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1. Welcome to Viva – Voce presentation on
Characterization of a mosquito larvicidal strain of
Bacillus thuringiensis subsp. israelensis/tochigiensis
(VCRC B-474), isolated from mangrove forests of Andaman and
Nicobar Islands
SUBMITTED TO
PONDICHERRY UNIVERSITY
Shankar K
Under the guidance of
Dr. A. M. Manonmani, Scientist - G (Retired),
ICMR – Vector Control Research Centre,
Pondicherry

2.  Mosquitoes represent the major arthropod
vectors of human disease worldwide transmitting
malaria, filariasis, dengue, chikungunya, Zika, yellow
fever, Japanese encephalitis etc.
 Vector control remains the main option for
containment of these diseases as development of
insecticide resistance & financial burden of
insecticide-based strategies have been major
setbacks for mosquito control programmes.

3.  Bio-control strategies have now emerged as
sustainable and eco-friendly approaches.
 Among bio-control agents, spore forming bacteria
enjoy top priority with Bacillus thuringiensis var.
israelensis (Bti) being recognized as the prime
candidate for use, due to its,
 Specific activity to mosquitoes.
 High activity against a broad spectrum of
mosquito larvae.
 Amenability to production using cheap raw
material.
 Safety to mammalian systems & safety to
non-target organisms.

4. Bacillius thuringiensis subsp. israelensis (VCRC
B17)
 An indigenous strain of Bacillus thuringiensis subsp.
israelensis isolated from soil sample of paddy field in
Uzhavarkarai, Puducherry, India, in 1980.
 It has been extensively studied, production technology
patented and licensed to 19 commercial firms till date.
 Presently, this is the only mosquito larvicide used in
operational mosquito control programmes and available,
commercially. Hence, it is wise to look for alternatives to
Bti.

5. Bacillus thuringiensis subsp. israelensis/
tochigiensis (VCRC B-474)
`
 The search program for new mosquitocidal
microbes from the soils of mangrove forest
ecosystem has resulted in a larvicidal
bacterium, VCRC B474
 This isolate shares the flagellar antigen of two
serotypes israelensis (H14)/tochigiensis (H19)

6. Mode of action

7. Broad objective
To characterize the isolate of B. thuringiensis
subsp. israelensis & tochigiensis (VCRC B474)
for its mosquito control potential

8. 1. To compare the efficacy of B. t. subsp.
israelensis/tochigiensis with B. thuringiensis subsp.
israelensis
2. To study the amenability of the strain for large scale
fermentation
3. To develop cost effective media for production of
mosquitocidal toxins
4. To develop water dispersible powder formulation
and evaluate under laboratory conditions
5. To develop tablet formulation and evaluate under
simulated field conditions

9. Objective 1
To compare the efficacy of B. thuringiensis subsp.
israelensis/tochigiensis (VCRC B 474) with the
standard mosquitocidal strain of Bacillus thuringiensis
subsp. israelensis (VCRC B 17)

10. GIS MAP
Uzhavarkarai, 1980 2005
B-17, Bacillus thuringiensis subsp. israelensis
B-474, Bacillus thuringiensis subsp. israelensis/tochigiensis

12. Morphological and microscopic examination of
Bti/t (VCRC B- 474)
Plate 1. Colony were white round,
shiny, raised at the centre
Plate 2. Stained micrograph (100X)
showing completely lysed spores and
crystals (s-spores, c-crystal)
s
c

13. Bioassay
Methodology
O/N
2%
5% 7h
72h
100 ml NYSM medium
Centrifugation
(10000 rpm for 15 min)
Pellet
10 ml NYSM broth
50 ml NYSM broth
Lyophilization
Inoculated one loopful of slant culture
(B-17, B-474)

14. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 6 12 18 24 30 36 42 48
ODat595nm
Time (h)
B-17 B-474
Fig 1. Growth kinetics of Bti and Bti/t
14

15. Mosquito
species
B-17
LC50(µg/ml)
B-474
LC50 (µg/ml )
Cx. quinquefasciatus 0.004 (0.003-0.005) 0.003 (0.002-0.004)
Ae. aegypti 0.010 (0.007-0.012) 0.008(0.005-0.010)
An. stephensi 0.015 (0.013-0.020) 0.018(0.009-0.022)
Table 1. Larvicidal efficacy of Bti and Bti/t against III
instar larvae of vector mosquitoes
• Figures within parenthesis indicates 95% fiducial limits.
• Both the strains were highly toxic and are statistically
indistinguishable

