The document discusses the role of gut microflora in the susceptibility of lepidopteran pests to Bacillus thuringiensis. It provides background on the diversity of gut microbes in different insect orders. Case studies show that certain gut bacteria can promote the insecticidal activity of Bt by cleaving Bt crystal proteins or having synergistic effects with Bt. The toxicity of Bt towards cotton bollworm was reduced when the insects were pre-treated with antibiotics, indicating that gut microflora influence Bt susceptibility.

1. Gut Microflora and Their Role in
Susceptibility of Lepidopteran Pests to
Bacillus thuringiensis Berliner
PRESENTED BY
K. PREMALATHA
Ph.D. Scholar
Department of Agricultural Entomology
Tamil Nadu Agricultural University
Coimbatore- 641 003

2. INTRODUCTION
 Insects are the most diverse and abundant animal clade, in numbers of
species globally (Basset et al.., 2012).
 Diversification and evolutionary success - relationships with beneficial
microorganisms.
Genta et al., 2006
host insect
morphogenesis,
food digestion,
nutrition,
antifungal toxin,
pheromone,
pH,
vitamins,
temperature tolerance,
parasitoid development,
detoxification

3. • Bacillus thuringiensis - Bombyx mori (L.) and Ephestia
kuehniella (Zeller), @ the beginning of the 20th century
(Ishiwata, 1901 and Berliner, 1915),
• Lepidoptera - insect model
• Types of microbes present in the midgut - new strategies for
pest and resistance management (Broderick et al., 2003).

4. Factors influencing microbial colonization
of the insect gut
(Dillon and Dillon, 2004)
• Bignell- 1982 (structure and microbial colonization)
• Simple, straight digestive tract – microbiota
• More complex structures - paunches, diverticula, and caeca microbiota (Tanada
and Kaya,1993)

5. Cont.,
• Hemiptera- lumen of the midgut caeca (Dasch et al., 1984)
• Coleoptera and tephritid flies- Midgut and caeca (Douglas and Beard, 1996)
• Migratory grasshoppers- peritrophic matrix- (Mead et al.,1988)
• Manduca sexta - gut lumen and hindgut epithelia- (Prestia and Hirshfield, 1988)
• Fruit fly Bactrocera tryoni - peritrophic matrix (Murphy et al., 1994)
• Wood- and litter-feeding termites – hindgut (Breznak, 2000).

6. • pH -selecting and enriching of bacteria
• Optimum pH -6-7
• Lactic acid bacteria -acidic pH.
• Create their own low pH- producing lactic acid by Streptococci
(Enterococci) in the gut of Lymantria dispar (Kodama and Nakasuji,
1971).
• Change in pH - buffering capacity of the gut contents versus the size and
metabolic activity of the microbiota.
• Secondary plant compounds have antimicrobial properties - midgut pH
alters the composition of the microbiota and able to detoxify secondary
plant compounds. (Walenciak et al., 2002)
Cont.,

8. Pathogenic interaction
• Negative interaction
• Microbes associated with host insects will affects the insect
host in terms of fitness, reproductive success, feeding and
influence of other symbionts.
• Producing variety of toxins and evade the host immune
system for the successful infection.
• Ex: Bacillus thuringiensis known to affect the host by
producing Cry proteins.

9. Symbiotic interaction
• Symbiosis is close and often long-term interaction between
two or more different biological species.
• In 1877, Albert Bernhard Frank used the word symbiosis to
describe the mutualistic relationship in lichens
• Symbiotic associations- commensalism, mutualism and
parasitism
• Commensalism: Relationship between two living organisms
where one benefits and the other is not significantly harmed
or helped (Paracer and Ahmadjian, 2000).

