1. The document discusses gut microflora and its role in susceptibility of lepidopteran pests to Bacillus thuringiensis (Bt). It covers types of insect-microbe interactions like pathogenic, symbiotic, and their characteristics. 2. Methods for analyzing gut microflora diversity are described, including gene targeting, molecular fingerprinting, fluorescent in situ hybridization, and metagenomics. Studies on the diversity of gut microflora in various lepidopterans are presented. 3. The role of gut microflora in influencing Bt susceptibility is explored through case studies on enzymes, siderophores, competition with pathogens, and synergistic activity with Bt. The document concludes

1. WEL-COME

2. 7
Gut micro flora and their role in susceptibility ofGut micro flora and their role in susceptibility of
Lepidopteran pests to B. tLepidopteran pests to B. t
Doctoral seminar - I

3. • Introduction
• Types of insect-microbe interactions
• Methods of gut microflora diversity analysis
• Diversity of gut microflora in lepidopterans
• Role of gut microflora
• Case studies
• Conclusion
OutlineOutline

4. Types of insect-microbe interactionsTypes of insect-microbe interactions
Pathogenic
Symbiotic

5. • Microbes associated with host insects will affects the
insect host in terms of fitness, reproductive success,
feeding and influence of other symbionts
• Displaying of potent virulence mechanisms, including a
variety of toxins, and the development of strategies to
evade the host immune system are required for the
success of the infection
Pathogenic interactionsPathogenic interactions

6. • Among the bacterial species that are able to cause
disease in insects some of them are opportunistic, such
as Bacillus thuringiensis, while others, as in the case of
Photorhabdus luminescens, are very specific and engage in
a complex lifecycle which involves being vectored by
nematodes

7. • 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 can be divided in commensalism,
mutualism and parasitism
• Commensalism occurs when the microbe, while doing no
apparent harm, benefits from the host but provides no
advantage in return
Symbiotic interactionSymbiotic interaction

8. • Mutualism is a less flexible association where the microbe and
insect mutually benefit each other
• Parasitism occurs when one species increases its fitness while the
other is harmed by the association
• Certain commensal microorganisms could be considered mutualists
• 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

9. 1. Gene targeting: gene-specific PCR
2. Molecular fingerprinting techniques
3. Fluorescent in situ hybridization
4. Meta genomics approach
Methods of gut microflora diversity analysisMethods of gut microflora diversity analysis

10. • 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 and
groups of unculturable microbes
Gene targeting: gene-specific PCRGene targeting: gene-specific PCR

11. These molecular fingerprinting techniques include
o Denaturing andTemperature Gradient Gel Electrophoresis
(DGGE/TGGE)
o Terminal Restriction fragment length polymorphisms (RFLP)
o Single strand conformation polymorphism (SSCP)
o Random amplified polymorphic DNA (RAPD)
• These techniques are usually used to analyze the sequence of 16s
rRNA from different microbial species, where both molecular
fingerprints and phylogenetic affiliation of microbial species can be
generated
Molecular fingerprinting techniquesMolecular fingerprinting techniques

12. Agarose gel electrophoresis of 16S rRNA PCR amplicon from six bacteria
isolates from gut of the larvae of laboratory populations of H. armigera

13. Bacterial species of 6 isolates from the gut of larvae of laboratory populations of
H. armigera determined with partial 16S rRNA gene sequence from BLAST
search

14. • 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 bacterial species or genus
• The approach has been shown to be particularly useful in studying
uncultivated microbes to observe the dynamics of microbiota
Fluorescent in situ hybridizationFluorescent in situ hybridization

15. Bacterial localization in the gut of S. littoralis larvae with
Fluorescent In Situ Hybridization
Tang et al., 2012

16. • Metagenomics can be defined as the study of the metagenome, the
whole genetic material of the microbial community existing in certain
eco-environments
• The ultimate goal of metagenomics is to acquire a global view of the
composition and function of the microbial community
• The proper methods for DNA extraction remain keys to reaching a
comprehensive and unbiased evaluation of metagenomes of the
community, particularly for the unculturable micro-organisms
Meta genomics approachMeta genomics approach

18. The order-based proportional composition of microbiota inThe order-based proportional composition of microbiota in
the DBM larval midgutthe DBM larval midgut
Xiaofeng Xia et al., 2013

19. Gut bacterial diversity of Gypsy moth (Lymantria dispar)
Broderick et al., 2004

20. 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
Diversity of gut bacteria in silkwormDiversity of gut bacteria in silkworm
Khyade and Marathe, 2012

