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.