 £ 10525.91 £ 10

P. Sharma et al. / Bioresource Technology 97 (2006) 1599–1604 1601

sure, respectively (Table 1). It was followed by Cte of thesame plant with LC50 30.80 and 21.18 ppm after treatmentof 24 and 48 h. The Cte and Pee of Ch. album were next inpotency with LC50 212.15 and 140.10 ppm for the formerand 332.75 and 254.56 ppm for the latter, respectively,after treating for 24 and 48 h. These were followed by theMee of A. annua, the LC50 being 425.60 and 414.48 ppmafter 24 and 48 h of exposure. The larvicidal eVect of Peeof Ch. album was fair with LC50 314.90£10 after 24 h butit was improved after 48 h with LC50 126.25£10 ppm. Incase of S. oleraceus, Cte, Mee and Pee showed poorlarvicidal activity with LC50 221.98£100 and 179.73£100 ppm, 742.93£10 and 591.90£10 ppm, and 557.95£10 and 537.03£10 ppm, respectively, after 24 and 48 h ofexposure. The larvicidal activity of these extracts weregraded according to Deshmukh (1982).

Table 2

3.2. Morphometric and developmental studies

As the Pee of A. annua was the most eVective withminimum LC50 values, it was further investigated for itseVect on the development of An. stephensi. At 7.50 and15.0 ppm, no ovicidal eVect was observed since 100%hatching occurred at these concentrations. However,hatching was reduced to 90% at 30 ppm, 80% at 60 ppmand 75% at 120 and 240 ppm (Table 2). In the aVectedeggs, egg shell was found broken and damaged, alongwith airXoats (Fig. 1). In some eggs, hatching was initi-ated but was followed by mortality of the larvae, whichgot trapped in the broken egg shell. The aVected eggs inmost cases increased in size from an average length of0.679–0.689 mm, indicating odema and hence, the deathof larvae (Table 3).

EVect of the petroleum ether extract of Artemisia annua on development and growth index of Anopheles stephensi

¤ Statistical evaluation carried out by using Student ‘t’ test (p < 0.001) compared to the control and untreated.

S. no. Concen-tration(ppm)

Eggstreated

Trans-formationof eggs intolarvae (%)

Averagelarvalperiod(days)

Larvalmortality(%)

Trans-formationof larvaeinto pupae(%)

Averagepupalperiod(days)

Pupalmortality(%)

Trans-formationof pupaeinto adults(%)

Trans-formationof eggsinto adults(%)

Averagedevelop-mentalperiod(days)

GrowthIndex¤

1. 7.50 20 100.00 8 20.00 80.00 2 19.00 81.00 65.00 10 6.502. 15.00 20 100.00 8 25.00 75.00 2 20.00 80.00 60.00 10 6.003. 30.00 20 90.00 10 45.00 55.00 2 38.00 62.00 30.00 12 2.504. 60.00 20 80.00 10 53.00 47.00 3 57.00 43.00 18.00 13 1.385. 120.00 20 75.00 10 55.00 45.00 3 57.00 43.00 15.00 13 1.156. 240.00 20 75.00 11 55.00 45.00 3 83.50 16.50 05.00 14 0.367. Control 20 100.00 8 15.00 85.00 2 22.00 88.00 75.00 10 7.50

Fig. 1. Anopheline egg under control (A) and treated with Artemisia annua (B). AF, air Xoat; SH, Shell; TL, trapped larva. Scale line: 1 mm £ 100.

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After successful hatching, many deformities developedas larval development progressed. Exoskeleton waspartly dechitinised specially in the abdominal region. Ali-mentary canal, haemolymphatic tissue, tracheal networkand fat bodies were signiWcantly damaged (Fig. 2). Aver-age body length of IV instar larvae reduced from 4.385 to4.162 mm. Head and the thorax were swollen to increasein length from 0.643 to 0.720 mm and 0.823 to 0.829 mm,respectively, while the abdomen was reduced from 2.902to 2.611 mm (Table 3). Larval period was not aVected atlower concentrations and remained eight days but waslengthened to 10 days at 30, 60 and 120 ppm, and up to 11days at 240 ppm. Some of the larvae were arrested at thelarval or larval–pupal intermediate stage. Only 80% oflarvae could successfully pupate at 7.50 ppm, while thiswas reduced to 75% at 15 ppm, 55% at 30 ppm, 47% at60 ppm and 45% at 120 and 240 ppm (Table 2).