21. Enteric bacteria in larvae of five lepidopteran species identifiedEnteric bacteria in larvae of five lepidopteran species identified
by 16S rRNA gene sequence analysisby 16S rRNA gene sequence analysis
Broderick et al., 2009

22. Spatial and Temporal DistributionSpatial and Temporal Distribution

23. Change of bacterial composition along the digestive tract of 5th-
instar larvae of artificial food-feeding S. littoralis
Xiaoshu Tang et al., 2012

24. Different gut bacterial community structures in S. littoralis
larvae of different instars feeding on artificial diet
Days

25. • Bacillus thuringiensis (or Bt) is a facultative anaerobic,
gram-positive, soil-dwelling bacterium, commonly used
as a biological alternative to a pesticide; alternatively, the
Cry toxin may be extracted and used as a pesticide.
• B. thuringiensis was first discovered in 1901 by Japanese
biologist Shigetane Ishiwata, most abundantly found in
grain dust from silos and other grain storage facilities
Role of gut micro flora on susceptibility ofRole of gut micro flora on susceptibility of B.tB.t

26. • In 1911, B. thuringiensis was rediscovered in Germany by
Ernst Berliner, who isolated it as the cause of a disease
called Schlaffsucht (excessive sleeping) in flour moth
caterpillars
• Bt first became available as a commercial insecticide in
France in 1938, and in the 1950s it entered commercial
use in the USA.

27. Bt subspecies can 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),
depending on their ICP composition.
Characteristics of B. tCharacteristics of B. t

28. During sporulation many Bt strains produce crystal proteins
(proteinaceous inclusions), called δ-endotoxins (Cry
proteins), which are encoded by cry genes, and have
insecticidal action
This has led to their use as insecticides, and more recently to
genetically modified crops using Bt genes.

29. • The sporulated Bt with ICP or spore-ICP complexes must be
ingested by a susceptible insect larva followed by solubilisation,
and processing from a protoxin to an activated toxin core in the insect
digestive fluid.
• The toxin core travels across the peritrophic matrix and the C-terminal
region binds to specific receptors called cadherins on the brush border
membrane of the gut cells, resulting in pore formation by the N-
terminal domain.
• Accumulation of toxin oligomers results in toxin insertion in the
membrane, pore formation, osmotic cell shock, septicaemia and
ultimately insect death.
Mode of Action of BtMode of Action of Bt

31. • Enzyme activity- Digestive enzymes of some insects might
be derived from the microbiota
• Siderophore synthesis and antagonistic activity
• Inhibition of pathogenic microbes through compitation
• Synergistic activity
Role of gut micro flora onRole of gut micro flora on

32. Case studiesCase studies

33. Enzyme activity of isolated and characterized bacterial strains.
Anand et al., 2009

34. Effect of the antibiotics chloramphenicol and tetracycline on the total viable bacteria (A)
and viable proteolytic bacteria (B) from the gut of the velvetbean caterpillar

35. Effect of the antibiotic tetracycline in the activity inhibition (%) of general proteases,
serine-proteinases , and lipases from the gut of the velvetbean caterpillar

36. Synergistic activity of antibiotic producing B. cereus on gut bacterial
abundance and reproduction of Btk in DBM
Confection treatment

37. Effect of coinfecting B. t. kurstaki (Btk rifR) with an antibiotic
producing strain of B. cereus BGSC 6A4 on the mortality of
diamondback moth larvae
B. cereus (BGSC 6A4—dark shading)
Antibiotic negative B. cereus strain
(ATCC 11778—light shading
Control insects (open bar)

38. Effect of antibiotic mixture concentration on the culturable midgut bacteria and
toxicity of B. t to Lymantria dispar
Broderick et al., 2006

39. Restoration of B. thuringiensis toxicity by an Enterobacter sp. After elimination of
detectable gut flora and B. thuringiensis activity by antibiotics.

40. Mortality induced by B. thuringiensis in droplet-feeding assays of third-
instar gypsy moth larvae that were treated with various antibiotics to
eliminate midgut bacteria
a n = 3 Replicates of 20 larvae each, b Mean cfu of entreric bacteria /larva ±SE (n = 10);
Enterococcus,1.8 ± 0.12 107, Staphylococcus, 1.2 ± 0.79 103. c 0.3 mg/ml, d 0.1
mg/ml, e 0.5 mg/ml each of gentamicin, streptomycin, penicillin, and rifampicin.
Frankenhuyzen et al., 2009

41. Contributions of gut bacteria to Bacillus thuringiensis-induced mortality
Broderick et al., 2009

42. Gut Bacteria Are Not Required for the Insecticidal Activity of
Bacillus thuringiensis toward the Tobacco Hornworm

43. ConclusionConclusion

44. Thank youThank you