Abstract: Bioinsecticide Bacillus thuringiensis var. kurstaki (Btk) was used for controlling the mosquito species (Anopheles stephensi and Culex quinquefasciatus) which gave a significant (p&lt;0.05) mortality in both species. The higher concentration of Btk was highly effective compared to the control ones. The controlling effect was dose and time dependent. Among the studied mosquitoes the C. quinquefasciatus (LC 50 0.154%) was more susceptible than A. stephensi (LC 50 0.372%) towards the bioinsecticide Btk

A Raja Rajeswari

A Senthil Kumar

M Moorthi

N Ramakrishnan

P Dhanapakiam

V K Ramasamy

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Journal of Environmental Biology May 2009, 30(3) 433-435 (2009)
©Triveni Enterprises, Lucknow (India) For personal use only
Free paper downloaded from: www.jeb.co.in Commercial distribution of this copy is illegal
Efficacy of Bacillus thuringiensis var. Kurstaki in the  control of two mosquito
species (Anopheles stephensi and Culex quinquefasciatus)
A. Senthil Kumar1, M. Moorthi1, N. Ramakrishnan1, P. Dhanapakiam*2, V.K. Ramasamy1 and A. Raja Rajeswari1
1Post Graduate and Research Department of Zoology, Sri Vasavi College, Erode - 638 316, India
2Post Graduate and Research Department of Zoology, J.K.K.College of Arts and Science for Women, Komarapalayam - 638 183, India
(Received: June 23, 2007; Revised received: September 28, 2007; Accepted: November 11, 2007)
Abstract: Bioinsecticide Bacillus thuringiensis var. kurstaki (Btk) was used for controlling the mosquito species (Anopheles stephensi and
Culex quinquefasciatus) which gave a significant (p<0.05) mortality in both species.  The higher concentration of Btk was highly effective
compared to the control ones. The controlling effect was dose and time dependent.  Among the studied mosquitoes the C. quinquefasciatus
(LC
50
  0.154%) was more susceptible than A. stephensi (LC
50
  0.372%) towards the bioinsecticide Btk.
Key words: Bacillus thuringiensis var. kurstaki (Btk), Mosquitoes, Anopheles stephensi, Culex quinquefasciatus
PDF of full length paper is available with author (*divya_vkr@rediffmail.com)
Introduction
Mosquitoes, the dipteran insects acts as pests and vectors of
many dreaded human diseases such as malaria, filaria, dengue/
dengue hemorrhagic fever (DHF) etc.  Among the anophelines,
A. stephensi is an important vector of malaria while among the
culicines, C. quinquefasciatus and Aedes aegypti are the vectors of
filaria and dengue/DHF respectively. Development of resistance to
various types of insecticides such as organochlorides,
organophosphates and carbamates (Singh and Bansal, 2001;
Bansal and Singh, 2002, 2007; Shanmugasundaram et al., 2008)
poses serious threat to the conventional control measures for vectors.
Such insecticides pollute water bodies, air and land. There is every
likelihood of development of pesticide resistant strains of mosquitoes
due to over dependence on chemical toxicants. In recent years,
several pathogenic strains of the Bacillus thuringiensis (Bt) and B.
sphaericus were reported to posses a high level of biological activity
against mosquito larvae in laboratory and field studies (Davidson et
al., 1981; Barjac and Sutherland, 1990).  Highly mosquitocidal strains
of B. sphaericus produce a protein binary toxin which is toxic to
mosquito larvae.  Many western country researchers have reported
biological activity of Bt to control the mosquito larvae both in field and
laboratory as a safer insecticidal alternative (Charles et al., 1996;
Nielsen-Le-Roux et al., 1997; Wirth and Georghiou, 1997; Berber
et al., 2004).  Although this bacterium is highly toxic to mosquito
larvae, but in India very few reports are available on this aspect
(Balaraman and Hoti, 1987; Vasuki et al., 1995).  The present study
