evaluation of solanum xanthocarpum extract as a synergist for cypermethrin against larvae of the...

Entomological Research

36

(2006) 220–225

© 2006 The AuthorsJournal compilation © 2006 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

Blackwell Publishing Ltd

RESEARCH PAPER

Evaluation of

Solanum xanthocarpum

extract as a synergist for cypermethrin against larvae of the filarial vector

Culex quinquefasciatus

(Say)

Lalit MOHAN, Preeti SHARMA and C. N. SRIVASTAVA

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

Correspondence

Dr 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. Email: [email protected]

Received 21 August 2006; accepted 31 August 2006.

doi: 10.1111/j.1748-5967.2006.00037.x

Abstract

Cypermethrin and crude extracts of


were both observed fortheir larvicidal activity against


. Petroleum ether extractwith lethal concentration (LC)

50

and LC

90

of 41.28 and 111.16 p.p.m. after 24 h andLC

50

38.48 and LC

90

80.83 p.p.m. after 48 h, respectively, was found to be the mosteffective, followed by carbon tetrachloride and methanol extracts. LC

50

and LC

90

forcypermethrin were 0.0027 and 0.0097 p.p.m. after 24 h and 0.0013 and 0.0092 p.p.m.after 48 h of exposure, respectively. Combined formulations were evaluated forsynergistic activity and a 1:1 ratio of cypermethrin and petroleum ether extract wasobserved to be more effective than 1:2 and 1:4 ratios. Combinations of

S. xanthocarpum

extracts and cypermethrin demonstrated higher larvicidal activity, indicating synergisticactivity. These results demonstrate the need for further studies on the effectivenessand toxicity to humans and animals, particularly aquatic forms.

Key words:

antagonism,

Culex

, cypermethrin, larvicide,

Solanum

, synergism.

Introduction

Integrated vector control is an effective and essential part ofany successful vector control program. Chemical control,although effective, is often used only as a temporary solutionto disease outbreaks. The over use of chemical control oftenleads to resistance to these chemicals, resulting in arebounding vector population and disease potential. Selectedbotanicals have been shown to be effective larvicides andadulticides and in some cases are more ecofriendly againstnon-target animals (Prakash & Rao 1997). Phytoextractshave been successfully tested for various biocontrolprograms (Markouk

et al.

2000; Jeyabalan

et al.

2003;Sharma

et al.

2004; Choochote

et al.

2004 Sharma

et al.

2006). Natural plant extracts provide potential for thedevelopment of botanical pesticides and synthetic analogs(e.g. pyrithrin). Identifying insecticides that are efficient,as well as being suitable and adaptive to ecologicalconditions, is imperative for continued effective vectorcontrol management. Synergistic activity between currenteffective pesticides and phytochemicals is a powerful tool fordeveloping insect control strategies (Bernard & Philogene

1993). The current study evaluates the synergistic actionof an Indian insecticidal herb,


(Mohan

et al.

2005), and cypermethrin against

Culexquinquefasciatus

, the filarial vector of India and other Asianpacific countries (Rahman

et al.

1989).

Materials and methods

Maintenance of mosquito colony

The mosquito

Cx. quinquefasciatus

was reared in ourlaboratory under the control conditions of 27

±

1

°

C, 85%relative humidity (RH) and a normal photoperiod from eggsinitially collected from the cyclic colony at the MalariaResearch Centre, Delhi. The eggs were immersed indechlorinated tap water in enamel basins of 30 cm diameter.The hatched larvae were fed brewer’s yeast. The transformedpupae were separated manually with a glass dropper into a500 mL beaker with water and introduced into adult cages of12 in

×

12 in

×

12 in for adult emergence. Adult mosquitoeswere fed a glucose meal (cotton soaked in 10% glucosesolution). Albino rabbits were used to provide a blood meal

Solanum

as a synergist for

Culex


36

(2006) 220–225

221

© 2006 The Authors. Journal compilation © 2006 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

for adult female mosquitoes after the second day of emergence,and every third day thereafter. Moist filter paper was keptin a beaker in the cages for mosquitoes to lay their eggs on.Eggs laid on the filter paper were immersed in larval basinscontaining water for the maintenance of the colony.

Phytoextract bioassay

Roots of

S. xanthocarpum

collected from areas adjacent tothe Dayalbagh Educational Institute, Agra, were washed,then dried in the shade. Dried roots were chopped into smallpieces of approximately 1 cm size by a Falcon stem cutter(Biocraft Scientific, India), and they were subjected toextraction with petroleum ether, carbon tetrachloride andmethanol in a Soxhlet apparatus (Borosil, Mumbai, India) for72 h each (Saxena

et al.

