A comprehensive note on Advances in silkworm disease.doc

A recent advances in silkworm diseases

1. Defence Mechanism of the silkworm, Bombyx mori L
By
Shabir Ahmad Bhat
(Associate Professor College of Temperate Sericulture SKUAST-K Mirgund)
The domestication of silkworm and rearing of them in colonial form have
probably made them most sensitive to environmental, nutritional and microbial factors,
resulting in various infectious and non infectious diseases leading to silkworm mortality
and cocoon crop loss all through the year. There are 2-kinds of healthiness, one is
resistance to diseases in which diseases does not occur by infection and another is
adaptability against adverse environments. These two aspects are controlled by
Nutritional, physiological, pathological, genetical factors besides environmental
conditions. Resistance is conditioned by specific major genes and is characterized by a
high level of resistance to specific strain of pathogen. There are two kinds of resistance :-
a) vertical when a variety is more resistant to a particular pathogen rather than others and
b) horizontal - not resistant to any particular pathogen but universal to all pathogens.
Rabinson (1969) has defined resistance as the ability of the host to hinder a
pathogen or diseases causing agent where as the tolerance is the ability of the host to
hinder the occurrence of the diseases even though it is attacked by the pathogen.
According to Schafer (1971), the proliferation of the pathogen or parasite is not
necessarily impeded in a tolerant variety where as in a resistant variety, the proliferation
of pathogen it self is impeded. Thus, the underlying mechanism it self is completely
different in a resistant and a tolerant variety. According to Russel (1978), the term
resistance may be qualified by such words as high, intermediate, or low because there
may be a complete gradation from external resistance to extreme susceptibility. It can be
achieved through the exploitation of diseases resistant breeds.
Defense system in silkworms
The mechanisms involved in disease resistance largely center around the immune
response exhibited by an organism. In higher organisms this mechanism is well
developed and the resistance exhibited by them have been observed to be heritable in
several instances. However in case of lower organisms including insects the immune
system is poorly developed and the immunity acquired is transitory and it is because of
this drawback disease exploitation has not shown success in lower organisms including
silkworms. Consequently, insects have to protect themselves from the threats during their
life span like abrasive food, toxic chemicals and invasive pathogens etc. The silkworm
being a monophagous insects feds and utilizes nutrients from mulberry leaves for its

2. growth and development. Hence it is expected that the silkworm like other lepidopterans
has adopted effectively to the presence of secondary metabolites obtained from the
mulberry by evolving various mechanisms to utilize these chemicals to protect itself
against the accumulation of toxins and pathogens. The silkworm is healthy if it is not
challenged by pathogen or it should be strong enough to fight against the infection in
presence of pathogens. Though they are definitely related the exact dietary requirements
and their interactions with physiological and pathological factors involved in making the
silkworms robust however, have not been resolved clearly. Insect immunologists carried
a lot of research to operate an alternative mechanism to counter the diseases. The inbuilt
mechanism is the primary basis for their resistance to various diseases. Silkworm has
developed a kind of immunity like other phytophagous insects by resorting to various
mechanisms. The defense mechanism of insects against pathogens generally involves:-
Role of Exoskeleton
It is the mechanical barrier prevents from the invasive pathogens via possible
structural barriers like rigid exoskeleton (cuticle) and peritropic membrane that line the
midgut epithelium, surrounds the food bolus and protects it. The cuticle provides a
physical barrier against the pathogens in the environment and water loss from the body.
The existence of pro-phenoloxidase enzyme in the cuticle which is transported from the
haemolymphy has been seen to participate actively in the defence system of the insect
when ever some injury to the skin occurs. These are highly active enzymes are
responsible for melanization / encapsulation reactions commonly seen against
microorganisms and parasites. It is usually stored in inactive pro- and are activated by
the bacterial peptidoglycan. Miranpuri et. al. (1992) observed increased phenoloxidase
activity in the virus affected grass hopper (Melanoplus sanguinipes). Watanabe et al.,
(1990) found many other gut enzymes like sucrose, cellolase and phasphatases have also
antiviral activity. The peritropic membrane provides an effective mechanical barrier
against the abrasive margins of cut leaves protecting the underlying epithelial layer of the
gut. Peritropic membrane acts as the first line of defense reducing or eliminating the
absorption of certain allelochemicals. Besides, an efficient detoxification system helps to
over come this problem (Ahmad and Pardini, 1990). In silkworm, a phenol UDP-
glucosyltransferase in the peritropic membrane of gut is reported in addition to another
previously known detoxification enzyme i.e, cytochrome P-450. The main function of
this enzyme is adding glycosyl group to the hydrophobic substances in mulberry leaf to
make them water soluble so that they can be efficiently excreted from the body. In