10. • Mutualism : Relationship between individual of different
species (microbe and insect) mutually benefit each other
• Parasitism occurs when one species increases its fitness while
the other is harmed by the association
• Example: insect gut microbes contributes to food digestion,
produces essential vitamins and keeps out potentially harmful
microbes by competing with them for nutrients
Contreras and Vlisidou, 2008

11. Methods of gut microflora diversity analysis
1. Gene targeting: gene-specific PCR
2. Molecular fingerprinting techniques
3. Fluorescent in situ hybridization

12. Dissection of insect gut and isolation of gut microbial DNA
Homogenized
1 ml 0.1 M phosphate buffer (pH 7.0)
Dissect (whole gut)
Rinsing with sterilised distilled water
5% (v/v) Sodium Hypochlorite (NaOCl) solution
Surface disinfected with 70% (v/v) ethanol
Starved for 24 h
Third instar larvae (10 No’s)

13. Gene targeting: gene-specific PCR
• Gene targeting techniques employ gene specific primers to
specifically amplify target genes, including conserved 16S
rRNA gene or a gene of specific functional interest from the
metagenomic DNA of insect gut symbionts.
• This approach has been widely applied to insect gut
symbiotic microbiota analysis and has revealed substantial
bacterial diversity
Brauman et al., 2001

14. Molecular fingerprinting techniques
DNA-fingerprinting?
Technique of determining
nucleotide sequences
of certain areas of
DNA which are unique
to each individual.

15. Fluorescent in situ hybridization
• Fluorescent in situ hybridization (FISH) is commonly used in
microbial ecology studies to visualize symbiotic bacteria in the
gut
• The application of FISH in insect gut microbial studies often
involves fluorescently labeled probes targeting 16s rRNA with
sequences specific for a bacteria.
Tang et al., 2012

16. 5 th instar L
Wash 3 times 70% ethanol and H2O
insects frozen at -20 °C
dissected
Gut was cut into 3 pieces
4% formaldehyde overnight.
washing 3 times (PBS)
embedded
5 µm thin section
mounted on SuperFrost Ultra Plus glass slide
lysozyme for 15 min at 37° C
washing
hybridized with 1.5 mM of probe
hybridization buffer @ 46°C for 4
hrs in Advalytix slide booster
50 ml washing buffer
20 min.
dried

17. Bacterial localization in the gut of S.
littoralis larvae with Fluorescent In Situ
Hybridization
A. Detection of Clostridium sp,
B. Clostridium sp. deep in the gut
lumen.
C. Enterococcus mundtii
D. E. casseliflavus.
E. Propionibacterium
F. E. coli
G. Klebsiella pneumonia.
Tang et al., 2012

18. Gut microbial diversity in insects
• Collembolan (Folsomia candida)-Erwinia amylovora, Staphylococcus
capitis, Pantoea agglomerans and Pseudomonas putida (Thimm et al.,
1998).
• Termite and cockroach- complex of micro organisms protozoan
spirochetes, gram positive and gram negative bacteria, archea and yeast
(Paster et al., 1996).
• Coleopteran- sugar fermenting bacteria belonging to Lactobacillus,
Clostridium, Bacillus and members of CFB (Cytophaga, Flavobacterium,
Bacteriodes) group.

19. Cont.,
• Drosophila - Gluconobacter, Acetobacter, Campylobacter, Pseudomonas,
Serratia, Klebsiella and Comomonas; Firmicutes like Lactobacillus and
Enterococcus.
• Fruit fly Certatitis capitata- Klebsiella, Enterobacter, Citrobacter and
Pantoea (Behar et al., 2008).
• Lepidopterans (Lymantria dispar)- Enterobacter, Pseudomonas,
Staphylococcus, Paenibacillus, Serratia, Pantoea , Micrococcus and
Bacillus (Broderick et al., 2004).

20. Diversity of gut microflora in DBM
• 97% of the bacteria were from three orders:
Enterobacteriales, Vibrionales and Lactobacillales
Xia et al., 2013

21. Diversity of bacterial flora in lepidopteran larvae
• Broderick et al., (2009) evaluated the enteric bacteria of six lepidopterans
by 16S rRNA gene sequence analysis out of six species, five species gut
bacteria were identified
Larval species
Bacterial species detected in
guts of larvae
Family Species
Nymphalidae Vanessa cardui Lactococcus lactis
Klebsiella sp.
Sphingidae Manduca sexta Enterobacter sp.
Klebsiella sp.
Pieridae Pieris rapae Enterobacter sp.
Pantoea sp.
Noctuidae Heliothis virescens none detected
Gelechiidae Pectinophora gossypiella Enterococcus casseliflavus
Lymantriidae Lymantria dispar Enterobacter sp. NAB3
Pseudomonas putida