1994). After removing the solventsfrom the plant extracts in a vacuum rotary evaporator, 6.38,2.21 and 60.25 g of viscous paste was obtained per kg of dryplant material for petroleum ether, carbon tetrachloride andmethanol, respectively. Residues (10 g) obtained for eachfraction were dissolved in 100 mL ethanol independently toobtain stock solutions of 100 000 p.p.m. in each case. Six testconcentrations were prepared by further diluting the stock inethanol ranging from 7500 to 20 000 p.p.m. for petroleumether and carbon tetrachloride extract, and from 30 000 to75 000 p.p.m. for methanol extract. For bioassay, 1.0 mL ofeach of these test concentrations was added to 249.0 mLdechlorinated tap water in 500 mL beakers to obtain workingtest concentrations of 30–80 p.p.m. for petroleum ether andcarbon tetrachloride and 120–300 p.p.m. for methanolfractional residues. Twenty third instar


larvae obtained from lab culture were exposed to theseworking test concentrations. Experiments were conducted intriplicate, and controls in each series (1.0 mL ethanol with249.0 mL dechlorinated tap water at 27

±

1

°

C, 85% RH)were performed in parallel to the experiments according tostandard WHO (1975) procedures. Mortality observationswere noted 24 and 48 h after treatment.

Cypermethrin bioassay

Cypermethrin (25% emulcifiable concentrate [EC]; RPG LifeSciences, Mumbai, India), purchased from a local market, wasdiluted in dechlorinated tap water to obtain a 20 p.p.m. stock.Different test concentrations ranging from 0.0015 to 0.05 p.p.m.were prepared by diluting this stock, and a bioassay against


larvae was conducted as for the phytoextracts.

Combined efficacy of phytoextract and

cypermethrin

For combination studies, 20 p.p.m. stock of cypermethrintogether with the most efficient phytoextract was prepared.

Keeping cypermethrin as the standard, its stock was mixedwith the stock of phytoextract in ratios of 1:1, 1:2 and 1:4.Test concentrations for each of the mixed formulation ratioswere prepared by further diluting the combination mixture inwater. Larval efficacy for each formulation was observed asabove and lethal concentration (LC)

50

as well as LC

90

weredetermined.

Data analysis of mortality response

Mortality data produced for phytoextracts and cypermethrinbioassays and for mixed formulations were analyzed byProbit Analysis (Finney 1971). The corrected percentmortality was calculated by Abbot’s formula (Abbot 1925)on mortality if it ranged from 5 to 20% in controls:

Corrected % mortality = [(T

−

C) / (100

−

C)]

×

100

where T is the percent mortality in the test concentration andC is the percent mortality in the control.

Regression equations, LC

50

and LC

90

were obtained alongwith standard error and fiducial limits at 95% confidencelevel. A co-toxicity coefficient (Sarup

et al.

1980) and a syn-ergistic factor (Kalayanasundaram & Das 1985) for mixedformulation experiments were calculated after calculatingLC

50

and LC

90

for each combination.

Co-toxicity coefficient (CTC) = [toxicity of insecticide (alone) /toxicity of insceticide with plant extract]

×

100

Synergistic factor (SF) = toxicity of insecticide (alone) / toxicity of insecticide with plant extract

A value of SF > 1 indicates synergism and SF < 1 indi-cates antagonism.

Results

Larvicidal activity of


The bioefficacy of root extracts against


larvae is shown in Table 1 (Fig. 1). The LC

50

and LC

90

ofpetroleum ether extract were 41.28 and 111.16 p.p.m. and 38.48and 80.83 p.p.m. after 24 and 48 h of exposure, respectively.The LC

50

and LC

90

of carbon tetrachloride extract were 64.99and 252.43 p.p.m. and 59.20 and 186.15 p.p.m. after 24 and48 h of exposure, respectively. The LC

50

and LC

90

of methanolextract were 248.55 and 578.25 p.p.m. and 215.52 and562.72 p.p.m. after 24 and 48 h of exposure, respectively.All of the values fit well within 95% confidence limits.

Bioassay of mixed formulations

The results of the bioassay of different ratios of cypermethrinand petroleum ether root extract combined against

L. Mohan

et al.