3. addition to this it is reported to have multiple role in defending the insect like adsorption,
ultra-filtration, polyanion exclusion of ingested toxic phytochemicals and anti-oxidant
activity against the free radicals produced from metabolic activities. Such variations are
genetically determined and the difference in tolerance to pathogen is related to the
expression of the host to physical and mechanical barriers as well as the environment.
Digestive juices and their role in defense mechanism
Lepidopteran larvae demonstrate several specialization of alimentary canal the
most active epithelial transport known, called a goblet cell and highest pH values known
to be generated by biological system which protects the host from the invading pathogen.
Several larval midgut proteases have been described from different species of lepidoptera
(Ponnuvel et al., 2007). The digestive enzymes of insects are active at extremely high pH
(10-12) observed in the midgut. The intestinal proteinase was first reported by Shinoda
(1930). Studies conducted on midgut proteases of the pharate adult of B. mori and
Antheraea pernyi (Euguchi and Iwamoto, 1973) suggested their utilization as source of
the cocoon digesting enzyme. The nature of midgut protease of pharate adult and
alimentary canal silkworm larvae differs from each other in respect of higher pH 11.2
prevailing in the alimentary canal compared to pharate adult enzyme pH at 9. Further it
was reported that protease in the midgut tissues and digestive fluid increase with the
increasing feeding from 1 to 5
th
instars. Change in the proteolytic activity in the midgut
and digestive fluid content were analysed in the silkworm. The activity of enzyme
disappears in the pupal stage and reaches the peak 1 or 2 days before emergence (Eguchi
and Iwaamato 1976). It has been known that silkworms in its 5
th
instar exhibit strong
alkaline proteolytic activity in the midgut tissues and digestive fluid. The characteristic of
the antiviral substances in the silkworm gut juice against BmNPV were described by
Funakoshi and Aizawa (1989) and correlation between alkaline protease activity and
higher tolerance against BmNPV was established (Datta et al., 200) and it was suggested
that antiviral effect of the gut juice was due to proteases enzymes. It is reported that
tolerance to BmNPV infection is positively correlated with alkaline protease activity and
that the enzymes is more active in the midgut of tolerant breeds.
Red fluorescent protein
Digestive juice of silkworm exhibit antimicrobial activity and the Red fluorescent
protein (RFP) produced in the larval gut juice was first reported by Mikai et al., (1969)
was found to posses antiviral activity against BmNPV. The RFP biosynthesis was
demonstrated in vitro from 3 compartments, a protein from midgut, chlorophyll-a and a

4. basic protein from the chloroplast of green leaves. The RFP consisting of chlorophyllid-a
derived from chlorophyll-a is reported plays essential role in antiviral activity against
BmNPV. The protein was observed to bind to the prosthetic group of the chlorophyll
known as chlorophyllid-A binding protein (ChBP) a new category of proteins from
lipocalin family known as pentadecacalin. The exact mechanism of antiviral property of
RFP is unknown however it is believed that it destroys the nucleocapsid of NPV or block
the multiplication of NPV or agglutinates the virus and excreted along with faeces.
Further it needs to know the exact mechanism of the protein in inhibiting the
multiplication of BmNPV and identify the gene /s responsible for production of RFP.
Experiments revealed that RFP is detected only in the digestive juice of silkworm fed
with mulberry leaves and not found in the silkworms fed with artificial diet with out
mulberry leaf powder since chlorophyllase from the live mulberry leaves is necessary for
RFP synthesis. It was also observed that larvae reared in continuous dark are more
susceptible to per oral infection with NPV than the larvae reared in light. The increased
susceptibility may be due to the absence or lower production of RFP in the gut juice of
larvae reared in darkness.
Serine proteases
Each group of insects has their own inbuilt defensive mechanism against the
epidemics. Serine proteases are a class of enzymes characterized by the presence of an
invariant catalytic triad consisting of three amino acids i.e., His, Asp, Ser which plays an
important role in the regulation of immune and coagulation responses by proteolytically
cleaving and activating subsequent members of theses pathways. Serine proteases are
important in the interactions of insect disease vectors and parasites (Paskewitz et al.,
1999). In silkworm two types of serine proteases namely digestive proteases and non-
digestive proteases were isolated. The digestive proteases of the gut juice are mainly
involved in the digestion of food at alkaline pH i.e., (pH 10). The alkaline protease
activity of in the gut juice of BmNPV tolerant and susceptible silkworm breeds and the
midgut region shows maximum alkaline protease activity as compared to foregut and
hindgut (Ponnuvel et al., 1999). Further the increased protease activity was observed in
the digestive juice of tolerant breeds as compared to susceptible breeds. The non digestive
serine proteases are group of proteins available in the haemolymph that regulate several
defense responses including haemolymph coagulation, antimicrobial peptide synthesis
and melanization of pathogen surface and are active at neutral pH (pH 7) (Gorman and
Paskewitz, 2001).

