This is my phd work which were done on Vector Control Research Centre, Pondicherry.

1. Contents lists available at ScienceDirect
Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica
WDP formulations using a novel mosquitocidal bacteria, Bacillus thuringiensis
subsp. israelensis/tochigiensis (VCRC B-474) – Development and storage
stability
Kannadasan Shankar, Gnanasundaram Prabakaran, Arulsamy M. Manonmani
⁎
Vector Control Research Centre (Indian Council of Medical Research), Indira Nagar, Pondicherry, 605006, India
A R T I C L E I N F O
Keywords:
Bacillus thuringiensis subsp. israelensis/
tochigiensis
SEM
SDS-PAGE
WDP formulations
Mosquitoidal activity
A B S T R A C T
A novel mosquito active strain, Bacillus thuringiensis (VCRC B474) sharing the antigens of 2 serotypes, namely
israelensis & tochigiensis was characterized by scanning electron microscopy and SDS-PAGE. The spherical and
ovoid crystals present in this strain was composed of major polypeptides the size of 28, 65, and 130 kDa re-
spectively. The sporulated cell mass was formulated into water dispersible powder (WDP) formulations with
different carrier materials and checked for activity against Culex quinquefasciatus larvae at monthly intervals for
up to a year. The formulation containing chalk was the most effective with LC50 values ranging between
0.274–0.523 μg/ml compared to the formulations containing bentonite (0.335-0.775) μg/ml and talc (0.348-
0.808 μg/ml). The decline in the activity of these formulations with storage period was as follows: 3 months
−14%, 22%, 20% respectively, 6 months - 25%, 35%, 37% respectively, 9 months − 39%, 50%, 47% re-
spectively and 12 months −52%, 43%, 40% respectively. This study demonstrated that wet biomass of bacterial
isolates could be simply mixed with carrier materials, dried and used for mosquito larval control without sig-
nificant loss of activity for up to 6 months at room temperature. Further, this strain of Bacillus thuringiensis var.
israelensis/tochigiensis (H14/19) can be a prospective candidate for use in mosquito control programs.
1. Introduction
More than one million people die every year due to vector borne
diseases including malaria, dengue, schistosomiasis, leishmaniasis,
Chagas disease, yellow fever, lymphatic filariasis and onchocerciasis,
globally (WHO Factsheet, 2014). It is a widely accepted fact that insect
control programmes should combine cost effectiveness, acceptable level
of population suppression and an environmentally friendly approach.
By using microbial agents, cost of control operations is reduced, but
also several ecological benefits are construed - accumulation of un-
wanted chemicals in the ecosystems is prevented, development of re-
sistance to pesticides is avoided and safety to non-target organisms and
to human population is ensured. Bacillus thuringiensis (Bt) is character-
ized by its ability to produce crystalline inclusions during sporulation
which also distinguishes it from other closely related bacterial species
namely B. cereus and B. anthracis. The crystalline inclusion bodies
which are made up of Cry and Cyt proteins, exhibit specific insecticidal
activity against different insect orders, including Lepidoptera, Diptera,
Coleoptera, Hymenoptera, Homoptera, Mallophaga, and Acari (Zhang
et al., 2012). The cry and cyt proteins of Bacillus thuringiensis var.
israelensis (Bti) are specific to larvae of noxious arthropod species order
Diptera, including mosquitoes, black flies and chironomid midges, but
do not affect the suborder Brachycera (Ben-Dov, 2014). This is the only
microbial agent being widely employed in mosquito control programs.
The different strains within each subspecies/serotype exhibit different
levels of toxicity against the same insect species and hence termed as
very high, high, moderate, poor and Non-toxic (Manonmani and
Balaraman, 2001). Subspecies like kurstaki (H3a:3b:3c), israelensis/to-
chigiensis (H14/H19) have been found to exhibit dual toxicity to mos-
quitoes and Spodoptera species (Balaraman, 2005). Though several
types of microbial formulations have been developed, water dispersible
powders are the most suited for Indian conditions. This necessitates
storage of bio-pesticides at tropical temperatures for long-periods.
