Silkworm has developed an efficient host defense mechanism against invading microorganisms through their immunological and genetic resistance. Immunological responses in silkworm B.mori are provided by circulating haemocytes which play an important role in innate immune mechanism such as phagocytosis, cellular encapsulation, phenoloxidase cascade and synthesis of antimicrobial proteins which are effectively engaged in defense reactions against invading pathogens . Antimicrobial proteins are the armament that insects have developed to fight off the pathogens. Several such antimicrobial proteins have been reported from silkworm B.mori like cecropins, attacins, lebocin, moricin, gloverins, lysozyme, defensins and hemolin. Insects in general are observed to respond differentially to infection by pathogens. Such differences are genetically determined and have been extensively studied in silkworm to develop resistant breeds.

1. Temperate Sericulture Research
Institute, Mirgud,SKUAST-KASHMIR

2. Disease
Resistance in
Mulberry Silkworm
Bombyx mori L.
Title:

3. Introduction
Insects are among the earliest and
most diverse taxon of animals on the
planet accounting for more species
than all other animals put together
because of their reproductive potential
and varied niche. (Purvas et al., 1992)
Insects are continuously exposed to potentially
pathogenic microorganisms like virus, fungi, bacteria,
microsporodians etc. But they have developed a
power mechanism to combat the invading
microorganisms through their innate immunity
comprising of cellular and humoral responses.
Mayhew 2007

4. • Like many other insects, silkworm is also susceptible
to a large no. of pathogenic organisms.
• The most important characteristic that determines the
commercial success of any silkworm breed is its
resistance to diseases.
• The primary defense of silkworm against pathogens
is the prevention of infection via possible structural
barriers like the integument , peritropic membrane and
midgut.
• Secondary defense is provided by the haemolymph
through cellular and humoral responses.
• The genetic resistance of silkworm to viral diseases is
mainly controlled by a polygenic mechanism.
Contd.
Watanabe ,2003

5. Avoiding Factors
Integument
Mid gut
Peritrophic
membrane
External Factors
Route of infection
Food quality
Enviroment
Immunological
Factors
( Haemocytes)
Cellular responses
Humoral responses
Genetic
Factors
 Polygenic
mechanism
Disease
resistance in
Bombyx mori

6. Expression of Resistance by B. mori
Larval Age:
Early instar larvae more susceptible than advanced
Degrees of tolerance in silkworm
Apparent tolerance
Real tolerance
Complete susceptibility
Intugmnent:
o waxy epicuticular layer (contains fatty acids)
o Epicuticular Lipids
o Epicuticular Cells (Cecropin synthesis)
(Kubera et al., 2005)
(Brey et al., 2001)

7. Peritropic membrane:
o Resistant to bacterial infection
o Prevents viral adsorption to midgut
o Barrier to entry of ingested virus to midgut
Midgut:
o Red fluorescent protein (midgut epithelium)
o Regenerative capacity of midgut cells (niddy layer of cells)
to replace infected cells.
Contd.,
(Hayaskiya et al.,1999)

8. Synthesis of RFP in silkworm mid gut
Chlorophyll -a Chlorophillidae-a
cholorophyllase
+ P252
Bm25RFP
(PROSTHETIC
GROUP)
(25 KDa
protein)
(antibacterial and antiviral)
(Chloroplast)
Ganesh et al .2008

9. Anti NPv activity of the anti-viral protein (RFP)
Set
numb
er
Treatment Set
number
of larvae
No. of larvae
severely
infected and
died (av. 0f 3
sets)
No. of larvae not
infected and
survived (av. of
3 sets)
Survival %
against NPV
infectivity
1 AVP+NPV 3×10 2 8 80
2 BSA+NPV 3×10 10 0 0
3 NPV+ Phosphate
buffer
3×10 10 0 0
4 AVP +Phosphate
buffer
3×10 0 10
5 BSA+ Phosphate
buffer
3×10 0 10
6 Phosphate
buffer
3×10 0 10
7 No. infection 3×10 0 10
Neelagand et al. 2011

10. Oral administration assay of AVP on silkworm
(pure Mysore)
S.No. Treated groups No. of silkworms
treated
% mortality % survival
1 Untreated 30×3 04 96
2 AVP 30×3 00 100
3 BmNPV polyhydra 30×3 98 02
4 BmNPV
polyhydra+AVP
30×3 00 100
Kalyankumar et al. 2010

11. External Factors Affecting Resistance / Susceptibility
 Route of infection
o Silkworm larvae are more susceptible to virus when
given subcutaneous injections than given per orally.
 Food quality
o Silkworm larvae reared on artificial diet containing autumn harvested
leaves are more susceptible to viral infection than artificial diet containing
spring harvested leaves.
Temperature
o Temperature much higher or lower than 25 o
C tend to act as stress
and increase the larval susceptibility to viral infections.

