Mosquitocidal activity of a native Bacil

European Review for Medical and Pharmacological Sciences

2011; 15: 149-155

Mosquitocidal activity of a native Bacillus
thuringiensis isolate Bt ReX02 from
Gunung Jerai Forest, Malaysia against
Culex quinquefasciatus and Aedes albopictus
A. PRAZANNA LAKXMY, R. XAVIER, C.M. REENA JOSEPHINE, Y.W. LEE*,
K. MARIMUTHU, S. KATHIRESAN, S. SREERAMANAN**
Department of Biotechnology, Faculty of Applied Sciences, Aimst University, Semeling, Kedah
Darul Aman (Malaysia)
*Coopers Lifesci Sdn-Bhd-WellTech Health care Group, Kuala Lumpur (Malaysia)
**School of Biological sciences, Universiti Sains Malaysia (USM), Penang (Malaysia)

Abstract. – Background and Objective:
To evaluate the mosquito larvicidal potential of
the native Bacillus thuringiensis isolate
BtReX02, which was isolated from a tropical rain
forest ecosystem in Malaysia. This study also
aimed at determining the phenotypic and biochemical characteristics of the isolate.

Materials and methods: The phenotypic characterization was carried out by growing the isolate in nutrient broth to observe the colonial
morphology, vegetative cells, sporulation, motility and haemolytic activity. The parasporal crystal morphology was determined by Coomassie
Brilliant Blue (CBB) staining of the sporulated
cells and then observed under light microscope.
The mosquito larvicidal assay was conducted
with the second instar larvae of Culex quinquefasciatus and Aedes albopictus to determine the
entomocidal potency of the isolate. The total
protein profile was determined by SDS-PAGE.
Results and Discussion: Preliminary phenotypic and biochemical characterization showed
that the isolate is motile, an indirect indication of
the virulence of the strain and exhibited hemolytic activity, an important feature of antidipteran Bacillus thuringiensis strains. Study
on the crystal morphology showed the presence
of cuboidal crystals, another characteristic feature of a mosquitocidal Bacillus thuringiensis
strains. Bioassay with the second instar larvae
of Culex quinquefasciatus, Aedes albopictus established the antidipteran activity of this native
Bacillus thuringiensis isolate. Protein profile
analysis revealed the unique pattern showing
high molecular mass as well as low molecular
mass proteins corresponding to the Cry and Cyt
proteins respectively. The protein profile is strikingly different from other mosquitocidal strains

such as Bacillus thuringiensis subsp.israelensis
and Bacillus thuringiensis subsp. jegathesan
Periodical introduction of such native strains
of Bacillus thuringiensis will add a new weapon
in the armoury to manage the vector borne diseases and also in the management of insect resistance.
Key Words:
Bacillus thuringiensis, Larvicidal activity, Culex quinquefasciantus, Aedes albopictus.

Introduction
Mosquito vectors continue to be a serious public health problem world over. The most important among are the genus Anopheles, Culex and
Aedes, which are responsible for transmitting the
pathogens of malaria, filariasis, dengue and
chikungunya. Mosquito control by application of
synthetic insecticides posed many serious problems including rapid development of insecticide
resistance among the mosquitoes. Currently, an
integrated vector control strategy has been advocated with major focus on utilization of biocontrol agents such as Bacillus thuringiensis (Bt) Bt
is a Gram-positive soil bacterium characterized
by its ability to produce proteinacious crystalline
inclusions during sporulation. These crystalline

inclusions also known as insecticidal crystal pro-

Corresponding Author: Rathinam Xavier, Ph.D; e-mail: rxavier77@yahoo.com

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A. Prazanna Lakxmy, R. Xavier, C.M. Reena Josephine, Y.W. Lee, K. Marimuthu, et al

teins (ICP), along with the spores, are widely
used as a biocontrol agent in agriculture, forestry
and public health to control the insect vectors1.
Cry proteins have been classified into classes and
subclasses according to their amino acid similarity2. These proteins are active against several orders of insects: lepidopteran specific (cry 1), lepidopteran and dipteran-specific (Cry2),
coleopteran-specific (Cry 3), and dipteran-specific (Cry4) proteins3. The cry2 genes encode 65
kDa proteins which occur in cuboidal
inclusions4. Cry2Aa protein is toxic to both lepidopteran and dipteran larvae, whereas Cry2Ab,
Cry2Ac and Cry2Ad are only toxic to lepidopteran insects1. In recent times several mosquitocidal
B. thuringiensis were isolated in different parts
of the world.
The objectives of the studies were to determine phenotypic and biochemical identity and its

mosquitocidal potency of the native Bt isolate
BtReX02 from forest ecosystem.