222


36

(2006) 220–225 © 2006 The Authors. Journal compilation © 2006 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd


larvae are shown in Table 2 (Figs 2, 3).The ratio 1:1 had LC

50

and LC

90

of 0.0055 and 0.0089 p.p.m.and 0.0050 and 0.0087 p.p.m. after 24 and 48 h of exposure,respectively. The LC

50

and LC

90

values for the ratio 1:2 ofcypermethrin and plant extract were 0.0058 and 0.0099 p.p.m.and 0.0066 and 0.0144 p.p.m. after 24 and 48 h of treatment,respectively; and for the ratio of 1:4 were 0.0054 and0.0088 p.p.m. and 0.0062 and 0.0126 p.p.m. after 24 and48 h of treatment, respectively.

For LC

50

values, the co-toxicity coefficient of the ratio1:1 was 49.09 and 26.0 and the combined factor was 0.49and 0.26 after 24 and 48 h of exposure, respectively:antagonism was seen at both time points. In the case ofLC

90

values, the co-toxicity coefficient was 108.99 and105.75 and the combined factor was 1.09 and 1.06 after24 and 48 h of exposure, respectively: synergistic actionagainst


larvae at both time points.For the ratio 1:2, the co-toxicity coefficient was 46.55 and24.07 and the combined factor was 0.47 and 0.24 for LC

50

values after 24 and 48 h of exposure, respectively, whichshows antagonism. For LC

90

values, the co-toxicity coeffi-cient was 97.98 and 104.55 and the combined factor was0.98 and 1.05 showing antagonism after 24 h and synergismafter 48 h of treatment. The ratio 1:4 had a co-toxicitycoefficient of 40.91 and a combined factor of 0.41 (antago-nism) after 24 h and 20.97 and 0.21 (antagonism) after48 h for LC

50

values, and for LC

90

values the co-toxicitycoefficient was 67.36 and the combined factor was 0.67(antagonism) after 24 h, and 73.02 and 0.73 (antagonism)after 48 h.

All three fractions of

S. xanthocarpum

demonstratedappreciable larvicidal activity after 24 and 48 h posttreatment. The petroleum ether extract showed greaterlarvicidal activity than the carbon tetrachloride andmethanol extracts.

Table 1 Efficacy of different root extracts of Solanum xanthocarpum against larvae of Culex quinquefasciatus

Solvent extract

Exposure(h)

Regression equation χ2

LC50 ± SE (fiducial limits)

(p.p.m.)

Relative toxicity irrespective of

time period


(p.p.m.)

Relative toxicity irrespective of

time period

Carbon tetrachloride 24 Y = 2.17X − 1.18 0.65 64.99 ± 9.48 (46.41–83.58)

3.82 252.43 ± 154.52 (50.44–555.30)

2.29

48 Y = 2.58X − 2.11 0.84 59.20 ± 6.56 (46.35–72.05)

4.20 186.15 ± 79.06 (31.20–341.10) 3.11

Methanol 24 Y = 3.61X − 7.26 0.42 248.55 ± 60.03 (130.90–366.20)

1.0 578.25 ± 34.08 (511.45–645.05) 1.0

48 Y = 2.99X − 4.97 5.79 215.52 ± 43.05 (131.15–299.90)

1.15 562.72 ± 72.84 (419.95–705.49) 1.03

Petroleum ether 24 Y = 2.98X − 2.79 0.27 41.28 ± 4.58 (32.30–50.26)

6.02 111.16 ± 26.30 (59.62–162.71) 5.20

48 Y = 3.98X − 5.28 0.60 38.48 ± 3.57 (31.48–45.47)

6.46 80.83 ± 10.51 (60.23–101.44) 7.15

Figure 1 Comparative efficacy of cypermethrin and root extracts ofSolanum xanthocarpum against Culex quinquefasciatus larvae for (a)lethal concentration (LC)50 values and (b) LC90 values. �, Petroleumether; �, carbon tetrachloride; , methanol; , cypermethrin.

Solanum

as a synergist for

Culex

Entom

ological Research

36

(2006) 220–225

223

© 2006 The A

uthors. Journal compilation ©

2006 The Entom

ological Society of K

orea and Blackw

ell Publishing A

sia Pty Ltd

Table 2

Combinatorial bioassay of cypermethrin (C) and petroleum ether root extract (E) of


against


larvae

TreatmentRatioC:E

Exposure action

period (h)Regression

equation χ2


(p.p.m.) CTC SF Type of action


(p.p.m.) CTC SF Type of action

Cypermethrin – 24 Y = 3.91 + 2.51X 0.24 0.0027 ± 0.0004 (0.0017–0.0033)

– – – 0.0097 ± 0.0021 (0.0056–0.0138)