was therefore undertaken to evaluate the efficacy of Btk for the
control of mosquito vectors in laboratory.
Materials and Methods
Fully fed female A. stephensi and C. quinquefasciatus were
collected early in the morning from sheep and cattle sheds in Erode
town with the help of mosquito collecting jar. Cages (32x22x21cm)
were used for rearing of mosquitoes, provided with cotton pads
soaked in 10% glucose solution. Enamel tray (15x15x6 cm) filled
with 500 ml water was kept in the cages for laying eggs. The eggs
were maintained in the laboratory at 25±1oC and 70% relative
humidity with a photoperiod of 12 hr.  The newly hatched larvae
were transferred into plastic rearing trays (21x15x9 cm) and fed on
40% brewers yeast and used for the trials.  Pupae were separated
from the trays and maintained separately. The B. thuringiensis var.
kurstaki (Btk) used in this study were purchased from Tamil Nadu
Agricultural Department at Thindal in Erode, TN, India.  Growth and
sporulation conditions were followed as described by Stewart et al.
(1981).
Preparation of sample: 4.42x 1010 spores in 2 milliliter of bacterial
samples 2.2x1010 spores per milliliter from cultures incubated for
48hr were transferred into sterile Eppendorf tubes and kept at -70oC
until use.  Experimental concentrations (2, 4, 6, 8 and 10%) were
prepared from the stock culture of Btk as per method described by
Berber et al.  (2004) for bioassay tests.  Control tests were also
conducted by adding only tap water.
Bioassays: The tests were carried out in the laboratory at a
temperature of 28±1oC and 70% RH.  The tests were repeated four
to five times to investigate variation and average was taken for
deriving inferences.
To each of the beaker containing different test
concentrations, 25 healthy, 4th instar larvae were released.  Percent
mortalities were calculated 8 hr later by counting both dead and
moribund larvae as per instructions given by WHO (1981).  Larvae
were considered moribund if they failed to flex head to siphon when
provoked with a glass rod.
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Kumar et al.
Statistical analysis: The results of larvicidal activity related to
different bacterial concentrations was determined at the 95%
confidence limit (p<0.05).  The LC
50
 and LC
90
 values were computed
using log probit regression analysis (Finney, 1971) and Chi-square
distribution for the expected mortalities (Fisher and Yates, 1963).
Results and Discussion
The results of comparative efficacy of Btk against two species
of mosquitoes (A. stephensi and C. quinquefasciatus) and the
larvicidal activities of five test concentrations of Btk after 8 hr are
given in Table 1.  In the present study, the author observed some
behavioural changes in the Btk treated mosquitoes like generally
ceased feeding, reduced activity, extreme sluggishness, paralysis
after ingestion and finally death.  The observed behavioural changes
were also observed by Chilcott et al. (1990) who have reported that
Bacillus thuringiensis (Bti) treated mosquito larvae generally cease
feeding within 1 hr, show reduced activity by 2 hr, extreme
sluggishness by 4 hr and general paralysis by 6 hr after ingestion.
From the Table 1 it is very clear that all the treated concentrations of
Btk are very effective even at very low concentration against both
mosquito species.  The larvae of C. quinquefasciatus proved more
susceptible than A. stephensi (Table 1).  Comparatively no significant
difference was observed in the larvicidal activities of Btk between the
mosquito species but their larvae development was significantly
controlled as compared to control group.  Burke et al. (1983) reported
that B. sphaericus 1543 lost its spore viability after 4 hr of UV exposure
while its larvicidal activity remained unchanged.  LC
50
 values of Btk
at 10% concentrations
 