16. Scanning electron micrograph of Bti/t
Plate 3. Spore crystal complex showing the size of crystals
C
S

17. Plate 5. SDS-PAGE profile
1 2 3
Lane 1. Molecular weight standard
Lane 2. B.t. subsp. israelensis
Lane 3 . B.t. subsp. israelensis /tochigiensis
220 kDa
100 kDa
60 kDa
45 kDa
30 kDa
20 KDa
135 kDa
65 kDa
27 kDa

18. To study the amenability of the strain for
large scale production

19. 100 ml of sample removed at 6
hourly interval
METHODOLOGY
Biomass Spore count Toxicity
O/N 5%
2%
Inoculated one loopful of culture
7h
48
50 ml NYSM broth
10 ml NYSM broth
600 ml NYSM broth
Production in 100 L fermentor using soya medium
5%7h
Slant culture

20. 6.71
6.9
6.5
5.78
5.23
5.1
5 5
0
1
2
3
4
5
6
7
8
6 12 18 24 30 36 42 48
DryweightofCelImass(g/l)
Time (h)
Maximum biomass observed by 12 h

21. Maximum toxin synthesis obtained by 24 h
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
12 18 24 30 30 42 48
0.04
0.021
0.005
0.007
0.01 0.01 0.01
LC50(µg/ml)
Time (h)

22. Table 2. Dynamics of spore count of Bti/t
Hours spores /ml
6 h NIL
12 h 6 X 105
18 h 2 X 107
24 h 3.3 X 109
30 h 3.2 X 109
36 h 3.2 X 109
42 h 3.1X 109
48 h 3.1 X 109
Maximum spores was observed at 24 h

23. To develop cost effective media for
production of bacterial toxins.

24. Development of cost effective
medium
1. Peanut milk 2. Egg yolk lyophilized
3. Hard boiled egg white 4. Egg mix

25. Methodology for media preparations
1. Peanut milk broth
 Kernel of raw peanuts were used. 2.5g, 5g & 7.5 g peanuts
crushed in a minimum amount of water
 Resultant slurry filtered with double layered cheese cloth
 The volume of the extract was made up to 100 ml each and
labeled as PM1, PM2 and PM3.
 pH of the medium was adjusted to 7.0
2. Egg yolk medium
 Egg yolk was separated from boiled eggs.
 Lyophilized, powdered and stored at 4˚C.
 0.12g, 0.25g & 0.37g was taken in 100 ml water and labeled as
EY1, EY2, EY3.
 pH of the medium was adjusted to 7.0

26. 3. Egg white medium
 Egg white was separated from boiled eggs and homogenised
 1g, 3g & 5g was taken in 100 ml water and labeled as EW1,
EW2,EW3.
 pH of the medium was adjusted to 7.0.
4. Egg mix medium
 Egg mix prepared by beating the whole egg contents in a mixer.
 0.5ml, 0.75ml & 1ml egg mix was taken in 100 ml water and
labeled as EM1, EM2 and EM3.
 pH of the medium was adjusted to 7.0.

27. Slant culture
First stage seed
Second stage seed
2.5 ml (PM1),
5.0 ml (PM2)
7.5 ml (PM3) and
NYSM
Significant difference
(p<0.05) in the cellmass,
sporulation and larvicidal
activity
Inoculate 0ne loopful of culture
5 % inoculum
2 % inoculum
Methodology for Production by Peanut Milk

28. 0
0.5
1
1.5
2
2.5
3
PM1 PM2 PM3 NYSM
Cellmassdryweight(g/l)
Production medium
Fig 4. Biomass production of Bti/t in PM1, PM2, PM3 and
NYSM medium
 The one way ANOVA of 4 media shows significant difference (F=(3,8)=
120.07, p < 0.05).
 A tukey post–hoc multiple comparison test, P < 0.05, indicated that PM2
had significantly higher biomass.

