Indian Journal of Biotechnology

Vol 2, April 2003, pp 268-270

Short Communications

Characterization of Mosquito Larvicidal Bacillus thuringiensis Isolated from Soils

of India
Banani Sur!", Nitu Nigam', A K Joshil and Vinod Biharil
IFermentation Technology Division,
Central Drug Research Institute, Lucknow 226 001, India
2Medical Genetics, Sanjay Gandhi Post Graduate Institute,
Lucknow 226 014, India
Received 4 February 2002; accepted 14 August 2002

It has been established now that insects are acquiring resistance to commercial products of Bacillus thuringiensis. So
there is a continuous search for bacteria producing new toxins. With this in view, an insecticidal B. thuringiensis was isolated from soil sample obtained from the vicinity of a penicillin factory in Vadodara, India. The isolate belongs to serotype
H-14. Phenotypic characters of the isolate were identical to
standard B. thuringiensis var. israelensis IPS-82. It was found
to possess good larvicidal activity against Anopheles stephensi
and Culex pipiens and exhibited high resistance for penicillin
(500 ug/ml), The electrophoretic protein profiles of purified
crystals with standard B. thuringiensis (IPS-82) were studied.
The isolate apparently showed the same protein profile as that
of B. thuringiensis var. israelensis. Soil with natural selective
pressure of antibiotic penicillin thus appears to be a good
ecological niche for the isolation of B. thuringiensis.
Keywords:

Bacillus thuringiensis, bioinsecticide, ecological

niche, parasporal

inclusion, serotype

The quest for non-hazardous

environmentally
compatible pest control measures has spurred the interest in Bacillus thuringiensis, which is now recognized as one of the most promising bioinsecticides. B.
thuringiensis is a spore forming bacteria, which produces insecticidal crystal proteins toxic to insect-pests
belonging to Lepidoptera, Diptera and Coleoptera. A
potent strain belonging to variety israelensis and active against mosquitoes and black fly larvae was discovered accidentally by Goldberg and Margalit in
1977 from the soil of mosquito breeding site in Israel.
Thereafter, many workers have reported the occurrence of highly mosquitocidal B. thuringiensis strains
belonging to serovars israelensis (Balaraman et ai,

* Author for correspondence:

Tel: 0522-2212411-18 ext 4356; Fax: 0522-2223405
E-mail: info@cdriindia.org.

1981; Abdel-Hameed et ai, 1990); kyushuensis (Ohba

& Aizawa, 1979) thompsoni and canadensis, respectively (Ragni et al, 1996). Soil is the natural reservoir
for insecticidal B. thuringiensis with moderate to low
toxicities (Ishii & Ohba, 1993; Ohba & Aizawa,
1979).
Because of possible development of resistance in
insects to the commercialized products based on
B. thuringiensis, there is a continuous search for bacteria producing new toxins. The present work aimed
to screen novel insecticidal isolate from soil reports
the isolation of an insecticidal isolate of B. thuringiensis.
Soil samples (4) were collected from grasslands,
orchards and urban lands in Lucknow and one soil
sample originated from a damp shadowed place near
the penicillin factory in Vadodara, Iridia. The samples
were collected by the removal of surface soil with a
sterile spatula and then collections were made from a
depth 2-5 ern below the surface and processed within
one week. Sample areas were devoid of spraying with
any commercial formulation of B. thuringiensis.
One gram of soil sample was placed in a tube (6x
W') containing 9 ml of sterilized distilled water and
was vortexed for 5 min and heated at 60C for 10 min
to allow only sporulating bacteria to survive. Finally
100 ul of the same was plated in starch nitrate medium (soluble starch, 2; yeast extract, 0.4; KN03. 0.2;
and agar, 2%; pH 7.0) and observed for 72 hrs for the
appearance of bacterial colonies. Colonies similar in
morphology of B. thuringiensis were selected and
streaked in Luria agar (HIMEDIA) plate fortified with
penicillin (0.005%) and were examined under a phase
contrast microscope for the presence of parasporal
inclusion bodies and spores.
All the isolates were grown in 10 ml of Luria broth
(HIMEDIA) in (6XY2") tube for 48 hrs with shaking
until sporulation. Qualitative bioassays were carried
on 3'd instar larvae of Anopheles stephensi using 25
numbers of larvae suspended in 50 ml of distilled
water in a beaker containing 100 ul of bacterial
growth.
Quantitative toxicity tests were performed only
with insecticidal isolate confirmed earlier by qualitative bioassays. Quantitative bioassay of 48 hrs-old
active culture was done by serial dilution. Six-fold

SUR et a/: ISOLATION OF B. THURINGIENSIS

serial dilution of the active culture was made in distilled water. Twenty 3rd instar larvae of either A.
stephensi or 4th instar larvae of Culex pipiens were
introduced at 10 ml of each dilution in beakers and
kept at 28C. The 24-hour mortalities were scored and
the LCso values were determined and compared with
LCso values of standard B. thuringiensis IPS-82. Toxicity was expressed in International Units (IU) using
the formula proposed by Lacey (1984).
LCso IPS 82, .ul ml'
--'--'-------xI5000=ICmr
LCso of test material