were 0.3719; 0.1541% and LC
90
 were 1.9943;
3.5019% for A. stephensi and C. quinquefasciatus respectively
(Table 1) which indicates that at higher concentration of Btk proved
more effective in knocking down the 4th instar larvae.  Complete
susceptibility of many mosquito species towards Bt have also been
observed by several authors (Laird, 1995; Chilcott and Wigley,
1993; Bansal and Singh, 2006).
Lethal toxicity data indicated that C. quinquefasciatus was
more susceptible (LC
50
 : 0.154%) at 10% concentration of Btk
compared to A. stephensi (LC
50
 : 0.372%).  The present study
was confirmed with the work of Ignoffo et al. (1981) who found that
B. thuringiensis israelensis (Bti) was more effective against some
species of lepidopterans.  The LC
50
 for Bti against Trichoplusia ni,
Heliothis zea and H. virescens were 109.6, 19.3 and 27.6 µg ml-1,
respectively.  Corresponding values for Btk were 15.9, 2.0 and 7.8
µg ml-1.
Among the five different concentrations studied in the present
investigation, minimum percentage of Btk are enough to kill the larvae
of C. quinquefasciatus than A. stephensi (Table 1).  It was clearly
showed that among the two species studied the C. quinquefasciatus
larvae are more susceptible with short period (Table 1) than the
A. stephensi.  It is evident from the study of Chen et al. (1984) on
Bt showed that the LC
50s
 for Culex  pipiens pallens, Anopheles
stephensi and Aedes albopictus species were 0.55, 2.05 and
6.37x104 spores ml-1, respectively.
Ali et al. (1981) studied the efficacy of Bti formulation (R-
153-78) and gave LC
90
 values of 4.56-9.84 ppm against the
midges and LC
50
 values of 0.13-0.24 ppm against mosquitoes.
This study clearly indicates that the Bt is more toxic against
mosquitoes than the midges. This study was further supporting
the present work.
In the present study, different percentage mortalities were
observed in the both mosquito species (A. stephensi and
C. quinquefasciatus) were treated against Btk (Table 1) at different
concentrations.  This was due to their mode of action of Btk involves
the synergistic interaction of four toxic proteins and the mosquito
larvae toxicity is due to the crystal protein formed during sporulation
than other known bacterial strains.
The 100% mortalities of mosquitoes in 10% concentration
of Btk  may be due to that Btk products contain the spores and
parasporal crystals of Btk H-3a, 3b serotype which must be ingested
by the larval stage of mosquito to cause mortality. Following
ingestion, the parasporal crystals are solubilised in the alkaline
larval midgut, followed by proteolytic activation of the soluble
insecticidal crystal proteins. The toxin binds to a receptor on the
midgut cell wall resulting in pore formation in the cell, which leads to
death of the larva.
Table - 1: The toxicological status and log probit analysis of the mortality of Bacillus thuringiensis var. kurstaki to the larvae of two species
Mosquito Concentra- No. of mortality Percent Percent Chi- LC
50
LC
90
species tions (%)  during the end experimental corrected Regression equation square (%)
 
(%)
of 8 hr  mortality  mortality
Anopheles 2 5 42 36.75 5.506+2.588(3.3010-3.595)
stephensi 4 8 67 64.01 5.506+2.588(3.6021-3.595)
6 10 83 81.46 5.506+2.588(3.7782-3.595) 0.1301 0.3719 1.9943
8 11 92 91.28 5.506+2.588(3.9031-3.595)
10 12 100 100 - -
Culex 2 7 58 54.20 5.718+1.861(3.3010-3.599)
quinquefasciatus 4 9 75 72.74 5.718+1.861(3.6021-3.599)
6 10 83 81.46 5.718+1.861(3.7782-3.599) 0.1369 0.1541 3.5019
8 11 92 91.28 5.718+1.861(3.9031-3.599)
10 12 100 100 - -
434
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Acknowledgments
The authors are thankful to the Principal, Sri Vasavi College,
Erode for providing the facilities and necessary equipments to carry
out this project work.
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Efficacy of Bacillus thuringiensis var. Kurstaki in the control of two mosquito species (Anopheles stephensi and Culex quinquefasciatus)

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Efficacy of Bacillus thuringiensis var. Kurstaki in the control of two mosquito species (Anopheles stephensi and Culex quinquefasciatus)

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