22. Diversity of gut bacteria in silkworm
• Digestive tract of multivoltine, cross breed silk worm, Bombyx mori (L)
(PM x CSR2)
Isolates Species identified
1 Bacillus circulans
2 Proteus vulgaries
3 Klebsiella pneumonia
4 Escherichia coli
5 Cittrobacter freundii
6 Serratia liquefaciens
7 Enterobactor sp
8 Pseudomonas fluorescens
9 P. aeruginosa
10 Aeromonas sp
11 Erwinia species

23. Gut microbiota of the Spodoptera littoralis
(A) The structure of the
alimentary canal.
(B) Relative abundance of
bacteria in the three
segments
(C) Rarefaction curves of
the bacterial diversity in
gut section I and section
III.
Tang et al ., 2012
Spatial Distribution

24. Temporal distribution
• Body length of S. littoralis larvae increases from 1.5 mm to 40
mm, and the diameter of its gut increases from 0.5 mm to 7
mm.
Tang et al ., 2012

25. Functions of insect gut bacteria
Nutritional symbioses
• Bacteria passing through the gut can simply be digested and
used for itself as nutrients (nutritional bacteria).
Schauer et al. (2012)
Digestion of recalcitrant plant polymers
• Asian longhorned beetle (Anoplophora glabripennis) and the
Pacific dampwood termite (Zootermopsis angusticollis) both
degrade lignin during the passage through the gut.
Geib et al. (2008).

26. Nutrient provisioning
• Gut symbionts Rhodococcus rhodnii , which provisions B vitamins to its
blood-feeding host Rhodinus prolixus (Eichler and Schaub, 2002)
• Gut bacteria in termites directly fix nitrogen from the atmosphere
(Thong-On et al., 2012).
Immunity and protection
• Axenic locusts - entomopathogenic fungus – Beauvaria and
Metarhizium- Pantoea agglomerans- phenolic repository (Dillon and
Charnley ,1995)
• The facultative endosymbiont Hamiltonella defensa protects the aphid
from attack by the parasitoid Aphididus ervi (Oliver et al., 2005)
• Serratia symbiotica helps the aphid tolerate higher temperature (Russel
and Moran ,2006)

27. Population changes and phenotype manipulation
Candidatus cardinium- host feminization and
parthenogenesis (Zchori-Fein and Perlman, 2004)
Phenotype manipulation -Wolbachia.
Infected females produce viable offspring.
Uninfected female - less fit reproduction
Wolbachia - thelytoky parthenogenesis
Hemiptera, lepidoptera, diptera, coleoptera,
hymenoptera
(Hoerauf and Rao, 2007)

29. Bacillus thuringiensis
 Gram-positive, soil-dwelling bacterium, commonly used as
a biological pesticide.
 Gut of caterpillars, moths and butterflies
 Leaf surfaces, aquatic environments, animal feces, insect-rich
environments, and flour mills and grain-storage facilities

30. • B. thuringiensis was first discovered in 1901 by Japanese
biologist Ishiwata, most abundantly found in grain dust from
silos and other grain storage facilities
• In 1911, B. thuringiensis was rediscovered in Germany by
Berliner, who isolated it as the cause of a disease called
Schlaffsucht (excessive sleeping) in flour moth caterpillars
• Bt- commercial insecticide in France in 1938, and in the
1950s it entered commercial use in the USA.

31. Characteristics of Bt
Bt synthesize more than one parasporal inclusion. The
parasporal inclusions are formed by different insecticidal
crystal proteins (ICP)
The crystals have various shapes (bipyramidal, cuboidal, flat
rhomboid, spherical or composite with two crystal types)

32. During sporulation Bt strains produce crystal
proteins (proteinaceous inclusions), called δ-
endotoxins (Cry proteins), which are encoded by cry
genes, and have insecticidal action
Genetically modified crops - Bt genes.