The pupae transformed exhibited many disruptionsincluding rupturing of the body wall, discarding of

bristles and disorganization of cephalothorax (Fig. 3).Active histolysis of haemolymphatic tissue started andcontinued while histogenesis was arrested. Size ofpupae was also reduced from an average of 3.998–3.712 mm. As in larvae, the abdomen of the pupaereduced from 2.993 to 2.100 mm while the cephalotho-rax swelled from 1.005 to 1.610 mm (Table 3). Pupalperiod was not aVected at 7.50, 15.0 and 30.0 ppmbut was prolonged up to 3 days at 60.0, 120.0 and240.0 ppm. Adult emergence was further reduced from88% at control to 81% at 7.50 ppm, 80% at 15.0 ppm,62% at 30.0 ppm, 43% at 60.0 and 120.0 ppm and16.50% at 240.0 ppm. The emergence of adults from theeggs treated, being 75% at control, reduced to 65%,60%, 30%, 18%, 15% and 5% at 7.50, 15.0, 30.0, 60.0,120.0 and 240.0 ppm, respectively. Developmentalperiod was not aVected at 7.5 and 15 ppm but extendedup to 12 days at 30 ppm, 13 days at 60 and 120 ppm and14 days at 240 ppm. Growth index was also eVected

Table 3Average length of eggs, larvae and pupae of Anopheles stephensi with their body division under control and after exposure to Pee of Artemisia annuafor 48 h

S. no. Plant extract Eggs (mm) Fourth instar larvae (mm) Pupae (mm)

Head Thorax Abdomen Total Cephalothorax Abdomen Total

1. A. annua (Pee) 0.689 0.720 0.829 2.611 4.162 1.610 2.100 3.712(§0.007) (§0.011) (§0.019) (§0.078) (§0.088) (§0.035) (§0.234) (§0.312)

2. Control 0.679 0.643 0.823 2.902 4.385 1.005 2.993 3.998(§0.006) (§0.029) (§0.094) (§0.484) (§0.258) (§0.110) (§0.205) (§0.125)

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

P. Sharma et al. / Bioresource Technology 97 (2006) 1599–1604 1603

being 7.50 at control and reduced to 6.50 at 7.5 ppm,6.00 at 15.0 ppm, 2.50 at 30.0 ppm, 1.38 at 60.0 ppm, 1.15at 120.0 ppm and 0.36 at 240.0 ppm.

4. Discussion

The Pee of A. annua was most eVective and had sig-niWcant inXuence on hatching and post-hatching devel-opment of the mosquito. Jarial (2001) reported similareVects with garlic extract against Aedes aegypti display-ing attached shell caps with trapped larvae within theshell and arresting the hatching eggs. Several morphoge-netic abnormalities as observed in this study, have beenobserved in An. stephensi larvae when treated with meth-anol extract of Ageratum conyzoides (Saxena and Sax-ena, 1992). Larval period was not aVected at lowerconcentrations but was lengthened to 11 days at240 ppm. This Wnding was in accordance with the phyto-extract-induced lengthening of Ae. aegypti larval perioddue to interference in normal hormonal activity (Supa-varn et al., 1974). The failure in adult emergence couldbe due to insuYcient availability of chitin during meta-morphosis resulting in death of larvae and pupae entan-gled in the weak integument. Similar phytoextract-induced deformities was noted in An. stephensi (Saxenaand Sumithra, 1985). Degenerative eVects on the lifecycle of An. stephensi as observed by us have also beenreported by Dhar et al. (1996) and also on the develop-ment of other insects by several other authors (Sherifet al., 1985; Parkman and Pienkowski, 1990).

The Wndings regarding larvicidal action of extracts ofA. annua and Ch. album with the degenerative eVects of

the Pee of A. annua on the development of An. stephensiindicated their appreciable larvicidal potential. Thiscould lead to better application of the botanical deriva-tives during the suitable developmental period andcould also be helpful in proper understanding of thebiology of the mosquito with reference to such naturalmosquitocide.

Acknowledgements

Authors are thankful to Prof. S.S. Bhojwani, Directorand Prof. K.K. Dua, Head, Department of Zoology forencouragement. We are also grateful to Council ofScientiWc and Industrial Research, New Delhi for Wnan-cial assistance.

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