Hence, formulations need to be designed such that the virulence of the
bacterial toxin is maintained on long-term storage. Further, the cost of
Bti along with the frequent treatment is relatively high per unit of
treated area where as chemical agents like methoprene are much
cheaper to deploy. If this is replaced with indigenously developed
products, the cost of mosquito control is likely to come down. In view of
this, we developed water dispersible powder (WDP) formulations using
https://doi.org/10.1016/j.actatropica.2018.12.023
Received 30 January 2017; Received in revised form 14 December 2018; Accepted 14 December 2018
⁎
Corresponding author.
E-mail address: manonmanivcrc@gmail.com (A.M. Manonmani).
Acta Tropica 193 (2019) 158–162
Available online 16 December 2018
0001-706X/ © 2018 Published by Elsevier B.V.
T

2. an indigenously isolated, novel mosquitocidal strain of B. thuringiensis
(VCRC B-474) sharing the antigens of two serotypes israelensis/tochi-
giensis (B.t.i/t) and cost effective carrier materials namely chalk, ben-
tonite, and talc. The mosquito larvicidal activity of these formulations
on storage at room temperature (RT) is reported in this study.
2. Materials and methods
2.1. Bacterial strain used
An indigenous isolate of B. thuringiensis subsp. israelensis/tochigiensis
(H14/H19) (VCRC B474) obtained from mangrove forests of Andaman
and Nicobar island of India was used in this study (Geetha et al., 2007).
2.2. Serological identification
Serotyping of the isolate was done as per the procedure of de Barjac
and Bonnefoi (1962). Antigen in the form of motile bacteria was de-
veloped by successive passages of the bacterium in soft Nutrient (0.2%)
agar. The motile cells were grown in nutrient broth for 5–8 h, for-
malized to a final concentration of 0.5%, stored overnight at 4 °C and
centrifuged at 8000 rpm for 15 min. The pellet was suspended in for-
malized saline and stored at 4 °C as H-antigen. This antigen was diluted
to approximately 105
cells/ml and 0.9 ml aliquots challenged with
0.1 ml aliquots of each antiserum (serial double dilutions). Antiserum
was provided by Dr. de Barjac, Pasteur Institute, Paris. Proper controls
were maintained for antigen & antiserum. The mixture was incubated at
37 °C in transparent tubes for 2 h and observed for agglutination, which
was identified by the presence of a clear supernatant and visible sedi-
ment at the bottom of a tube. The identity of the isolate was further
confirmed by serotyping performed by Dr. Michio Ohba, Kyushu Uni-
versity, Japan.
2.3. Scanning electron microscope
Bti/t strain was grown in NYSM medium at 30 °C for over 72 h until
sporulation was completed as determined by light microscopy. The
spores and crystals were pelleted by centrifugation at 4 °C at 9000 g for
10 min and lyophilized. Sample preparation was achieved by sprinkling
the lyophilized powder lightly on a double side carbon tape for
mounting on aluminum stub and viewed on Quanta 200 FEG scanning
electron microscope (SEM), at a voltage of 20 kV.
2.4. Analysis of crystal proteins
Sodium Dodecyl Sulfate, Polyacrylamide gel electrophoresis (SDS-
PAGE) was performed as described by Laemmli (1970) using 10% se-
parating and 4% stacking gels on mini protean cell slab vertical appa-
ratus (Bio-Rad, USA). The proteins were extracted from the cell mass of
Bti/t according to the procedure of Lecadet et al. (1992) and 15 μg of
protein was loaded on to the gel. After electrophoresis, the gels were
stained in a solution containing 50% (v/v) ethanol, 10% (v/v) acetic
acid and 0.1% (w/v) Coomassie brilliant blue R250 for 40 min, and
then destained in a solution containing 6.75% (v/v) glacial acetic acid
and 9.45% (v/v) ethanol. The molecular weight of the proteins were
determined by using protein molecular weight standards (Sigma).