– – –

48 Y = 4.81 + 1.73X 0.19 0.0013 ± 0.0004 (0.0005–0.0021)

– – – 0.0092 ± 0.0016 (0.0061–0.0123)

– – –

C with E 1:1 24 Y = 6.10X + 12.67 0.71 0.0055 ± 0.0007 (0.0042–0.0069)

49.09 0.49 Antagonistic 0.0089 ± 0.0027 (0.0036–0.0143)

108.99 S. 1.09 Synergistic

48 Y = 5.40X + 12.01 0.42 0.0050 ± 0.0005 (0.0040–0.0061)

26.00 0.26 Antagonistic 0.0087 ± 0.0026 (0.0036–0.0138)

105.75 1.06 Synergistic

1:2 24 Y = 5.52X + 11.82 0.63 0.0058 ± 0.0009 (0.0040–0.0061)

46.55 0.47 Antagonistic 0.0099 ± 0.0037 (0.0026–0.0172)

97.98 0.98 Antagonistic

48 Y = 6.18X + 12.82 0.88 0.0054 ± 0.0005 (0.0045–0.0063)

24.07 0.24 Antagonistic 0.0088 ± 0.0018 (0.0052–0.0123)

104.55 1.05 Synergistic

1:4 24 Y = 3.81X + 9.49 1.06 0.0066 ± 0.0008 (0.0050–0.0082)

40.91 0.41 Antagonistic 0.0144 ± 0.0051 (0.0044–0.0244)


48 Y = 4.16X + 10.03 1.24 0.0062 ± 0.0007 (0.0049–0.0075)

20.97 0.21 Antagonistic 0.0126 ± 0.0036 (0.0056–0.0196)


CTC, co-toxicity coefficient; SF, synergistic factor.

L. Mohan et al.

224 Entomological Research 36 (2006) 220–225 © 2006 The Authors. Journal compilation © 2006 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

Discussion

Economical and efficient mixtures of toxicants have beenused in pest control over the years. When certain pairs oftoxicants are administered together, the resultant effect maybe greater than, equal to or less than what might be expectedfrom the sum of the activity of the toxicants when administeredseparately. In “synergism”, the joint toxic action of mixturesis significantly greater than that which can be predicted fromthe individual action of the components; conversely, toxic“antagonism” is the toxic action resulting from a mixture oftwo or more components in which the resulting response issignificantly less than the minimum effect predictable fromthe separate action of each component (Sarup et al. 1980).

In the present study, extracts of S. xanthocarpum werefound to act synergistically as well as antagonistically. Thecombination of cypermethrin and the plant extract wasgreatly enhanced at different ratios. However, the larvicidalactivity was minimal when the mixed formulation containedan equal amount of both the constituents (i.e. a 1:1 ratio). The

co-toxicity coefficient and synergistic factor for ratio 1:1 werehigher than for the other ratios of the mixed formulationstested. In this ratio, the plant component decreases the toxicityof cypermethrin earlier but later increases its toxicity againstCx. quinquefasciatus larvae by degrading the detoxifyingenzymes in the mosquito, and thus it exhibits synergism asobserved by Thangam and Kathiresan (1991) in Aedes aegypti.The present study is favourably supported by the finding ofBansal and Singh (2006). Cypermethrin, an approved insectide,is effective for the control of Cx. quinquefasciatus. Furthermore,Thangam and Kathiresan (1991) noticed the synergisticproperties of Rhizophora apiculata, Caulerpa scalpelliformis

Figure 2 Comparative efficacy of cypermethrin and a combinationof cypermethrin and petroleum ether root extract of Solanumxanthocarpum against Culex quinquefasciatus larvae for (a) lethalconcentration (LC)50 values and (b) LC90 values. �, Cypermethrin (C);�, 1:1 C : petroleum ether extract (E); , 1:2 C:E; , 1:4 C:E.

Figure 3 Comparative efficacy of petroleum ether root extract ofSolanum xanthocarpum and a combination of cypermethrin andpetroleum ether extract against Culex quinquefasciatus larvae for(a) lethal concentration (LC)50 values and (b) LC90 values. �, Petroleumether extract (E); �, 1:1 cypermethrin (C) : E; , 1:2 C:E; , 1:4 C:E.

