disease resistance in silkworm -ppt
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Temperate Sericulture Research Institute, Mirgud,SKUAST-KASHMIR
Disease Resistance in Mulberry Silkworm
Bombyx mori L.
Title:
IntroductionInsects 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
• 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
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
Expression of Resistance by B. mori
Larval Age:Early instar larvae more susceptible than advancedDegrees of tolerance in silkworm Apparent tolerance Real tolerance Complete susceptibility
Intugmnent:o waxy epicuticular layer (contains fatty acids)o Epicuticular Lipidso Epicuticular Cells (Cecropin synthesis)
(Kubera et al., 2005)
(Brey et al., 2001)
Peritropic membrane:o Resistant to bacterial infectiono Prevents viral adsorption to midguto 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)
Synthesis of RFP in silkworm mid gut
Chlorophyll -a Chlorophillidae-acholorophyllase
+ P252
Bm25RFP
(PROSTHETIC GROUP)
(25 KDa protein)
(antibacterial and antiviral)
(Chloroplast)
Ganesh et al .2008
Anti NPv activity of the anti-viral protein (RFP)Set
number
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
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
External Factors Affecting Resistance / Susceptibility
Route of infectiono Silkworm larvae are more susceptible to virus when given subcutaneous injections than given per orally.
Food qualityo Silkworm larvae reared on artificial diet containing autumn harvested leaves are more susceptible to viral infection than artificial diet containing spring harvested leaves.
Temperatureo Temperature much higher or lower than 25 oC tend to act as stress and increase the larval susceptibility to viral infections.
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 Effecto 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)
Immunological Responses in B. mori
(Mohande et al., 2010)
Small Oenocytoid
Prohaemocyte Round Plasmatocyte Oval Plasmatocyte
Irregular Plasmatocyte Granulocyte Spherulocyte
Oenocytoid
Properties of Haemocytes in B. mori
(Ling et al., 2005)
Haemocyte Differentiation in B. mori
(Ling et al., 2005)
Role of Haemocytes in Defense Mechanisms
Haemocytes
Cellular Responses
Humoral Responses
•Phgocytosis•Encapsulation•Nodule Formation•Haemolymph Coagulation•Melanization
• Anti microbial protein synthesis•Humoral Factors:
Phagocytosis
Ingestion(Pseudopodia/ membrane
invagination
Disposal(Degranulation)
Digestion(Lysozyme)
Exocytosis
Destruction(Phagocyte)
Recognition
(Gotz & Bomann, 1985)
Process of Phagocytosis
(Salt, 1970)
(Gotz & Bomann, 1985)
Destruction of Bacteria by Lysosome
Phagocytosis of Bacteria by granulocyte
Gupta 1991
Phagocytosis of Bacteria by Plasmatocyte
Gupta 1991
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)
Inactivation of Bacteria by Encapsulation
Koizumi et al, 2002
Combined Action of Granulocyte & Plasmatocyte
Sakamato et al, 2011
Nodule Formation
Multicellular defense mechanism (Granulocytes & Plasmatocytes)
Large Doses of Bacteria
Entrapping of PathogensAggregation of Haemocytes
Destruction of Pathogens
Multicellular Sheath Formation
Release of Factors by Haemocytes
Inactivation of Bacteria by Nodule formation
Koizumi et al, 1991
Haemolymph Coagulation
Major Immune ReactionGranulocytes & Oenocytoids
Coagulation of haemolymph
Proclotting Enzyme(zymogen)
Clotting Enzyme (seriene protease)
Clotting Protein(coagulogen)
Coagulin(clot)
Polymerization
(wound healing)
(wound Site)
(wound healing)
Trapping of bacteria by Haemolymph coagulation
Boman, 1999
Melanization
Activation of Prophenoloxidasees (zymogen)(synthesized in haemocytes)
Release of Phenoloxidases into haemolymph
Phenoloxidase (Tyrosine & dopa)
Oxidation & Polymerization
Melanin deposition( A tanned insoluble material)
Inactivation of Fungus by Melanization
Cerenius & Soderhall, 2004
Humoral Responses in Silkworm B. mori
Cecropins Attacins Lebocins MoricinGloverins Defensins Lysozyme
LectinsHemolinPhenoloxidases
Antimicrobial Proteins Other Factors
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, 1995Furukawa 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 +veGram –ve
Wen et al, 2009
Lysozyme Hydrolyse bacterial cell wall
Gram +ve Gandhe et al, 2007
Tanaka & Yamakawa 2011
Antimicrobial proteins from B. mori
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
Humoral Factors
Lectins Hemolin Phenoloxidases
Two types(260 KDa, 280 KDa)
BMLEL-1BMLEL-3
BMLEL-2Recently reported
Takase et al, 2009
4 KDa Polypeptide
Tanaka et al, 2008
Ashida et al, 1998
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
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
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.
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
Breed/Progeny
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
Breed/Progeny
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
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
Breeding process of Bivoltine breed DR-1 resistant to BmNPVGeneration 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
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
disease in sericulture.