5. Lipase
The lipase having strong antiviral activity isolated from gut the digestive juice of
silkworm and its expression confirmed in midgut and not in any other tissues (Ponnuvel
et al., 2003). It is designated as Bmlipase which suggest that an insect digestive enzyme
has potential as physiological barrier against BmNPV at the initial site of viral infection.
Some antiviral factors are known to contain fatty acids and phospholipids hence are
lipoproteins (Koizumi et. al., 1997) though generally antiviral and antibacterial factors are
proteins. A glucose binding lipoprotein is also identified from the haemolymph of pupae
which is involved in selef defence system (Ujita et. al., 2002).
Role of Nutrition
The mulberry leaf is the sole nutriment of silkworms hence its quality directly
influences its survival, growth and over all development. The chemical composition of
mulberry leaves varies with the variety, season, leaf maturity and environmental factors
apart from agrononmical practices. Phytophagous insects accumulate the potentially
harmful chemicals like phenolic, peroxides, iron etc, however the presence of
carbohydrate implants can mask the unpalatable taste of some allelochemicals and induce
feeding. When nutritional quality is poor or deficient in any one of the major nutrients, it
induces more intake resulting in the accumulation of toxic chemicals from the plant.
Peritropic membrane acts as the first line of defense reducing or eliminating the
absorption of certain allelochemicals. Besides, an efficient detoxification system helps to
over come this problem (Ahmad and Pardini, 1990). In silkworm, a phenol UDP-
glucosyltransferase in the peritropic membrane of gut is reported in addition to another
previously known detoxification enzyme i.e, cytochrome P-450. The main function of
this enzyme is adding glycosyl group to the hydrophobic substances in mulberry leaf to
make them water soluble so that they can be efficiently excreted from the body. Sugar
conjugation is a major pathway for the inactivation and excretion of both endogenous and
exogenous compounds. This newly identified enzyme is known to have wide substrate
specificity like flavonoids, coumarins, terpenoids and simple phenols (Lique et al., 2002).
In another efficient excretory mechanism oxalic acid in mulberry leaf is combined with
calcium to form calcium oxalate less toxic to silkworms which is deposited on the
integument of newly molted larvae as yellowish substance. Water content in the mulberry
leaf influences ingestion, digestion and absorption of nutrients but too high content is

6. known to affect digestion by diluting the haemolymph in addition it alters pH and normal
metabolic processes. The soluble sugar content has direct impact on the thickness of the
wax layer preventing water loss from the larval body higher the content in the leaf thicker
the wax layer will be and prevent the water loss from the organisms body. It is evident
that all the major macro and micronutrients influence the disease resistance in the
silkworm directly or indirectly. If the nutrients are deficient in leaf during rearing then the
chances of diseases particularly viral and bacterial diseases are more. It is also reported
that mulberry receives excessive nitrogen application or less sunshine contain high
nitrates and free amino acids like arginine that affect larval metabolism and decreases the
tolerance in silkworms, however higher content of aspartic acid in the mulberry leaf is
known to increase the ingestion in the silkworms.
Among the vitamins, vitamin B series and vitamin C are indispensable for silkworm
growth. Vitamin C is phagostimulant and is known to enhance the survival. Minerals like
Potassium and Calcium, Megnesium, Iron and Phasphorus are important to silkworm
nutrition and their content in the feed is known to influence susceptibility to diseases. The
contents of potassium and phasphorus in haemolymph are important as these ions help to
maintain the blood pH at optimum levels ranging from 6.2-6.8. It is also experimentally
proved that high protein content in the mulberry leaf induces the protease activity in the
host gut which inactivates nuclearpolhedrosis virus. The contents of organic acids in the
mulberry leaf is known to vary and higher contents of oxalic acid, succcinic acid etc are
detrimental to health of the silkworms. Poor hygrothermic conditions and combined with
poor quality feed ( low in proteins, sugars and high in cellulose contents) are reported to
be one of the predisposing factors which render silkworm susceptible to bacterial
flacherie and cytoplasmic polyhedrosis (Ono et. al., 1969; Ebihara, 1966).The
quantitative requirements of nutrients are as important as its quality. It is reported that
silkworms fed with mulberry leaf low in protein, sucrose but high in fiber render
silkworms susceptible to diseases (Watanabe et. al., 1990). The larvae become
susceptible to disease like bacterial flacherie and grasserie due to malnutrition and
starvation leading to crop losses even in ideal conditions. The starvation and / or infection
with pathogens decreases the pH of haemolymph which makes them susceptible to
diseases.
Role of Phytochemicals (Secondary metabolites)
Recent development on the role of secondary metabolites in insect nutrition and
survival have thrown light on many interesting facts about plant insect interaction. Plant