Materials and Methods
Bacterial Strains
B. thuringiensis isolate BtReX02 was obtained
from Molecular Microbiology Laboratory,
AIMST University, Malaysia, which was previously isolated from a forest (Gunung Jerai)
ecosystem in Malaysia. B. thuringiensis var israelensis was obtained from Institute for Medical
Research, Kuala Lumpur, Malaysia.
Phenotypic Characterization
Morphological Features
The Bt isolate Bt ReX02 was inoculated in 20
ml of nutrient broth. The colonial morphology
was studied by plating a 20 µl culture drawn after
48 h, onto nutrient agar and incubated at 32˚C for
36 h. The colonies appeared were studied for
colonial morphology and vegetative cell morphology.
Characterization of Parasporal Inclusions
The Bt isolate BtReX02 was grown in nutrient

broth at 30˚C for 72-96 h or until more than 90%
of the cells had lysed, releasing spores and crystals. Spore-crystal mixtures were washed (10,000
g for 10 min) once in 1.0 M NaCl and then twice
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in cold sterile water and resuspended in 1.0 M
NaCl. A straight inoculating wire was used to
transfer an aliquot of the spore-crystal suspension on to a microscopic slide. The slide was
then heat fixed and stained with Coomassie Brilliant Blue dye (0.133% Coomassie Brilliant Blue
(CBB) stain in 50% acetic acid), rinsed with distilled water, dried and observed under light microscope using 100x oil immersion objective5.
The presence of parasporal bodies were clearly
observed as dark-blue staining objects.
Motility Test
Motility of Bt isolate was determined by the
growth pattern on nutrient agar plates. The isolates were streak-inoculated onto the middle of
the agar plate from top to bottom and incubated
overnight at 30˚C. If a colony was to spread out
from the inoculation site, the strain was scored as
motile; otherwise it was scored as non-motile6.
Haemolytic Assay

The haemolytic activity of Bt isolates was tested against human erythrocytes. The blood agar
was prepared by gently mixing 5ml of blood with
100 ml of nutrient agar. The prepared blood agar
was then poured onto Petri plates and allowed to
solidify. Once the blood agar has completely solidified, the Bt isolate from nutrient broth culture
were streak inoculated and incubated at 37ºC.
The haemolytic activity was visually examined
by denoting the clear zone formed after 24 h.
Protein Electrophoresis by SDS-PAGE
The Bt isolate was grown in nutrient broth at
30˚C at 200 rpm for 72 to 96h or until sporulation and 90% of autolysis of the cells. The culture was harvested by centrifuging at 10000g at
4ºC (Beckman Coulter, Avanti J-25, Straits Scientific, Ampang, Selongor, Malaysia) for 15 min.
The spore-crystal suspension was washed 3 times
with 1M NaCl solution to remove proteases and
was harvested by centrifugation. Protein concentration in the cell extracts was determined by the
protein assay method of Bradford7. The total protein profile of spore-crystal mixture was determined by SDS-PAGE analysis, as described by
Laemmli8 using 10% acryl amide separating gels.
Twenty five µl (75 µg/ml) of the total cell extract
was mixed with 25 µl of sample buffer (50 mM
Tris HCl (pH 6.8), 100 mM dithiothreitol, 2%

w/v sodium dodecyl sulphate (SDS), 0.1% bromophenol blue and 10% v/v glycerol) and boiled
at 80ºC for 10 min.

Mosquitocidal activity of a native Bacillus thuringiensis isolate Bt ReX02 from Gunung Jerai Forest

The electrophoresis was performed at room
temperature at 30 mA for 2 h. The gel was
stained in a solution containing 50 % (v/v)
ethanol, 10 % acetic acid, and 0.1 % (wt/vol)
Coomassie Brilliant Blue R250 for 60 min and
destained with a solution containing 6.75 %
(v/v) glacial acetic acid and 9.45 % (v/v)
ethanol. The masses of the proteins were determined by comparison with protein standard.
Mosquito-Larvicidal Assay
Second-instar larvae of Culex quinquefasciatus and Aedes albopictus were used for mosquito-larvicidal assays. Bt isolate BtReX02 was
grown in nutrient broth at 30ºC for 4 days or until 90% lysis of the cells. Bioassay to test the toxicity of the spore-crystal mixture of the isolate
was conducted at 25ºC in 60-70% humidity with
a 16 h light and 8 h dark cycle. All the tests were
performed in triplicate.
Ten second instar mosquito larvae were placed

in 25 ml of beaker with 10 ml of seasoned water
with known concentrations of spore crystal mixture. Distilled water was used as negative control. Totally 11 concentrations were used such as
150 µg/ml, 170 µg/ml, 190 µg/ml, 210 µg/ml,
230 µg/ml, 250 µg/ml, 270 µg/ml, 290 µg/ml,
310 µg/ml, 330 µg/ml and 350 µg/ml. The larval
mortality were recorded for every 12 h for 2
days, the number of dead larvae in each beaker
were counted. Then the LC50 was calculated for
24 h and 48 h observation for each species using
probit analysis.
Statistical Analysis
Statistical evaluation was carried out to determine the LC50 by probit analysis with Statistical
Package of Social Science (SPSS, Chicago, IL,
USA) 11.0 for windows software.