34. Mode of Action of Bt

35. Mechanism of Cry protein toxicity.
A: Ingestion
B: In the midgut, endotoxins are
solubilized from Bt spores
(s) and inclusions of crystallized
protein. (cp).
C: Cry toxins are proteolytically
processed to active toxins in the
midgut.
D: Cry toxins aggregate to form pores in
the membrane.
E: Pore formation leads to osmotic lysis.
F: Heavy damage to midgut membranes
leads to starvation or septicemia.
Whalon and Wingerd, 2003

36. Role of bacteria in the mode of action of Bt
• Gut bacteria -promoting insecticidal activity of Bt (Mason et
al., 2011).
• Gypsy moth larvae- elimination of the gut microbial
community abolished Bt insecticidal activity (Broderick et al.,
2009).
• Bacteria must be cause septicaemia.
• B. thuringiensis -enable the enteric bacteria (Enterobacter
spp. and E. coli ) to the gut epithelium (Broderick et al.,
2006).

37. • Velvetbean caterpillar, Anticarsia gemmatalis Hubner.
• Bacillus subtilis, Bacillus cereus, Enterococcus gallinarum,
Enterococcus mundtii, and Staphylococcus xylosus - proteolytic
bacteria .
• Insects host plants- protease inhibitors.
• Cleaving the Bt crystal proteins toxin
Visotto et al. (2009)

38. • B. thuringiensis + chitinolytic bacteria synergistic
insecticidal activity against Spodoptera littoralis (Boisduval).
Sneh et al. (1983)
• Cry1C bacterial endochitinase
(Serratia marcescens) synergistic activity -S. littoralis
Regev et al. (1996)
Cont.,

39. Case studies

40. Enzyme activity
• Case study 1
• Velvetbean caterpillar, Anticarsia gemmatalis
Visotto et al., 200940

41. Case study 2: Midgut bacteria required for Bt
insecticidal activity
Broderick et al., 2006 41

42. Restoration of B. thuringiensis toxicity
by an Enterobacter sp.
Growth of Bt, Enterobacter sp. NAB3, and E. coli ECE52 in tryptic soy broth (Left) and L. dispar
hemolymph (Right)
42

43. Midgut Microflora of H. armigera Populations From Different Locations
27 species
24 Species
7 species
2 species
1 species
AllLocations
41.8%totalmidgutflora
Case study 3: Antibiotics influence the toxicity of the delta endotoxins of Bt towards the
cotton bollworm, H. armigera
Paramasiva et al., 2014
43

44. Bt formulation
LC90 - 10.00 % (250 and 500 μg antibiotics)
LC90 - 83.33% (without antibiotics)
Cry1Ab
LC90- 6.67% (250 and 500 μg antibiotics)
LC90- 86.67% (without antibiotics)
Cry1Ac
LC90- 3.33 % (250 and 500 μg antibiotics)
LC90- 93.33% (without antibiotics)
Paramasiva et al., 2014
Effect of antibiotics in the artificial diet on mortality of Helicoverpa armigera larvae
due to Bt
44

45. Effect of antibiotics on the mortality of H. armigera larvae due to Bt toxins across
three generations
Biolep
F1 to F3  6.67 to 3.33 (250 μg antibiotics +
0.15% Bt)
F1 to F3  60.00% to 30.00% (without
antibiotics + 0.15% Bt)
Cry 1Ab
F1 to F3  13.33 to 3.33% (250 μg
antibiotics + 12 μg Cry 1Ab)
F1 to F3  60.00% to 30.00% (without
antibiotics + 12 μg Cry 1Ab)
Cry 1Ac
F1 to F3  6.67 to 3.33 (250 μg antibiotics +
12 μg Cry 1Ac)
F1 to F3  70.00% to 46.67% (without
antibiotics + 12 μg Cry 1Ac)
Paramasiva et al., 2014Paramasiva et al., 2014
45

46. Case study 4: Contributions of gut bacteria to Bt-induced
mortality of different Lepidopteran species
Larval species Bacterial species detected in guts of larvae
Family Species sterile artificial diet diet with antibiotics
Nymphalidae Vanessa cardui Lactococcus lactis
none detected
Klebsiella sp.
Sphingidae Manduca sexta Enterobacter sp.
none detected
Klebsiella sp.
Pieridae Pieris rapae Enterobacter sp.
none detected
Pantoea sp.
Noctuidae Heliothis virescens none detected none detected
Gelechiidae
Pectinophora
gossypiella
Enterococcus
casseliflavus
none detected
Lymantriidae Lymantria dispar Enterobacter sp. NAB3
none detected
Pseudomonas putida
Broderick et al. (2009)
46