7. allelochemicals are non-nutrients but can interact with essential nutrients to play a role in
their assimilation (Reese, 1978), apart from influencing survival of phytophagous insects.
Phytochemicals of mulberry leaf like phenolic acid, tannins, flavonoids, terpenoids etc
don not posses direct nutritive value but they are known to act as anti-oxidant compounds
hence are important for maintenance of health of the insect (Felton and Summers, 1995).
A strong correlation is shown between levels of certain phytochemicals and susceptibility
to viral infection in phytophagous insects (Duffey et al., 1995). Felton et al., (1989) have
attributed the phytochemicals which are not major nutrients, a multifunctional role in the
growth and survival of insects. The primary low molecular weight antioxidants in plants
and animals are phenolic acids, ascorbic acids, -tocopherol and tripepetide glutathione
ᾱ
(Barbehenn, 2003). One of the phenolic acids is cholorogenic acid which is known to be a
precursor of antibacterial protein in silkworms (Ito, 1978). The activity of anti-
streptococccus protein in the digestive fluid was influence by the presence of caffeic acid
in mulberry (Xu et al., 1994). The phenolic compounds in mulberry also acts as feeding
stimulant / deterrents as reported by Uno (1982) who found that the addition of methyl
caffeate, ethyl -resorcylate, -resorcylic acid and caffeaic acid to artificial diet increased
ᵝ ᵝ
feeding efficiency however 5-7-dihydroxychromone known to be anti-microbial in nature
but had feeding deterrent effect.
The three complex concepts in defense mechanism against viral diseases
involving phytochemicals are as:-
Multiplicity: Viral disease is simultaneously influenced by more than one phytochemical
each exerting its effect simultaneously.
Interactivity: A chemical does not influence viral disease independently of other
chemicals. It interacts with other dietary components of the feed.
Multifunctionality: A chemical often exhibits multiple mechanisms of action in
combination with other chemicals or with baculoviruses.
Role of supplementation to enhance the survival and metric traits
Nutritional quality of feed not only plays important role in the robust growth and
development of silkworms, improvement of the commercial characters, reproductive
potential but also plays vital role in tolerance. Good nutrition favors tolerance to infection
while poor nutrition renders the host more susceptible to the disease. The major leaf
components such as nitrogen and water contents decide largely the growth of an insect, if
any deficiency occurs in the quality and quantity of mulberry leaves, imbalance in
silkworm physiology, growth and development occurs that weakens the larvae and makes

8. them prone to infection.Number of studies are available on increasing the yield through
nutrient supplementation but studies to increase the tolerance against diseases in
silkworms through chemicals / botanicals supplementation to the mulberry leaves are
very limited. Krishanaswami et al., (1980) reported chloramphenicol fortified leaf
increased larval weight and cocoon weight besides the survival. Supplementation of low
amounts of urea (0.2%) and ascorbic acid (0.05%) increased the leaf consumption and
survival where as higher dose of vitamin C is reported to cause ,mortality (Javeed and
Gondal, 2002). Similarly the supplementation of higher doses of nicotiamide increased
larval mortality due to hypervitaminosis and indicates that one should be careful in
deciding the dose required to avoid the deleterious effects of vitamins and minerals
which are required in micro quantities for the silkworm. Analogues of Benzimidazole and
Benlate, Bavistin, Derosal, Fumidil - B or Fumagillin, Methylthiophanate, Ethyl
thiophanate and Anisomycin have been reported to enhance the survival in the silkworm
infected with pebrine disease. Of the several therapeutic drugs, 0.2 percent Bavistin or 0.2
percent Benlate when fed to silkworms with mulberry leaf twice a day have been found
effective in controlling N. bombycis infection (Chandra and Sahakundu, 1993). Different
quinine derivatives viz., Chloroquine, Primiquine, and Sapraquine were also found
effective in increasing the survival to the extent of 96.46 percent in silkworm.
Benzimidazole derivatives viz., Albendazole and Mebendazole have also been found
effective against microsporodiosis at 0.25-1 percent concentration when fed to infected
silkworms during 5
th
instar on alternate days through mulberry leaf till onset of spinning,
reduced the larval mortality to the extent of 100 percent but the infection at the moth
stage has not been found eliminated completely (Bhat, 2006). Benzimidazoles are
effectors of microtubule polymerization being unique in selective toxicity to
microsporidians. The -tubulin subunit has been identified as the primary target of
Benzimidazole such as albendazole that acts by disrupting microtubules through -tubulin
binding and six different amino acid residues have been implicated in benzimidazole
susceptibility by mutational analysis. The microsporidia viz., E. hellem and E. cuniculi -
tubulins include His-6, Phe-167, Glu-198, Phe-200, and Arg-241, predicting that these
microsporidia are susceptible to benzimidazoles (Katiyar et al., 1994). Ultra- structure
evidences of the infection of carbendazim have shown an adverse effect of the drug on
both merogonic and sporogonic stages of the microsporidian spore in the midgut and in
the silk gland of the silk worms. The drug causes elongation, vaculation and deletion of
cytoplasmic contents of the meronts, spronts, sporoblasts and other spore stages of the N.