Results
The morphological studies of the native Bt
isolate BtReX02, exhibiting the typical colonial
morphology as that of the wild type Bt. The fully developed colonies were round, white, with
regular margins 9. Examination of the culture

grown in nutrient broth after 36 h under light
microscope confirmed the morphological appearance of thin slender rods in short chains.
Further examination of the culture after 72 h indicated the presence of spores.Then the sporulat-

ed culture of the native Bt strain BtReX02 was
subjected to Coomassie Brilliant Blue (CBB)
staining. The phase-contrast microscopic studies
on the crystal protein revealed that the inclusions are cuboidal in shape (Figure 1), a characteristic feature of cry 2 genes4.
The motility studies indicated that BtReX02 is
motile (Figure 2) and exhibit haemolytic activity
against human erythrocytes (Figure 3).
The larvicidal bioassay against second instar
larvae of Culex quinquefasciatus and Aedes albopictus showed that BtReX02 exhibited a striking larvicidal activity (Table I). It is understood
from the results that there is a higher larvicidal
potency of the Bt isolate toward Aedes albopictus as compared to of Culex quinquefasciatus
(Figure 4).
The protein profile study showed that the isolate has a major protein of 101.77 kDa and 94.11
kDa, in addition to other minor peptides including 64.76 kDa, 32.68 kDa and 17.44 kDa. The
protein profile of BtReX02 is strikingly different
from the reference strain B. thuringiensis var. israelensis (Figure 5).

Discussion
In spite of great improvements in health care
system globally, mosquito-borne diseases including malaria, filariasis, dengue and the viral encephalitides pose a major health threat world
over. For example, malaria still remains a major
public health problem, responsible for the death
of one million people each year and threatening
more than three billion people in 107 countries10.
Globally concerted efforts are directed toward
identifying new agents with novel activities to
manage the vector-borne diseases. Hence, Bt has

Table I. Toxicity of BtReX02 against Culex quinquefasciatus and Aedes albopictus second instar larvae.

Mosquito species

LC50 (µg/mL) LC50 (µg/mL)
at 24 hours at 48 hours

Culex quinquefasciatus

797.848

268.863

Aedes albopictus

398.006

208.996

LC50: 50% lethal concentration (in µg) of wet spore–crystal
complex per milliliter after 24 and 48 hours. The data are
the total of three assays as determined by Probit analysis.

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A. Prazanna Lakxmy, R. Xavier, C.M. Reena Josephine, Y.W. Lee, K. Marimuthu, et al

Figure 1. A, Colonial morphology of BtReX02. B, Phase-contrast microscopy of vegetative and sporulating cells (magnification x 1000). C, Light microscope photograph of BtReX02 spores and crystal (magnification x 1000).

received considerable attention among the research community for its insecticidal properties
against a range of insect pests and vectors of human diseases. Insecticidal preparation based upon various strains of Bt currently account for 8090 % of all biological pest control agents sold

worldwide, making it the most successful type of
bioinsecticide11. Currently VectoBac and Teknar,
which are based on B. thuringiensis subsp. israelensis, and VectoLex, a product based on B.
sphaericus, has been widely used to combat the
mosquito vectors12.

Figure 2. Experiment to show the motility of BtReX02.

Figure 3. Haemolytic activity of BtReX02.

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Mosquitocidal activity of a native Bacillus thuringiensis isolate Bt ReX02 from Gunung Jerai Forest

Figure 4. Larvicidal potency of BtReX02 against Culex quinquefasciatus and Aedes albopictus at 24 hours and 48 hours.

Natural Bt strains are periodically isolated
worldwide for novel activities to control insect
pests especially vectors of human diseases. Bt is
widely distributed in natural environments. However, it is the most abundant in soil13. In the present study a Bt isolate BtReX02 was isolated
from the forest land soil. B. thuringiensis subsp.
israelensis was the first mosquitocidal Bt isolated from Israel14. From then onward a number of
Bt isolates which are active against different
species of mosquitoes have been isolated from
different environments.