29. 0
2
4
6
8
10
12
14
16
PM1 PM2 PM3 NYSM
Numberofspores(108/ml)
production medium
 The one way ANOVA of 4 media shows significant difference across the mean
spore count (F=3, 8)= 24.46, p < 0.05).
 The tukey post-hoc multiple comparison test, (p>0.05) indicate no significant
difference in PM2 and NYSM, whereas the spore count in PM1 and PM3 were
significantly lower (p< 0.05).
Fig 5. Sporulation of Bti/t in PM1, PM2, PM3 and NYSM

30. Fig 6. Toxicity of Bti/t grown in PM1, PM2, PM3
and NYSM medium
0
0.005
0.01
0.015
0.02
0.025
PMI PM2 PM3 NYSM
0.021(0.018-0.026)
0.012(0.009-0.013)
0.025(0.022-0.030)
0.007(0.014-0.018)
LC50(µg/ml)
Production medium
 Maximum toxicity was noticed in PM2 medium and
conventional medium (NYSM)

31. Fig 7. Dynamics of biomass production of
Bti/t in PM2 and NYSM medium
0
1
2
3
4
6 12 18 24 30 36 42 48
DryweightofCellmass(g/l)
Hours
PM2 NYSM
 Maximum biomass production seen by 12 h in
PM2 and NYSM media

32. Fig 8. Dynamics of toxin synthesis of Bti/t in PM2 and
NYSM medium
 Maximum toxin synthesis was observed at 18 h in PM2
medium and 24 h in NYSM Medium
0
0.01
0.02
0.03
0.04
0.05
0.06
0
0.01
0.02
0.03
0.04
0.05
0.06
12 18 24 30 36 42 48
LC50(µg/ml)
LC50(µg/ml)
Cultivation time (h)
NYSM Medium PM2 Medium

33. Table 3. Dynamics of sporulation in PM2 and NYSM medium
Hours PM2 (spores /ml) NYSM (Spores /ml)
6 h NIL NIL
12 h 7 X 10 5 NIL
18 h 1.5 X 109 3 X 105
24 h 1.3 X 109 1.2 X 109
30 h 1.5 X 109 1.1 X 109
36 h 1.5 X 109 1.2 X 109
42 h 1.4 X 109 1.0 X 109
48 h 1.5 X 109 1.2 X 109
 The highest spore count obtained in PM2 and NYSM were
1.5 x 109 (CFU/ml) and 1.2 x 109(CFU/ml) respectively at 18
and 24h.
 The mean difference in spore count between two media was
not significant (student’s t-test for independent samples,
t=0.923, d.f=46, p=0.92).

34. Slant culture
2% inoculum
5 %
inoculum
Inoculate one
loopful of culture
EY1 (1.25g)
EY2 (2.5g)
EY3 (3.75g)
EM1 (5g)
EM2 (7.5g)
EM3 (10g)
EW1 (10g)
EW2(30g)
EW3 (50g)
Biomass (g/l)
Toxin synthesis (µg/ml)
Cost analysis (INR)
10 ml of NYSM
Medium
50 ml of NYSM
Medium
Methodology for production using Chicken Egg Medium
NYSM
medium

35. Fig 9. Biomass production and toxin synthesis in Egg
white and NYSM medium
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
EW1 EW2 EW3 NYSM
Dryweightofcellmass(g/l)
Production medium
Egg white
(Hard Boiled)
LC 50
(µg/ml)
EW1 0.012(0.010-0.014)
EW2 0.007(0.004-0.009)
EW3 0.026(0.020-0.049)
NYSM 0.006(0.003-0.009)

36. Fig 10. Biomass Production and Toxin synthesis in Egg yolk and
NYSM medium
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
EY1 EY2 EY3 NYSM
Dryweightofcellmass(g/)l
Production medium
Egg yolk
(Hard boiled and
Lyophilized)
LC 50 (µg/ml)
EY1 0.012 (0.010-0.015)
EY2 0.011 (0.009-0.012)
EY3 0.019 (0.015-0.029)
NYSM 0.006 (0.003-0.009)

37. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
EM1 EM2 EM3 NYSM
Dryweightofcellmass(g/l)
Production medium
Fig 11. Biomass production and toxin synthesis in Egg mix
and NYSM medium
Egg mix LC 50 (µg/ml)
EM1 0.011 (0.010-0.013)
EM2 0.008 (0.005-0.010)
EM3 0.013 (0.012-0.016)
NYSM 0.006 (0.003-0.009)

38. Table 4. Cost comparison for production of Bti/t in the various media
Culture media and
components (g/l)
Toxin synthesis
(µg/ml)
Dry
biomass(g/l)
Total cost for
production of
l kg of dry
biomass (INR)
Difference in
net Cost ratio
Peanut milk medium
PM1 (2.5g)
PM2 (5 g)
PM3 (7.5)
0.021
0.012
0.025
1.71
2.70
1.53
877/-
1111/-
2941/-
1: 26
1: 20
1: 7
Media ‘’EW’’ ( Hard boiled)
EW1 (10 g)
EW2 ( 30 g)
EW3 ( 50 g)
0.012
0.007
0.026
0.27
0.70
1.26
2962/-
3428/-
3174/-
1:7
1:6
1:5
Media “EY”
(Hard boiled and lyophilized)
EY1 (1.25 g)
EY2 (2.5 g)
EY3 (3.75 g)
0.012
0.011
0.019
0.42
0.75
0.94
476/-
533/-
638/-
1:48
1:43
1:36
Media”EM” (Egg Mix)
EM1 (5.0 ml)
EM2 (7.5 ml)
EM3 (10 ml)
0.011
0.008
0.013
0.43
0.64
0.95
930/-
937/-
842/-
1:25
1: 24
1:27
Nutrient Yeast Salt medium
(5 g glucose, 5 g peptone, 5 g Nacl, 3 g beef
extract, 0.05 g yeast extract (0.203 g MgCL2,
0.102 g Cacl2, 0.010 g Mncl2 (sigma))
0.006 1.46 23, 280/- 1

39. Comparsion of biomass, toxicity and cost-analysis in the
optimal medium from four different group
2.7
0.7 0.75 0.64
1.46
0
0.5
1
1.5
2
2.5
3
PM2 EW2 EY2 EM2 NYSM
Drybiomassg/l
0.012
0.007
0.011
0.008
0.006
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
PM2 EW2 EY2 EM2 NYSM
LC50µµg/mi
1111/- 3428/-
533/-
937/-
23,280/-
1kg biomass
PM2 EW2 EY2 EM2 NYSM

40. To develop water dispersible powder
formulations and evaluate under
laboratory conditions

41. Methodology for development of water dispersible
powder formulations
Chalk powder
Talc powder
Bentonite powder
Wet biomass
1 3 4 5
678
2
Slant culture Seed flask Pre-seed fermentor Production Fermentor Centrifugation
WDP formulations
Stored at RTMonthly bioassay

42.  Initial activity of WDP formulations against late III
instar larval stages of Culex quinquefasciatus.
CODE Formulation
(5 % a.i.+ 95 % filler)
LC50
(µg/ml)
WDP 1 B474 + Chalk 0.274
WDP 2 B474 + Bentonite 0.335
WDP 3 B474 + Talc 0.348

43. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 2 3 4 5 6 7 8 9 10 11 12 13
LC50(µg/ml)
Months
Chalk Bentonite Talc
Fig 12. Storage studies with WDP formulations
 ANOVA test indicated that there is one significant
difference among the mean LC50 values of the 3
formulations, F (2, 36) = 7.140, p<0.05.
 In addition, a post hoc (LSD) test showed that WDP (chalk)
had significantly higher activity than the other formulation
at the .05 level of significance

44. Fig 13. Storage stability of WDP at 3 months interval
for a year
 Chalk based WDP formulation was found to retain the activity
for a longer period of time.
86%
75%
61%
52% 78%
65%
50%
43%
80%
63%
53%
40%
3M 6M 9M 12M
Talc
(WDP-3)
Bentonite
(WDP-2)
Chalk
(WDP-1)

45. To develop tablet formulation and
evaluate under simulated field
conditions

46. -------------------------------------------------------------------------------------
Components* T1 T2 T3
-------------------------------------------------------------------------------------
Bti/t (mg) 0.010 0.020 0.030
Sodium starch glycollate (mg) 0.500 0.500 0.500
Lactose (mg) 9.390 9.380 9.370
Magnesium stearate (mg) 0.100 0.100 0.100
--------------------------------------------------------------------------------------
* For preparing 20 tablets