2.5. Beta-exotoxin determination by house fly bioassay method
Procedure: The supernatant of the Bacillus thuringiensis var. israe-
lensis/tochigiensis (VCRC B-474) and the standard strain (IPS 82) were
placed on a shaker at 220 rpm at 28 °C for 4 h. The suspensions were
centrifuged at 3000 rpm for 20 min, then autoclaved at 121 °C at 15 lbs
for 20 min and used for bioassay. 2 ml of supernatant and standard
respectively were added to 18 gm of housefly diet (wheat bran 7.0 gm,
water 6 ml and milk powder 5 ml), mixed well and infested with 25 2
day old housefly (Musca domestica) larvae. For the control experiment,
2 ml of the water was added instead of Bacillus thuringiensis var. israe-
lensis/tochigiensis supernatant. Tests were performed in 200 ml jars and
three replicates were kept for each test does. Jars were closed tightly
with coarse muslin cloth and put into a constant temperature and hu-
midity cabinet at 26 °C +/− 2 °C until adults emerged (8–10 days). The
number of adults emerged in normal condition were counted and the
percentage mortality was calculated. Mortality of housefly larvae in the
test experiment more than that in the control was taken as indicative of
the presence of Beta exotoxin.
2.6. Pilot scale production
The production medium consisted of 2.5% soybean flour (Glycine
max), which was dissolved in distilled water and the pH adjusted to 7.0
prior to inoculation. Bacterial seed culture was prepared by inoculating
10 ml of NYSM broth with one loopful of culture and incubating the
tube in a rotary shaker at 30 °C, 180 rpm for a period of 7 h. Second
stage seed was prepared by inoculating 5% of the first seed to 2 num-
bers each of 250 ml flask containing 30 ml of medium and incubating
on a rotary shaker at 30 °C, 180 rpm for a period of 7 h. The seed thus
prepared was transferred to 2 numbers of 2 l Erlenmeyer flask con-
taining 600 ml of the appropriate medium and incubating in rotary
shaker at 30 °C, 180 rpm for a period of 7 h. This seed culture was used
to inoculate a pilot fermentor (100 l capacity) containing 60 l medium
at 2% level and was set to control fermentation variables automatically.
The fermentor was run for 24 h at 29 ± 1 °C with an agitation of
200 rpm. pH controller was connected only to 2N NaOH and the pH was
never allowed to drop below 7.0. Dissolved oxygen (DO) was main-
tained between 20% and 30% saturation by controlling the airflow that
was set at 1 L/L/min. Antifoam A (SIGMA) was used as an anti-foaming
agent (Prabakaran and Balaraman, 2006). The biomass was harvested
using Hitachi high speed refrigerated centrifuge on continuous mode
and formulated into Bti water dispersible powder (WDP) formulations.
2.7. Preparation of water dispersible powder formulation
Three water dispersible powder formulations were prepared using
the wet biomass of B.t.i/t as active ingredient and chalk, bentonite, and
talc as carrier materials. The carrier materials were initially sieved
through 45 μm test sieve and kept aside. After ensuring toxicity of the
bacterial biomass (active ingredient), formulations were prepared by
mixing the active ingredient with each of the three carriers using pla-
netary ball mill, in the ratio of 5: 95. The formulations were air dried,
powdered, code named as WDP 1, WDP 2, and WDP 3 stored at room
temperature in screw capped vials. The larvicidal activity was de-
termined immediately after preparation of the WDP and later at
monthly intervals.
2.8. Preliminary dose fixation and LC50 determination
The LC50 value of the WDP’s was determined as follows: stock so-
lution was prepared by taking 10 mg of WDP in 10 ml of sterile distilled
water and thoroughly blending in a homogenizer. The stock solution
was further diluted 10 times and used for determining the larvicidal
activity. The formulations were bioassayed on the same day. Twenty
five late third instar larvae of Culex quinquefasciatus were added to
paper cups containing 100 ml of chlorine free tap water (WHO, 2005).
Initially, a wide range of test concentrations of the 3 formulations,
ranging from 1 to 100 μg/cup were tried to obtain mortality values
between 10–90%. based on the mortality obtained during the pre-
liminary dose fixation, 5 doses for each of the formulation what would
give values 10–90% mortality were selected. The bioassay cups were
held at room temperature with a photoperiod of 12 h light: 12 h dark.