Insecticidal isolate (B-5) was sent to Pasteur Institute of Paris for serological confirmation and preservation.
A loopful of sporulated culture grown on Luria
agar (HIMEDIA) was subcultured in 200 ml of nutrient broth (HIMEDIA) and shaken at 250 rpm for 5
days at 30C to ensure sporulation and complete
autolysis. The spore crystal mixture was thoroughly
shaken in 1M NaCI containing 0.01 % Triton X-IOO
and sedimented by centrifugation at 12,000 g for 10
min. Sedimented crystals were then solubilised at
Table I-Standard

identification card of strain 8-5

No. 8-5 Soil

Origin: India, 1997

60C for 3 hrs in 50 mM NaOH and 10 mM EDTA.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method
of Laemmli (1970) by using 10% running and 4%
stacking gels. Standard strain of B. thuringiensis var
israelensis (IPS-82) was used for comparison of protein profile.
A total of 50 isolates recovered from five soil samples were analyzed for mosquito larvicidal assay
against Anopheles stephensi. Only one isolate (B-5)
originated from the vicinity of penicillin factory
proved to possess mosquitocidal activity. Organism
had the typical morphology of B. thuringiensis strain,
centrally occupied elliptical spore in non swollen sporangia and measured 1.25 x 5-7.5 us. International
Unit (IU) was determined by quantitative bioassay
using IPS-82 as standard. The isolate was found to
possess 8,000 IV against Anopheles stephensi and
10,000 IU against Culex pipiens. It was highly resistant to penicillin and sugar arabinose, xylose and
mannitol were not fermented to produce acid (Table 1), which is one of the confirmatory test for B.
thuringiensis (Buchanan & Gibbons, 1975). Acid
production from salicin represents strain variation (de
Barjac & Bonnefoi, 1968).
During the present survey, recovery of B. thurstrain with mosquitocidal activity is 2% (l
in 50) probably due to use of selective isolation medium. B. thuringiensis is known for hydrolyzing
starch and reducing nitrate salts to nitrite. So the use
of starch nitrate medium preferentially selects B.
thuringiensis culture to grow. In the present survey
insecticidal assay did not include coleoptera and lepidoptera. Probably incidence of insecticidal B. thuringiensis could be more from Indian soil provided
coleoptera and lepidoptera larvae were also included
in this study. It would be apparent that non-toxic isolates were more frequent in soil. Earlier researchers
also reported non-toxic isolates from soil of Japan
belonging to serotype 14 (serovar israelensisi (Kim et
al, 1988; Ohba et al, 1988). In B. thuringiensis var
israelensis (H-14) insecticidal toxicity is attributed to
72 mDa transmissible plasmid (Gonzalez & Carlton,
1984). Possibly in soil due to adverse environmental
condition loss of plasmid is more frequent resulting in
dominance of non-toxic isolates in the soil. On the
other hand, it was observed that the soil with natural
selection pressure of penicillin is more fertile for B.
thuringiensis
probably due to advantageous environment.
ingiensis

Spores: elliptical, in

Gram: +

non-swollen sporangia

Size: (5-7.5) x (1.25-2) urn

Parasporal inclusion bodies: +

Motility: +

Nutrient agar: Smooth

Acid from:
Adonitol: -

Methyl red: +

Inositol: -

Nitrate reduction: +

Xylose: -

Starch hydrolysis: +

Mannitol: -

Egg-yolk lecithinase: +

Arabinose: -

Esterase: +

Dextrose: +
Lactose: -

Alkaline phosphatase: +

Salicin: +

Indoline (in urea-indole): -

Trehalose: +

AMC (Voges-Proskauer):

Sucrose: -

MIC to penicillin: 500 ug/rnl

Endotoxin:

Agglutination: H-14

+ (Anopheles stephensi, Titre: 10,000 (IU)

Culex pipiens)

IC pipiens
8000 (IU)
IA. stephensi

Exotoxin: -ve
AMC, Acethyl-methyl-carbinol.
centration

MIC, Minimum

inhibitory con-

269

INDIAN J BIOTECHNOL,

270
a

65-KDa

APRIL 2003

also provided evidence that B. thuringiensis strains

with low to moderate mosquitocidal activities are
common in natural environments as reported earlier
by other researchers (Ohba & Aizawa, 1979; Ishii &
Ohba, 1993).
References

29-KDa
19-KDa .
_~_

Fig. I-Protein profile of Bacillus thuringiensis alkali-solubilized

toxins on SDS-PAGE: lanes a SIGMA MW-SDS-70L standard
protein b, B-5, C, IPS-82.

Purified parasporal inclusion of the type strain of

serovar israelensis (IPS-82) and the isolated B-5
strain were examined for their protein component by
SDS-PAGE (Fig. 1). Parasporal inclusion of the
strains consisted of highly heterogenous multiple
protein components with molecular masses ranging
from 14 to 65 kDa in B-5 and IPS-82. In both the
strains, 29 kDa and 65 kDa protein were the major
protein component as evidenced by SDS-PAGE
(Fig. 1). A number of other minor protein bands lesser
than 29 kDa were more prominent in isolate B-5.
At present, little information is available regarding
the actual mosquitocidal toxin in the isolate B-5
strain. However, it is likely that 65 kDa protein is
primarily responsible for mosquitocidal activity because the protein of this class is mostly insecticidal in
strains of B. thuringiensis var israelensis (H-14) (Aronson et al, 1986).
It is well accepted that 25 to 29 kDa proteins having mosquito specific activity in B. thuringiensis var.
israelensis are cytolytic for vertebrate and insect cells
(Thomas & Ellar, 1983; Knowles et ai, 1992). In the
present study, protein of the size, 29 kDa is also detected in B-5 isolate. However, 29 kDa protein of the
isolate, whether haemolytic or not will be the subject
of future work. Further peR analysis will foretell the
crystal type gene in the isolate.
The study concludes that the soil with natural selective pressure of antibiotic like soil near the antibiotic producing factory or medical colleges may prove
a good ecological niche for the organism, as B. thuringiensis is naturally resistant for penicillin. It has

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