9. bombycis (Jyothi et al., 2005).The exposure of microsporidia spores to albendazole has
shown clumping of chromatin in the nuclei, inhibition of spindle formation, and
enlargement of nuclei, disruption of the nuclear membrane and malformation of spores of
N. bombycis. The mode of action of albendazole and the related benzimidazole
derivatives have also been seen to prevent microtubule assembly which in the case of
susceptible microsporidian species inhibits the formation of intranuclear spindle, the only
known case of microtubule formation in microsporidians. These drugs bind with subunits
of RNA polymerase and block the transition from the chain initiation phase to the
elongation phase. The drug streptomycin specifically inhibits chain elongation by the
microsporidia RNA polymerase.Aqueous / Solvent extracts of different locally available
botanicals are also reported to increase the economic traits and also decrease the mortality
caused by different pathogen in silkworms (Patil, 2005, Bhat and Kamilli, 2014,
Nayeema and Bhat, 2015). The aqueous extracts of ajowan have been found effective not
only in preventing larval mortality and suppression of infection at moth stage but also
help in improving the economic characters of the Lamerin silkworm breed (Bhat, 2006).
Thymol an active ingredient of ajowon is reported to be effective in the control of
microsporidian disease in Apis mellifera (Honey bee), Rice, (2001). However, it is
reported that the chemical or botanical treatment do not eliminate the infection
completely in one generation and may require 10-12 generations to control the infection
completely. Solvent extraction of Misteltoe Viscum album-a semi-parasitic plant of
temperate himalaya are known to increase various economic parameters viz., fifth instar
larval duration, total larval duration, average larval weight, cocoon yield by number,
cocoon yield by weight, single cocoon weight, single shell weight, shell ratio, pupation
rate and reduced mortality when fed to the Bombyx mori nuclear polyhedrosis virus
infected silkworms. The plant is easily available, environment friendly, cheap in
production and safe to user. Aqueous leaf extracts of Morus alba L., Tribulus terrestris
L., Vetiveria zizanoides Nash and Psoralea coryleifolia L. were found to enhance the
economic characters in B. mori (Rajashekhargouda, 1991). Plants like P. coryleifolia and
T. terrestris (Sameul Manoharraj, 1994), Acacia suma, Caesalpinia coriaria and
Terminalia tomentosa (Manoharan, 1996) were found to enhance the tolerance against the
grasserie when the aqueous extracts aree applied on mulberry leaves before feeding to the
silkworm. These botanicals may not contain major nutrients but certainly contain some
phyochemicals / allelochemicals which might influence the larval metabolism or inhibit
the pathogen development thus enhances the survival.

10. Pathological studies
Different factors in which the silkworm breed is reared including pathogenic load, age
and stage of silkworms may also affect the host tolerance. The lethal doses cause
mortality in shorter time however the sub lethal doses may prolong the mortality similarly
young age silkworms are less tolerant as compared to late age worms owing to their
softer mesenchymal tissues. Young age larvae (Chawki worms) are highly sensitive to
sub-lethal doses of infection as compared to late age worms which may sustain the sub-
lethal doses and spin cocoons also. European silkworm breeds are more susceptible to
infection followed by Japanese and Chinese breeds (Govindian et al., 1998). Bivoltines
have been reported to be more susceptible as compared to multivoltines. Among various
bivoltine breeds studied, NB7 has been found more susceptible followed by NB4D2 and
KA (Patil and Geethabai, 1989). The pathogen multiplied at the same rate in the larvae of
susceptible and resistant breeds but the infected columnar cells are discharged during
moult and replaced by regenerated cells but the degree of epithelial regeneration varied
with sustable and resistant strain of B. mori and dosage of virus/ pathogen. Kobara et al
1967 showed the susceptibility to CPV was greatest immediately before and after moult
but decreased considerably during the intervening period.
Genetic basis for tolerance against diseases
Insects in general are observed to respond differentially to infection by microbial
pathogens (Weiser, 1969). Such differences are genetically determined and have been
studied extensively in silkworm to develop resistant breeds (Tanada and Kaya, 1993). In
silkworms, large difference exists among various strains in their tolerance to infections of
BmNPV, BmCPV, BmIFV, BmDNV, Beauveria bassiana, and Nosema bombycis (Aruga
and Watanabe, 1964, Aratake, 1973,Watanabe, 1966, Baig et al., 1991; Nataraju, 1995).
The difference in susceptibility among the most susceptible and highly tolerant/resistant
races ranges from 875, 2000, and 10,000 fold for BmNPV, BmCPV and BmDNV
infections respectively (Liu Shi Xian, 1984). However, all the silkworm breeds have
been found more or less susceptible to microbial attack and no breed has been found
resistant to the infection. Chinnaswamy and Devaiah, (1984), have reported large
difference among various breeds in their tolerance to infections. The mechanisms
involved in diseases resistance largely center on immune responses exhibited by the
organisms. In the higher group of animals these mechanisms are sufficiently developed