Figure 5. SDS-PAGE profile of BtReX02. M: Molecular
weight standards; Lane 1: B. thuringiensis subsp.israelensis;
Lane 2: BtReX02.

Comprehensive analysis of the morphological,
biochemical and mosquito larvicidal toxicity of
BtReX02 was performed. Experimental results
indicated that the isolate haemolytic, motile with
spherical crystal protein and exhibited antidipteran activity.
Generally, Bt is a motile bacterium. However,
non-motile Bt had also been reported 15. The
motility of Bt is an indirect indicator of virulence
and biological activity of the Bt strains. Experiments showed that a transposon mediated mutant
Bt strain that lacked flagella, was defective in its
ability to swarm as well as in the secretion of
both hemolysins and phosphatidylcholin-preferring phospholipase (PC-PLC)16. The transposon
insertion was localized in the flh A gene, a flagellar class II gene involved in the type III export of
flagellar components. The FlhA flagellar basal
body protein is also required in flagellum assembly and swarm cell differentiation. Further the flhA mutant showed decreased virulence against
Galleria mellonella larvae. Thus, FlhA appears
to be an essential virulence factor 17. The motility
study also forms the basis for sero typing with
flagellar antigens.
The light microscopic study of CBB stained Bt
isolate showed the presence of cuboidal crystals.
The CBB staining is an improved method, has
two major advantages over convention use of
phase contrast microscopy. The first, unlike
phase-contrast microscopy, very small parasporal
bodies were readily evident. Second, the presence of stained crystalline bodies are striking and
instantaneously recognizable, much more so than
phase-contrast microscopy5. Presence of cuboidal
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A. Prazanna Lakxmy, R. Xavier, C.M. Reena Josephine, Y.W. Lee, K. Marimuthu, et al

crystal is a characteristic feature of cry2 genes4,
thus indicating the presence of cry2 related genes
in this Bt isolate. Cry 2Aa protein is toxic to both
lepidopteran and dipteran larvae, whereas
Cry2Ab, Cry2Ac and Cry2Ad are toxic only to
lepidopteran insects1. This could be attributed to
BtReX02 harbouring cry2A genes as indicated by
its mosquitocidal activity.
Mosquitocidal Bt species produce an array of
insecticidal crystal proteins (ICP). These crystals
are predominantly comprised of one or more
crystal (Cry) and Cytolytic (Cyt) toxins. Cyt proteins are parasporal inclusion proteins from Bt
exhibits hemolytic activity. For example B.
thuringiensis subsp. israelensis produces four
proteins ranging from 135-27 kDa18; in the subsp. kyushuensis proteins ranging from 140 to 25
kDa19; in subsp. fukuokaensis proteins of 90 to
27 kDa 20; and in subsp. medellin proteins of 100
to 30 kDa were reported21 . In all these isolates a
cytolytic toxin, generally 25 to 30 kDa, is also
produced22. The protein profile of BtReX02 also
showed the presence of a protein of 32.68 kDa
size, indicating the possible presence of cytolytic
toxin. Insecticidal crystal proteins from B.
thuringiensis subsp. jegathesan contain seven
major polypeptides, with molecular masses of
80, 70-72, 65, 37, 26 and 16 kDa which are not
related to those produced in B. thuringiensis subsp. israelensis. From the protein profile of
BtReX02, it is evident that this isolate produces a
complex mixture of proteins; such an array of
proteins may be beneficial in delaying the onset
of insect resistance23.
The bioassay results indicated that the native
Bt isolate is comparatively more effective against
Aedes albopictus than Culex quinquefasciatus. In
both the species maximum mortality occurred
within 24 hours at a toxin concentration of 398
µg/ml for Aedes albopictus and 797.85 µg/ml for
Culex quinquefasciatus, respectively. In terms of
potency, which inverse of LC50 indicated the elevated mosquito larvicidal potency of BTReX02
against Aedes albopictus. Further larvicidal assays with other medically important mosquito
vector will provide a good insight on the host
range of this native Bt isolate. Thus, a detailed
study on the molecular aspects including the cry
and cyt gene profile will reveal the full entomocidal potential of this isolate.
In summary, for the first time we have isolated a native strain of Bacillus thuringiensis,
BTReX02 from a forest ecosystem and demonstrated the mosquito larvicidal efficacy against
154

Culex quinquefasciatus and Aedes albopictus.
Such new a isolate can be best utilized in combating the vectors in an effective manner. Further the commercialization of this isolate may
play a major role in the integrated vector control programs and to manage the insect resistance.

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––––––––––––––––––––
Acknowledgements

The Authors are grateful to Mr. Patchamuthu and Vector Control Unit, School of Biological Sciences, University Sains Malaysia (USM), Penang, Malaysia, for
the facilities provided.

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