47. 1. Bt+ No anti. lethal
(all 6 sps)
2. Bt +Anti 5 sps
mortality reduced
3. Bt63-100%- Vc,
Ms, Pr & Hv
4. Anti+ Bt 0-10%-
Vc, Ms, Pr & Hv
5. Cry 1Ac+ No
anti50% Ld
6. Cry 1Ac+ Anti11%
Ld
7. Cry 1Ac+ Anti33-
75% Pg
Broderick et al. (2009)
47

48. Case study 5: Gut bacterial influence on susceptibility of lepidopteran pests to
Bacillus thuringiensis subsp. kurstaki
 Cocktail- streptomycin and
rifampicin
 300 μg/ ml- S. litura
 400 μg/ ml- H. armigera,
 1mg/ml - P. xylostella
 300 μg/ - Crocidolomia binotalis.
Gadad et al., 2016
Spodoptera litura Helicoverpa armigera
Plutella xylostella Crocidolomia binotalis48

49. Influence of gut bacteria on susceptibility of lepidopteran pests to the Bacillus
thuringiensis subsp. kurstaki
Gadad et al., 201649

50. Case study 6: Benzylideneacetone, an Immunosuppressant, Enhances
Virulence of Bt Against Beet Armyworm (Lepidoptera: Noctuidae)
 Benzylideneacetone (BZA) metabolite of Xenorhabdus nematophila
 Enzyme inhibitor - phospholipase A2 (PLA2).
 Phospholipase A2 eicosanoid biosynthesis.
 Eicosanoid- Cellular immune reactions of hemocyte microaggregation
and phagocytosis etc.,
Kwon and Kim, 2008
50

51. 50 µl hemocyte suspension + 10
µl control solvent
50 µl hemocyte suspension + 10
µl (4 ppm Bta suspension)
50 µl hemocyte suspension + 10
µl (10 µM BZA)
50 µl hemocyte suspension + 10
µl (4 ppm Bta +10 µM BZA)
Kwon and Kim, 2008
51

52. Enhancement of B. thuringiensis virulence
in a mixture with BZA against the 5th instars
of S. exigua
A. BZA (250µM) and Bta (5.27 X
103 cfu/g) 100 ppm
B. BZA (250µM) and Btk (3.0 X
1010 cfu/g) 1,000 ppm
Kwon and Kim, 2008
52

53. Case study 7: Ecological consequences of ingestion of Bacillus cereus on Bt
infections and on the gut flora of a lepidopteran host
Bt + strains of B. cereus synergistic - gypsy moth (Broderick et al., 2000).
Zwittermicin A - suppression of the larval gut flora.
A reduction in the number of competitors, increase the proliferation of Bt in the
gut and cadaver, improve the binding of crystal toxins to the gut surface, or reduce
the functioning of a damaged gut.
Raymond et al., 2008
53

54. Antibiotic producing strain of B. cereus (BGSC 6A4- dark
shading) or an antibiotic negative strain (ATCC 11778-
light shading)
B. cereus synergist (strain
6A4) on cadaver
Raymond et al., 2008
54

55. Case study 8: Synergistic Effect of Entomopathogenic Bacteria (Xenorhabdus sp.
And Photorhabdus temperata ssp. temperata) on the Pathogenicity of
Bt. ssp. aizawai Against S. exigua (Lepidoptera: Noctuidae)
 Xenorhabdus and Photorhabdus  -Ve bacteria Fy: Enterobacteriaceae
 Steinernema and Heterorhabditis- deliver bacteria into target insect
hemocoel - kill insect by septicemia. (Park and Kim 2000)
 Bt - Xenorhabdus and Photorhabdus bacteria to infect the insect hemocoel
by oral application
Synergistic Effect
Jung and Kim, 2006 55

56. 68 %
65 %
X. sp. (2,000 g/ml) + Bt (1,000 g/ml)
Ptt. (2,000 g/ml) + Bt (1,000 g/ml)
Jung and Kim, 200656

57. Jung and Kim, 200657

58. Case study 9: Enhanced Toxicity of Bt. kurstaki
and aizawai to Black Cutworm Larvae (Lepidoptera: Noctuidae)
with Bacillus sp. NFD2 and Pseudomonas sp. FNFD1
• Chafer- Cyclocephala borealis  killed by B. t. japonensis
• NFD2 and FNFD1
58