11. and the resistance exhibited by them has been observed to be heritable in several
instances (Samson and Chandrashekaraiah, 1998). However, natural resistance / tolerance
to certain pathogenic agents may have been seriously reduced or even lost due to human
intervention for a highly domesticated species such as the silkworm. And if not
completely lost, the breeders may still be able to select representative individuals in the
right gene combination for strengthened resistance or tolerance to a certain disease or
diseases. Then, the gene combination can be incorporated into genetic backgrounds of
commercial varieties for cocoon production.
Host parasite interaction is a dynamic equilibrium. Under normal conditions, the host
and the microorganisms exist in balance but any decrease in the host tolerance or increase
in the pathogen load can result in the development of the diseases.The genetic flexibility
of pathogenic agents remains as one of the most difficult barriers in developing and
maintaining resistant or tolerant breeds successfully in long run (Lea, 1993). Any
resistance of polygenic nature may require considerable time, effort and resources before
it can be fully utilized in commercial cocoon production. Resistance against silkworm
viral diseases (BmNPV, BmCPV, BmIFV) are controlled by polygenes with exception to
BmDNV (Watanabe and Maeda, 1981) that is controlled by recessive monoges. Most
silkworm strains are non-susceptible to BmDNV1 while others are susceptible
(Watanabe and Maeda, 1981). The non-susceptibility of silkworm to BmDNV1 infection
is determined by a recessive gene (nsd-1) which is located on the 21st chromosome at
8.3 positions (Watanabe, 1980). A dominant gene (Nid – 1) controlling the non-
susceptibility of silkworm to BmDNV1 is also recorded (Eguchi et al, 1998). The non-
susceptibility of silkworm to BmDNV2 is controlled by a recessive gene, nsd-2 (Seki,
1984). The nsd-2 gene is located on a different chromosome and on the one, which
contain either the nsd-1 or Nid-2 gene. The non-susceptibility to the Chinese isolate,
BmDNV-Z is controlled by recessive gene nsd-Z located on the 5
th
chromosome. Several
silkworm genetic resources in India, China and Japan have been screened for resistance to
BmDNV and genetic resource materials have been identified. With this it is possible to
evolve breeds resistant to BmDNV controlled by single gene (Yiuan et al 1999). In Japan
and hybrid race, Taisei, resistant to BmDNV1 have been evolved crossing BmDNV1
resistant breed C 150 and high yielding N203. A strain of silkworm, Diazo is also
resistant to BmCPV infection; where in the susceptibility to the pathogen is controlled by
a dominant major gene (Watanabe, 1965, 1967). There are reports of difference in
susceptibility of silkworm breeds to white muscardine caused by Beauveria bassiana

12. (Aratake, 1961). The major genes for resistance to the pathogen are located on the 7 and
11
th
chromosomes. The comparative studies of DNA linkage map with conventional
phenotypic map indicated that the resistance is linked to chitinase genes especially
‘calcina’ and ‘muscardine’ gene. It is probable that the chitinase gene is responsible for
the resistance to Beauveria fungus with linkage between the chitinase genes and fungus
resistance (Shimada, 1999). Gene responsible for bacterial toxicosis and
microsporidiosis are yet unknown, though it is reported a silkworm breed “Baipidan is
said to have some resistance to N. bombycis infection (Nataraju et al., 2005). Another
silkworm breed “Lamerin” from North Eastern part of India survives with microsporidia
infection for the past several generations without eliminating the breed (Bhat and
Nataraju, 2007).Young and weak silkworms are more susceptible to the disease and show
high mortality and the worms coming out of moult are also most susceptible to the
infection because the peritrophic membrane of midgut is sloughed off during moulting
leaving the larvae more vulnerable to infection. Various reasons given below have been
attributed to the difference in tolerance. Inadequate digestion of seal covering the polar
filament and host resistance to infection by actively destroying the microsporidian during
its migration to tissues or by elimination of infected cell by the host, failure of spores to
germinate in the digestive tract because of unsuitable pH and enzymes (Weiser, 1969).
Pure Mysore, a silkworm race infected with microsporidia has been found to survive for
longer duration than other infected silkworm races. This is attributed to high regenerative
capacity of midgut of Pure Mysore to recover from the infection (Fujiwara, 1993). Wild
silkworm, Antherea pernyi and Platysamia cercropia are also comparatively resistant to
microsporidia infection (Weiser, 1969).
Table 1: Genetic basis and nature of resistance to pathogens infecting silkworm,
Bombyx mori L
Pathogen Type Inheritance Genes Variations
BmNPV QuantitativePolygenic - Small
BmCPV QuantitativePolygenic - Small
BmIFV QuantitativePolygenic - Large
BmDNV1 Complete Monogenic(recessive) Nsd-1 Absolute
BmDNV2 Complete Monogenic(recessive) Nsd-2 Absolute
BmDNVZ Complete Monogenic(recessive) Nsd-zAbsolute
BmDNV-Ind Complete Monogenic (recessive) Nsd-1 Absolute
B. thurinigiensisQuantitative - - -
B. bassiana QuantitativeDominant Major calcina and
muscardine
-
N. bombycis (Unknown) - - -