12. Chemicals
o Silkworm larvae treated with sumithion were more susceptible to per oral
infection with NPV or CPV than control.
o Larvae treated with DDT show increased susceptibility to NPV
 Synergistic Effect
o Silkworm larvae that have been exposed to bacteria show an increased
susceptibility to viral infection
o IFV and DNV have a synergestic effect on silkworm B. mori
(Watanabe , 2003)

13. Immunological Responses in B. mori
(Mohande et al., 2010)
Small Oenocytoid
Prohaemocyte Round Plasmatocyte Oval Plasmatocyte
Irregular Plasmatocyte Granulocyte Spherulocyte
Oenocytoid

14. Properties of Haemocytes in B. mori
(Ling et al., 2005)

15. Haemocyte Differentiation in B. mori
(Ling et al., 2005)

16. Role of Haemocytes in Defense Mechanisms
Haemocytes
Cellular
Responses
Humoral
Responses
•Phgocytosis
•Encapsulation
•Nodule Formation
•Haemolymph Coagulation
•Melanization
• Anti microbial protein
synthesis
•Humoral Factors:

17. Phagocytosis
Ingestion
(Pseudopodia/ membrane
invagination
Disposal
(Degranulation)
Digestion
(Lysozyme)
Exocytosis
Destruction
(Phagocyte)
Recognition
(Gotz & Bomann, 1985)

18. Process of Phagocytosis
(Salt, 1970)

19. (Gotz & Bomann, 1985)
Destruction of Bacteria by Lysosome

20. Phagocytosis of Bacteria by granulocyte
Gupta 1991

21. Phagocytosis of Bacteria by Plasmatocyte
Gupta 1991

22. Encapsulation
Multicellular
Defense
Aggregation of Haemocytes
(Granulocytes)
Attraction of other Haemocytes
(Plasmatocyte / Oenocytosis)
Release of coagulum,
Formation of Capsule (melanin synthesis)
Destruction of pathogen
Multicellular layer formation
Lyse & release of factors
(chemical signals)

23. Inactivation of Bacteria by Encapsulation
Koizumi et al, 2002

24. Combined Action of Granulocyte & Plasmatocyte
Sakamato et al, 2011

25. Nodule Formation
Multicellular defense mechanism
(Granulocytes & Plasmatocytes)
Large Doses of
Bacteria
Entrapping of Pathogens
Aggregation of Haemocytes
Destruction of Pathogens
Multicellular Sheath Formation
Release of Factors by Haemocytes

26. Inactivation of Bacteria by Nodule formation
Koizumi et al, 1991

27. Haemolymph Coagulation
Major Immune Reaction
Granulocytes & Oenocytoids
Coagulation of haemolymph
Proclotting Enzyme
(zymogen)
Clotting Enzyme
(seriene protease)
Clotting Protein
(coagulogen)
Coagulin
(clot)
Polymerization
(wound healing)
(wound Site)
(wound healing)

28. Trapping of bacteria by Haemolymph coagulation
Boman, 1999

29. Melanization
Activation of Prophenoloxidasees (zymogen)
(synthesized in haemocytes)
Release of Phenoloxidases into haemolymph
Phenoloxidase
(Tyrosine & dopa)
Oxidation & Polymerization
Melanin deposition
( A tanned insoluble material)

30. Inactivation of Fungus by Melanization
Cerenius & Soderhall, 2004

31. Humoral Responses in Silkworm B. mori
 Cecropins
 Attacins
 Lebocins
 Moricin
Gloverins
 Defensins
 Lysozyme
Lectins
Hemolin
Phenoloxidases
Antimicrobial Proteins Other Factors

32. AMP Type Main activity Effective
against
Reference
Cecropins 4 KDa Polypeptide
(40 amino acid residue)
Form ion channels in
bacterial cell
membranes
Gram +ve
Gram–ve
Steiner et al,
1981
Attacins 20 KDa Polypeptide
(32 amino acid residue)
Inhibit synthesis of
bacterial outer
membrane proteins
Gram –ve Hultmark 1983
Lebocin 3 KDa Polypeptide
(32 amino acid residue)
Bacterial cell lysis (ion
leakage)
Gram –ve Furukawa et al,
1997
Moricin 4 KDa Polypeptide
(42 amino acid residue)
Attacins bacterial cell
membrane
Gram –ve Hara et al,
1995
Furukawa et al,
1999
Gloverins 20 KDa Glycine rich Gram +ve Kawoka et al,
2008
Defensis 3KDa Polypeptide
(30 aminoacids)
cysteine rich
Act on cytoplasmic
membrane form ion
channels and cause
cell lysis
Gram +ve
Gram –ve
Wen et al,
2009
Lysozyme Hydrolyse bacterial
cell wall
Gram +ve Gandhe et al,
2007
Tanaka & Yamakawa 2011
Antimicrobial proteins from B. mori

33. Antimicrobial proteins from insects
Insect AMPs
Drosophila Drosocin , attacins,defensins,
metchikowins,drosomycin,andropin,royalisin.
Honey bee Apisimin,hymenoptaecin,apidaecins,abaecin,
definsins.
Dipterian
insects
Dipteracins, defensins,cecropins,attacins.
Lepidiopterans Defensins, attacins, cystosins, gallysins,
gloverins, lysozyme, cecropins.
Ravi et al. 2011