Solanum as a synergist for Culex

Entomological Research 36 (2006) 220–225 225© 2006 The Authors. Journal compilation © 2006 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

and Dictyota dichotoma individually and with DDT. Moreo-ver, the larvicidal potential of Vinca rosea, Leucus aspara,Pedalium murax, Clerodendron inerme, Turnera ulmifoliaand Parthenium hysterophorus in combination withphenthoate and fenthion against Anopheles stephensi hasbeen observed by Kalayanasundaram and Das (1985) andsignificant synergism was noted with fenthion: the synergisticfactor was 1.40, 1.31, 1.61, 1.48, 1.38 and 2.23, respectively.The inferences regarding the larvicidal potential of crudeextracts of S. xanthocarpum suggests its suitability as anecofriendly, effective larvicide in the management of mos-quito populations and in limiting the outbreak of variousvector borne epidemics. In addition, the results concerningthe mixed formulation of the extract and cypermethrin mayprove helpful in finding an effective combination of naturaland synthetic products against mosquito larvae, which maybe an alternative to existing, conventional chemical insecti-cides and provide a solution to combat the ever-increasingproblem of the world’s mosquito population.

Acknowledgments

We are thankful to Professor V. G. Das, the Director of theDayalbagh Educational Institute, Agra, India and ProfessorK. K. Dua, Head, Department of Zoology at the Institute, forencouragement. We are also grateful to the Department ofScience and Technology, Ministry of Science and Technology,Government of India, New Delhi, for financial assistance.

References

Abbot WS (1925) A method of computing of the effectiveness ofan insecticide. Journal of Economic Entomology 8: 265–267.

Bansal SK, Singh KV (2006) Laboratory evaluation for compara-tive insecticidal activity of some synthetic pyrethroids againstvector mosquitoes in arid region. Journal of EnvironmentalBiology 27: 251–255.

Bernard CB, Philogene (1993) Insecticide synergists: role, impor-tance and perspectives. Journal of Toxicology and EnvironmentalHealth 38: 199–223.

Choochote W, Tuetun B, Kanjanapothi D et al. (2004) Potential ofcrude seed extract of celery, Apium graveolens L., against the

mosquito Aedes aegypti (L.) (Diptera: Culicidae). Journal ofVector Ecology 29: 340–346.

Finney DJ (1971) Probit Analysis, 3rd edn. Cambridge UniversityPress, Cambridge.

Jeyabalan D, Arul N, Thangamathi P. (2003) Studies on effects ofPelargonium citrosa leaf extracts on malaria vector, Anophelesstephensi Liston. Bioresources Technology 89: 185–189.

Kalyansundaram M, Das PK (1985) Larvicidal and synergisticactivity of plant extracts for mosquito control. Indian Journal ofMedical Research 82: 19–21.

Markouk M, Bekkouche K, Larhsini M, Bousaid M, Lazrek HB,Jana M (2000) Evaluation of some Moroccan medicinal plantextracts for larvicidal activity. Journal of Ethnopharmacology73: 293–297.

Mohan L, Sharma P, Srivastava CN (2005) Evaluation of Solanumxanthocarpum extracts as mosquito larvicides. Journal of Envi-ronmental Biology 26: 399–401.

Prakash A, Rao J (1997) Botanical Pesticides in Agriculture.Lewis Publishers, Bocaraton, Florida.

Rahman SJ, Sharma SK, Rajagopal R (1989) Manual on Entomo-logical Surveillance of Vector Borne Diseases. NICD, NewDelhi.

Sarup P, Dhingra S, Agarwal KN (1980) Newer dimensions forevaluating the synergistic effect of non-toxic chemicals in themixed formulations against the adults of Cylas formicariusFabricius. Journal of Entomological Research 4: 1–14.

Saxena RC, Jayashree S, Padma S, Dixit OP (1994) Evaluasion ofgrowth disrupting activity of Ageratum conyzoides crudeextract on Culex quinquefasciatus (Diptera: Culicidae). Journalof Environmental Biology 15: 67–74.

Sharma P, Mohan L, Srivastava CN (2004). Larval susceptibilityof Ajuga remota against anopheline and culicine mosquitoes.Southeast Asian Journal of Tropical Medicine and PublicHealth 35: 608–610.

Sharma P, Mohan L, Srivastava CN (2006) Phytoextract-induceddevelopmental deformities in malaria vector. BioresourceTechnology 97: 1599–1604.

Thangam TS, Kathiresan K (1991) Mosquito larvicidal activity ofmarine plant extracts with synthetic insecticides. BotanicaMarina 34: 537–539.

World Health Organization (1975) Instructions for determiningthe susceptibility or resistance of mosquito larvae to insecticides.Mimeographed document WHO/VBC/75: 583.

evaluation of solanum xanthocarpum extract as a synergist for cypermethrin against larvae of the...

Documents