13. Development of disease resistance in silkworms
Diseases resistance is an important complementary objective of silkworm
breeding because high degree of susceptibility will generally results in decreased yield
and quality. Resistances to diseases result in stability of crop performance and increases
in productivity both qualitatively and quantitatively but susceptible to diseases is
considered to be inferior. It can be achieved through exploitation of diseases resistant
breeds. Therefore, screening of different breeds based on the magnitude of such
responses exhibited by them could serve as base for breeding resistant breeds in an
economically important insects like silkworms. Utilization of resistance/tolerance,
prevailing in genetic resources, having moderate to high economic traits, is an
economical and eco-friendly approach in management of diseases in agriculture and
veterinary field. The approach consciously avoids, altogether the susceptible varieties
hence the disease occurrence will be nil or below economic injury level. Several
epidemiological and genetical studies demonstrated that the silkworm races have
different level of susceptibility / tolerance to various pathogens. Several silkworm
indigenous resources in India, China, Japan, Iran and Thailand have been screened for
their resistance to silkworm viruses viz., BmDNV, BmCPV and BmIFV, bacteria, fungi
and microspodians. The response varies from one breed to another and are classified into
high, medium/ intermediate or low depending upon the magnitude of resistance. The
selection of tolerant breed can be made by direct selection or indirect (batch) selection. In
direct selection the breed is inoculated with known dose of pathogen load and the
survived individuals will be continued till emergence and egg laying. The larvae from
these laying are again given sub-lethal doses till the apparent tolerance is achieved. This
kind of selection is lengthy, difficult and some time there is risk of losing the tolerant
stock if the pathogen dose becomes higher. The indirect or batch selection is that type
where identification of breed tolerance through per oral inoculation and selection batches
from corresponding untreated population and continuation for further breeding. There is
no risk involved in this selection but the improvement in tolerance may be minimal. The
tolerant breed is always hardy and inferior in economic characters. Then the tolerant
(Donor) breed is crossed with the productive (Recurrent) ones and the gene combinations
of the two could be incorporated in single breed.

14. The inheritance controlled by polygenes could be developed after prolonged
selection under exposure to the respective virus for several generations. The process of
selection changes the genes frequency for viral resistance in the population and causes it
to increase. This could be achieved by batch selection and individual selection. In the
batch selection, sample larvae from each batch are tested for susceptibility to a virus. As a
result, the batch showing the highest resistance to the virus is selected for further
breeding. In the individual selection, the larvae from mixed population are exposed to the
pathogen and the surviving individuals are further exposed to the increased concentration
of virus. A selection pressure of 70- 80% is preferable. However there is risk of loosing
the strain in breeding due application of high selection pressure. Selection of silkworms
resistant to BmIFV and BmCPV was attempted successfully by individual selection
method for several generations by several workers (Uzigawa and Aruga, 1965; Funada,
1968; Watanabe, 1967). 10-16 fold increase in resistance was reached in breed
resistant to BmCPV. High hybrid vigor was obtained in crosses among selected breeds.
There had been several attempts to select silkworm strains tolerant to BmNPV infection.
Silkworm strain resistant to the induction of BmNPV was obtained after 19 generation of
selection under cold stress (5°C) treatment. A race No. 209A resistant to BmNPV was
developed following the low temperature treatment (Aizawa et al, 1961). Attempts have
also been made to induce resistance in silkworm against specific infections and the
attenuated viruses and anti- Virus serum have been observed to induce resistance to
infection in silkworm. Anti- BmNPV serum and attenuated BmNPV suppresses the
development of nuclear polyhedrosis (Nataraju, 1995). The response is short lived and
triple vaccination using attenuated BmNPV viruses, once at each instar suppressed the
nuclear polyhedrosis to an extent of 81.85% (Nataraju et al., 2000) in silkworm
population. Sivaprasad et al. (2003) studied relative tolerance of various geographically
different silkworm breeds against BmNPV and classified them into apparent and real
tolerant breeds on the basis of their relative tolerance. Yao et al. (2003) studied the
tolerance of the silkworm, B. mori to BmNPV and reported that silkworm strains having
high tolerance to BmNPV have improved yield traits.
Aratake (1973) succeeded in evolving a strain resistant to IFV by secondary
inoculation followed by selection of tolerant ones and Nataraju et al (2001) succeeded in
introducing the medium level of resistance in KA (which was highly susceptible to
BmNPV) by utilizing another diapasusing stock which was found to have moderate level
of resistance and evolved a breed DR1 resistant to BmNPV.