59. Mashtoly et al., 2011
59

60. Case study 10: Synergistic Activity of a Bt d-Endotoxin and a Bacterial
Endochitinase against S. littoralis Larvae
 Peritrophic membrane- chitin embedded in a protein-carbohydrate matrix -
physical barrier against mechanical damage and invasion of microorganisms
(Terra, 1990)
 Chitinolytic bacteria affect - peritrophic membrane
 Endochitinases - perforation in peritrophic membrane & increase accessibility of
the §-endotoxin molecules to the epithelial membranes.
 B. thuringiensis + chitinase  insecticidal effect - Choristoneura fumiferana
larvae. Smirnoff (1977)
 Low conc. B. t subsp. entomocidus + chitinolytic bacteria  synergistic-
Spodoptera littoralis larvae
Eg:
 Pseudomonadaceae, Corynebacterium, Arthrobacter group, Streptomyces,
and Bacillus
60

61. (Sneh, 1983).
61

62. CONCLUSION
 Gut bacterial community is playing important role in the
biological activity of the Bt .
 Insecticidal activity was abolished by eliminating the
detectable midgut bacterial community.
 Insecticidal activity was restored by reintroducing an
Enterobacter spp., a member of the normal gut community.
 Management of crop pests through Bt insecticide by
exploiting the gut bacterial community of host insects.
62

63. Broderick, N. A., Raffa, K. F. and Handelsman, J. 2006. Midgut bacteria required for Bacillus
thuringiensis insecticidal activity. Proc. Natl. Acad. Sci., USA, 103:15196-15199.
Broderick, N. A., Robinson C. J., McMahon M., Holt J., Handelsman J. Raffa K. F. 2009.
Contribution of gut bacteria to Bacillus thuringiensis-induced mortality vary across a range
of Lepidoptera. BMC Biol., 7: 11.
Gadad, H., Vastrad, A. S. and Krishnaraj, P. U. 2016. Gut bacterial influence on susceptibility of
lepidopteran pests to Bacillus thuringiensis subsp. Kurstaki. International Journal of Environment,
Agriculture and Biotechnology (IJEAB), 1 (3): 581-585.
Jung, S. and Kim, Y., 2006. Synergistic effect of entomopathogenic bacteria (Xenorhabdus sp. and
Photorhabdus temperata ssp. temperata) on the pathogenicity of Bacillus thuringiensis ssp. aizawai
against Spodoptera exigua (Lepidoptera: Noctuidae). Environmental entomology, 35(6): 1584-1589.
Kwon, B. and Kim, Y. 2008. Benzylideneacetone, an Immunosuppressant, Enhances Virulence of Bacillus
thuringiensis Against Beet Armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 101(1): 36- 41.
Selected references
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64. Cont.,
Mashtoly, T. A., Abolmaaty, A., El-zemaity, M. E., Hussien, M. I. and Alm, S. R. 2011. Enhanced
Toxicity of Bacillus thuringiensis Subspecies kurstaki and aizawai to Black Cutworm Larvae
(Lepidoptera: Noctuidae) With Bacillus sp. NFD2 and Pseudomonas sp. FNFD1. Journal of
Economic Entomology, 104(1):41-46.
Paramasiva, I., Shouche, Y., Kulkarni, G. J., Krishnayya, P.V., Akbar, S.M. and Sharma, H.C., 2014.
Diversity In Gut Microflora Of Helicoverpa armigera populations from different regions in
relation to biological activity of Bacillus thuringiensis δ‐endotoxin Cry1Ac. Archives of
insect biochemistry and physiology, 87(4): 201-213.
Paramasiva, I., Sharma, H. C. and Krishnayya, P. V. 2014. Antibiotics influence the toxicity of the
delta endotoxins of Bacillus thuringiensis towards the cotton bollworm, Helicoverpa
armigera. BMC Microbiology, 14(200): 1-11.
Visotto, L. E., Oliveira, M. G. A., Guedes, R. N. C. and Ribon, A. O. B. 2009. Contribution of gut
bacteria to digestion and development of the velvetbean caterpillar, Anticarsia gemmatalis.
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64