The 5 doses were tested having four replicates for each dose along with
appropriate controls. Larval mortality was scored 24 h post treatment
K. Shankar, et al. Acta Tropica 193 (2019) 158–162
159

3. and corrected to control mortality, if any using Abbott’s formula
(Abbott, 1925). Each experiment was repeated thrice on different days.
Probit regression analysis was carried out to calculate LC50 values
(Finney, 1971). The mean LC50 values between the three WDP for-
mulations performed every month for a period of one year were taken
as replicates. A one way ANOVA was carried out to see the significant
variation in LC50 values between the formulations. Monthly mean LC50
data were repeated as replicates. Pair wise comparison of the for-
mulations was done using the post-hoc multiple comparison test based
on least significant difference (LSD).
3. Results
3.1. Identification
Serotyping of this B.t strain showed that the isolate shared flagellar
antigens of two serotypes i.e., israelensis and tochigiensis (H14/H19).
Scanning electron microscopy of the sporulated cell mass of B.t.i/t
showed crystal inclusions completely separated from the spores. The
crystals were spherical in shape and uniformly sized. The size of the
crystals ranged between 0.7-0.8 μm in diameter (Figs. 1 and 2). SDS-
PAGE analysis of the proteins extracted from the cell mass of B.t.i/t
showed proteins of molecular weight 130, 65, and 28 kDa to be present
in major quantities (Fig. 3).
3.2. Activity of WDP formulations
The WDP formulations were stored at room temperature (RT),
28 ± 2 °C and LC50 values determined at monthly intervals for up to
1 year. The LC50 values of the WDP 1, WDP 2, WDP 3 prior to storage
were 0.274, 0.335, 0.348 μg/ml (Table 1). After 3 months of storage,
the LC50 values were 0.317, 0.429, 0.433 μg/ml respectively, indicating
a loss of activity of 14, 22, and 20%. By the end of 6 months of storage,
the dose required for inciting 50% mortality was 0.369, 0.515, and
0.549 μg/ml respectively while the values obtained by 9 months in-
dicated a further reduction in the activity of the WDP’s. After a period
of 12 months of storage at RT, WDP 1 lost 48% activity only while WDP
2 & 3 lost 57% and 60% of the activity. Decoding of the sample labels
showed that WDP 1, 2, and 3 had chalk, bentonite and talc as carrier
materials respectively. ANOVA test indicated that there is a one sig-
nificant difference among the mean LC50 values of the three formula-
tions (Table 1), F (2, 36) = 7.140, P < 0.05. In addition, a Post hoc
(LSD) test showed that WDP 1 had significantly higher LC50 values than
the other two groups at the 0.05 level of significance. All other com-
parisons are not significant. These carrier materials have helped in
maintaining nearly 52%, 43%, and 40% of the activity of the WDP
formulations.
Fig. 1. Scanning electron microscope of agglomerates of spores of Bti/t.
Fig. 2. Scanning electron microscope of spherical crystal and ovoid spores with
diameter in nm. Scale = 1 μm.
Fig. 3. SDS-PAGE analysis of parasporal inclusion from Bti/t.
Table 1
LC50 values of the water dispersible powders of Bacillus thuringiensis subsp. is-
raelensis/tochigiensis at different intervals of storage at room temperature.