34. Humoral Factors
Lectins Hemolin Phenoloxidases
Two types
(260 KDa, 280 KDa)
BMLEL-1
BMLEL-3
BMLEL-2
Recently
reported
Takase et al, 2009
4 KDa
Polypeptide
Tanaka et al, 2008
Ashida et al, 1998

35. Pathogen Inheritance Genes
BmNPV Polygenic
(Diazo) Dominant
BmCPV Polygenic
(TX) Dominant
BmIFV Polygenic
BmDNV1 Monogenic (recessive)
Major dominant
nsd - 1
Nid-1
BmDNV2 Monogenic (recessive) nsd - 2
B. bassiana Dominant / major recessive cal and mus
N. bombycis unknown
Sudhakar Rao, 2006
Genetics of Disease Resistance in B. mori

36. Genetic mechanism in silkworms controlling
susceptibility to viral diseases
virus Resistant breed Susceptible
breed
Genetic
mechanism
Reference
CPV Diazo Okuso Dominant major
gene
Watanabe 1965
IFV NG H4 Recessive major
gene
Funada 1968
DNV C-124 N-124 Recessive major
gene
Watanabe and
Maeda 1978
DNV Diazo N-124 Recessive major
gene
Wantnabe and
Maeda 1981
DNV 908 J-124 Dominant major
gene
Eguchi et al.
1986
Samson and Chandrashekariah 2001

37. Disease Resistance through
Breeding
• Screening of silkworm races / lines for disease
resistance.
• Induction of diseases and selection.
• Exposure to stress conditions and selection
• Cross breeding / hybridization and selection.

38. Susceptibility of different silkworm races to NPV
Races % Gracesserie
(Natural)
% Gracesserie (artificially induced)
NB1 4.33 56.33
NB18 1.66 45.33
NB4D2 2.66 48.66
NB3C1 5.00 55.66
NB2 D1 2.50 68.66
K A 4.16 39.50
MS 2.33 52.00
PM 0.33 18.50
CB 5.83 57.50
EG 1.33 45.50
DF 6.33 46.50
CA 4.33 54.50
Baig et al, 1991

39. Breed/Prog
eny
No. of larva
inoculated
No. of
larvae
survived
No. of
larvae died
Survival % Mortality %
TX-R 400 337 63 84.25 15.75
HM-S 400 53 347 13.25 86.75
F1 400 373 27 93.25 6.75
F2 1325 1004 321 75.77 24.22
BCS 1675 897 793 53.55 47.34
BCR 1890 1688 202 89.31 10.69
Inheritance of resistance to Bm NPV in Silkworm
Nataraju et al, 2001

40. Breed/Prog
eny
No. of larva
inoculated
No. of
larvae
survived
No. of
larvae died
Survival % Mortality %
NB4 D2-S 100 0 100 0 100
C-Nichi-R 100 89 11 89 11
F1 100 88 12 88 12
F2 536 399 137 74.44 25.5
BCS 523 270 253 51.62 48.38
BCR 493 449 44 91.07 8.93
Inheritance of resistance to BmDNV-1 in Silkworm
Nataraju et al, 2001

41. Near isogenic lines of productive CSR breeds in response to BmNPV
Breed Original Stock NIL
CSR2 51 27
CSR12 68 24
CSR13 74 24
CSR4 44 31
Nataraju et al, 2001

42. Breeding process of Bivoltine breed DR-1 resistant to BmNPV
Generation Concentration of BmNPV
(POB/ml)
Larval age at inoculation Survival % of the selected batches
KA(Parent 1) 1 x 104
1st
instar -
G 133 (Parent 2) 1 x 104
1st
instar -
F1 (KA X G 133) 1 x 104
1st
instar >50
F2 1 x 104
1st
instar >80
BF2 (F2 X KA ) 1 x 105
1st
instar >50
G4 1 x 105
1st instar >70
G5 1 x 106
1st
instar >65
G6 1 x 106
1st
instar >70
G7 1 x 106
1st
instar >75
G8 1 x 106
1st
instar >75
G9 1 x 106 1st instar >75
G10 1 x 106
2nd
instar >80
G11 1 x 106
2nd
instar >80
Nataraju et al. 2001

43. CONCLUSION
Silkworm Bombyx mori has developed an efficient host defense mechanism
against invading microorganisms. However, there is paucity of information
concerning genetics of resistance to silkworm diseases especially to non-viral
diseases. Further studies are required to explore the genetic mechanism
controlling non- viral diseases of silkworm.
The indigenous Indian tropical polyvoltine races( pure mysore, nistari) showed
more resistance to diseases than temperate bivoltine races. It may be an ideal
approach to compare the expression level of anti microbial genes in hardy
polytine races with temperate bivoltine races at molecular level.
Under the existing circumstances, the use of silkworm breeds resistant to
diseases is one of the most attractive approaches for prevention of loss due to