47. Simulated field evaluation of tablet formulation
Mosquito species: Aedes aegypti
Larval stage: Late third instars
No. of larvae: 50 numbers
Water: 40 litres
Coded as : T1 (0.5 mg)
T2 (1.0 mg)
T3 (1.5 mg)

48. Fig 14. Efficacy of tablet formulation on the immature
stages of Aedes aegypti in cement tanks
• T1 and T2 concentration gave 80% mortality for the first three days, where as T3
showed activity for one more day.
• As mortality level of 80% or above is crucial for disease suppression, the
application of T3 at an interval of 4 days would be the ideal dosage.
0
20
40
60
80
100
1 2 3 4 5
Mortality%
Days
T1 T2 T3 Control

49. 1. Strain of VCRC B474 (Bti/t) and VCRC B17 (Bti)
showed similarity in shape and size of spore
crystals, protein profile, growth pattern and toxin
synthesis.
2. The isolate of Bti/t was ameniable to large scale
production using soya medium in 100 L
fermenter

50. 3. Among the various media studied, namely peanut and
egg based medium, the egg yolk based production
medium was found to be 43 times cost effective than the
conventional medium, NYSM.
4. Among the three WDP formulations, chalk based
formulation (WDP1) was found to be the most effective
& helped in retaining 75% of activity for about 6 months.
5. As mortality level of above 80% is crucial for disease
suppression. Among the three tablet formulations tested
formulation containing 1.5 mg (T3) of Bti/t would be
required at an interval of 4 days.

51. Conclusion

52. Publications
 Kannadasan Shankar, Gnanasundaram Prabakaran, Arulsamy M. Manonmani.,
2019.WDP formulations using a novel mosquitocidal bacteria, Bacillus
thuringiensis subsp. israelensis / tochigiensis (VCRC B-474) - Development and
storage. Acta trop., 193, 158-162
 Shankar, K., Prabakaran, G., Manonmani, A. M., 2016. Cost-effective medium
for the production of mosquitocidal toxins from a novel strain Bacillus
thuringiensis subsp. israelensis/tochigiensis (H14/H19). Eur. J. Biotechnol.
Biosci., 4, 12-16.
Conference presentation
International Conference on Modern biology and Environmental management Jan
27 -28th, WDP Formulations using a novel mosquitocidal bacteria, Bacillus
thuringiensis subsp. israelensis/tochigiensis – Development and storage stability,
Department of Zoology, Vivekanandha College of Arts and Science for Women
(Autonomous), Elayampalayam, Tiruchengode -637205

53. Acknowledgement
Director Dr. Ashwani kumar, Msc., Ph.D
Scientist - G
ICMR – Vector Control Research Centre
Pondicherry
Former DIRECTOR Dr. P. Jamblingam, MSC., Ph.D
Scientist – G (Retd.)
ICMR – Vector Control Research Centre
Pondicherry
Guide Dr. A.M. Manonmani, MSc., Ph.D
Scientist-G (Retd.)
ICMR – Vector Control Research Centre
Pondicherry
Doctoral Committee Member DR. S. L. Hoti MSc., Ph. D
Director in charge
Scientist-G
ICMR- National Institute of Traditional Medicine
Belagavi

54. Technical officer Mr. A. Mathivanan Msc.
Mrs. Rengankumari (retd)
Mr. Chakravarthy (retd)
Mrs. K. Vijaylakshmi
Lab assistant 1 Mr. S.Paneerselvam
Mrs. P.G. Geetha devi
Mr. M.K. Prakasam
Mr. K. Ramachandran
Technician-A Ms. V. Shakila
Mr. Rajavel
Multi tasking staff Mr. V. Anthonisamy (late)
Department of vector biology and Control
HOD Dr.K. Gunasekaran, Msc, Ph.d
Scientist ‘G’
Dr. C. Sadanadane Msc, Ph.d
Scientist “D’
Senior Technician -2 M. Stalin
S. Kamalasekaren

55. Thank you
Shankar . K