Months Relative
Humidity
(RH %)
Chalk
WDP 1 ((μg/ml)
Bentonite
WDP 2 (μg/ml)
Talc
WDP 3 (μg/ml)
LC50 ± SD LC50 ± SD LC50 ± SD
0 62.5 0.274 ± 0.025 0.335 ± 0.041 0.348 ± 0.056
1 60.6 0.298 ± 0.025 0.370 ± 0.055 0.361 ± 0.054
2 73.0 0.309 ± 0.095 0.388 ± 0.032 0.394 ± 0.022
3 75.5 0.317 ± 0.046 0.429 ± 0.019 0.433 ± 0.062
4 84.5 0.330 ± 0.026 0.460 ± 0.040 0.459 ± 0.100
5 84.0 0.341 ± 0.111 0.498 ± 0.010 0.506 ± 0.038
6 85.0 0.369 ± 0.027 0.515 ± 0.083 0.549 ± 0.056
7 81.5 0.394 ± 0.025 0.571 ± 0.091 0.562 ± 0.100
8 80.0 0.405 ± 0.084 0.626 ± 0.049 0.618 ± 0.085
9 76.5 0.442 ± 0.082 0.669 ± 0.049 0.649 ± 0.060
10 76.5 0.477 ± 0.027 0.667 ± 0.030 0.677 ± 0.079
11 78.0 0.508 ± 0.062 0.720 ± 0.041 0.738 ± 0.049
12 68.0 0.523 ± 0.109 0.775 ± 0.081 0.808 ± 0.023
K. Shankar, et al. Acta Tropica 193 (2019) 158–162
160

4. 4. Discussion
Serotyping of this B.t.i/t isolate showed that this strain shared the
flagellar antigens of two serotypes i.e., israelensis and tochigiensis (H14/
H19). The mosquitocidal efficacy of serotype israelensis has been agreed
by several workers. (Balaraman et al., 1981; Goldberg and Margalit,
1977) However, only two reports exist on the usage of serotype tochi-
giensis against mosquitoes. Ohba and Aizawa (1986) found this serotype
to be totally ineffective while Lonc et al. (2000) reported moderate
activity against Aedes aegypti. An isolate of B.t.i/t isolated from a similar
ecosystem, namely mangrove swamps of Japan and sharing the anti-
gens of israelensis and tochigiensis has been reported to be mosquitocidal
(Maeda et al., 2001). Bioassay results of Beta exotoxin showed that
absence of Beta exotoxin.
Crystal inclusions produced during sporulation are the only phe-
notypic characteristic of Bt that distinguish it from taxonomically clo-
sely related Bacillus cereus species (Bravo et al., 1998). The morphology,
size and number of parasporal inclusions is known to vary among B.
thuringiensis serotypes (Onyancha, 2016). Crystal morphology is known
to provide valuable information on the insecticidal activity spectrum of
Bt strains. Dipteran specific Bt isolates have been found to possess
spherical shaped crystals as seen in Bti, a highly potent mosquitocidal
bacterium (Maeda et al., 2001; Obediat et al., 2004). Bt strains isolated
from three different habitats of Bangladesh showed mostly spherical
shaped crystals, with all isolates exhibiting toxicity to dipterans (Shishir
et al., 2012). It is interesting to note that this strain harbours spherical
parasporal crystal, which are specific to the serotype israelensis unlike
the rhomboidal crystals of B. thuringiensis subsp. tochigiensis (H19)
(Ohba and Aizawa, 1986). The cry and cyt gene profile have been
studied earlier by our group (Geetha et al., 2007). It is interesting to
note that this isolate though sharing the antigens of two different ser-
otypes possessed the same protein, cry and cyt gene profile as B.t.i.
SDS-PAGE profiles of the solubilized cell mass of this Bti/t isolate
(VCRC B 474) exhibited protein bands of size 135, 65, and 27-Kda re-
spectively. The same pattern of proteins has been observed in another
isolate of Bti/t obtained from Japan (Maeda et al., 2001). Further, there
is a high similarity between this isolate and a reference strain of the Bti
in the SDS-PAGE profile of proteins (Ibarra and Federici, 1986). The
130 kDa protein of israelensis is reported to be a member of the class of
Cry4 proteins, possessing high mosquitocidal activity (Hofte and
Whiteley, 1989). Usually, inclusions of Diptera-specific B. thuringiensis
strains contain broad-spectrum cytolysins (Cyt proteins) of 25–30 kDa
(Drobniewski and Ellar, 1989; Yu et al., 1991; Koni and Ellar, 1993;
Ishii and Ohba, 1994; Orduz et al., 1994), which are known to enhance
the mosquitocidal activity of the other co-existing Cry proteins in is-
raelensis (Wu and Chang, 1985). The presence of Cyt protein of size
27 kDa in this strain of Bti/t may be another reason for the high level of
activity to mosquito species, as also reported by Wu and Chang (1985).
Hence, the shape of the crystals, its protein profile, cry, and cyt gene
composition (Geetha et al., 2007) were similar to that found in the
highly mosquitocidal serotype namely, B.t.i.
Materials used in the powder formulations have important function
as biopesticide diluents and carriers (Subramanyam and Hagstrum,
2000). Several authors have observed sharp decrease in activity of dry
preparations when stored under humid tropical conditions. The in-
secticidal activity of Bti formulations (WDP) against fourth instar larvae
of C. pipiens molestus was found to reduce drastically from 13 days at
25 °C to 2.6 days when stored at 45 °C (Farghal and Darwish, 1988).
Ignoffo et al. (1982) recorded high loss of insecticidal activity when
wettable powder formulations of B. thuringiensis subsp. israelensis were
stored at 50 °C for 28 days, whereas another study showed that it could
be stored for 30 weeks at −40 °C, 8 °C and 30 °C without significant loss
of activity (Balaraman and Hoti, 1984). Talc and bentonite have been
used as carrier in WDP formulations of B. thuringiensis var. kurstaki
while chalk powder has been incorporated in Bacillus sphaericus based
WDP formulations (Prasad et al., 2006; Yadav et al., 2010). Our earlier
studies with WDP formulations prepared using indigenous isolates of Bti
and plaster of Paris as carrier material showed that the mosquito lar-
vicidal activity was retained for a long period of time when stored at
room temperature (RT) (Manonmani et al., 2008). Removing the water
content from the wet biomass by mixing with the carriers to the max-
imum possible level, and then drying for 48 h at room temperatures
ensured the minimum loss of activity for up to six months as observed
in this study. Storing the material at RT is desirable as cold storage
facilities are not available in developing countries where vector borne
diseases are most prevalent. In the present study, the chalk based WDP
retained 86% of activity for up to 3 months, 75% of its activity for up to
6 months, 61% of activity for 9 months and 52% of its activity for up to
a year when stored at RT. Chalk is a solid, powdered, mineral auxiliary
which has found use as an anti-blocking agent for pesticide wettable
and atomizable powder formulations as well as in the preservation of
microorganisms (Morales and Rochling, 1998; Sauer, 1997).
It is known that the selection of the most potent bioinsecticide
formulation is based on the ratio of active material and inert in-
gredients. Pesticides are rarely used in their pure or technical form. The
carrier materials serve a variety of functions – to enhance stability,
reduce toxicity, improve efficacy, easy transportation of product and
application in the mosquito breeding sites (Yap, 1990). The present
work examines the survival and shelf life of B.t.i/t, a novel strain of B.
thuringiensis sharing the antigens of 2 serotypes. When formulated with
chalk, bentonite, and talc and stored at ambient temperature, the for-
mulaton lost 48%, 57%, and 60% activity after 12 months. Based on the
LC50 value, it is seen that chalk powder was a better carrier when
compared to bentonite and talc. The activity of the WDP formulation
prepared using chalk as a carrier is appreciable for about 6 months
when stored at RT retaining 75% of its activity. However, results from
our study strongly suggest storing WDP formulations at RT for retaining
the activity for longer periods of time which is in agreement with the
observations of many other authors in their studies with water dis-
persible powder formulations (Manonmani et al., 2008; Farghal and
Darwish, 1988). This strain of B.t.i/t (H14/19) appears to be a pro-
spective candidate for use in mosquito control programs. Further work
such as simultaneous comparison of mosquito larvicidal activity with
commercial product by laboratory bioassay, container compatability
and applying the formulation in solution through ULV spraying, cold
mist or thermal fog need to be studied.
Conflicts of interest statement
The authors have no conflicts of interest to declare.
Acknowledgements
We thank Dr. I. Geetha, Microbiology division for her valuable
suggestions and Dr. S. Subramanian for his help in statistical work and
the analytical service of DST (SAIF, IIT) for scanning electron micro-
scope. We are grateful to Dr P. Jambulingam, Director, Vector Control
Research Centre for the support and the facilities provided.
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