15. The gene combination identified from the hardy breeds can be incorporated into
genetic backgrounds of commercial varieties through crossing followed by selection. The
work will be undertaken for its effective conclusion by following the below mentioned
standard methodology:-
Screening of silkworm breeds for disease tolerance (BmNPV)
Induction of diseases and selection of tolerant ones
Short-listing of tolerant / hardy breeds
Evaluation and identification of promising ones
Screening of silkworm breed for disease tolerane: Before initiating the breeding
experiment, a number of breeds should be collected to have more choice of selection.
While collecting, importance should be given to local material. After collection, the
different inbred lines are screened for their relative susceptibility by artificially disease
induced method in order to get desired breed or line which can be utilized in the
evolution of disease resistant breed /s. A number of studies have been carried out by
various workers (Aratake., 1973. Nataraju et. al., 2001., Sivaprasad et al., 2003., Yao et
al., 2003., Bhat et. al., 2016).
Induction of diseases and selection: For artificially induced method, the pathogens are fed
to silkworms perorally so as o induce the disease for identifying the disease resistant lines.
The same treatment is repeated for successive generations till the desired result is achieved.
In this method the insect acquire immunity / resistance after several generations. It has been
reported that Pure Mysore race acquire 12% more resistance for Bacillus thurengiensis after
seven generations of continuous rearing with sub-lethal doses. Japanese scientists succeeded
in producing a flacherie disease resistant breed after five generation and reported 20-30%
change in resistance.
Exposure to stress condition and selection: An epidemic of the pathogen has close link
with climatic factors such as temperature, humidity, air current and also food the environment
to which the insect is exposed also plays an important role in the manifestation of resistance.
It is easy to test the resistance under adverse environment than the good conditions,
nutritional deficiencies, air-tight rearing room and exposure to certain chemicals. Aizawa and
co-workers treated B. mori under stress conditions like cold, starvation, certain chemicals etc
which induce the development of an apparently latent viral infection in the larvae that had not
been fed with virus. Selection of a line for resistance to such resistance induction were
undertaken by subjecting the larvae to cold stress for 24h before each moult and the breeding
from the survivors which did not succumb to the induced virus. After 11 generations

16. consistently, only few fewer larvae died from the viral infection in the selected line than the
corresponding control line.
Cross breeding / hybridization and selection: Hybridization and selection is also one of the
means to develop a disease resistant breed. The parental stocks used as breeding resource
material must be disease resistant or comparatively resistant. While breeding the breeder has
to take into account the relationship between the economic characters and resistance to
disease and adverse conditions. The parental stocks, which are disease resistant, are always
associated with low quality and low input of silk. When such types of stocks are selected as
one of the parents, another stock with middle level disease resistance and better silk quality
should be the minimum requirement for crossing. These two stocks are complementary in
cross breeding and the resultant will be a new breed with combination of disease resistance
and high quality silk. Aratake (1973) succeeded in evolving a strain resistant to IFV with
the cross N131x C132 and subsequently selected for 10generations. Nataraju et al (2001)
have chosen a breeding progamme with recurrent back crossing with adapted parent and
exposure of the progeny to the pathogen to select the resistant progeny and was able to
introduce the medium level of resistance in KA which is highly susceptible to BmNPV by
utilizing another diapasusing stock which was found to have moderate level of resistance and
evolved a breed DR1 which was utilized as a substitute to KA.
Similarly Nataraju et al., (2001) attempted to introduce resistance in the productive
CSR breeds which are excellent in their cocoon characters but highly susceptible to BmNPV
and environmental fluctuations. By crossing with BmNPV resistant stock TX as a donor
parent and six back crosses with respective CSR stocks followed by the selection of
homozygous stocks emerged after three generations of sib matting, evolved the near isogenic
lines (NIL) of the CSR breeds. Selection of the resistant progenies was done by exposing
them to the pathogen and selecting the survival ones.
The general techniques / precautions used to breed disease resistant silkworms has three
steps:-
Inoculation to genetic stock should be done under favorable conditions like dosage,
highly purified inoculum, genetic stock with resistance to particular pathogen or
mixture of races for which resistance is required.
Each breed / line may be given a score according to the survival
The most resistant breed / s are selected and separate test is conducted on offspring
for each line
Follow the strict rules of sanitation
Good rearing conditions and quality feed

17. Time must be considered as an important factor in breeding for disease resistance.
Bio Technology approaches for development of Transgenic BmNPV resistant silkworm
hybrids
The conventional breeding method may have limitation but a multi-disciplinary
approach utilizing the recent advances in biotechnology could be a better alternative. Most of
the economic traits of silkworm are under polygenic control. The DNA profile of silkworm
stocks showing consistent phenotypes for productivity and quality were analyzed and the
DNA markers were identified and progeny selection for both high productivity and high
fecundity carried out by DNA marker assisted selection. After identification of DNA
markers linked to the targeted traits, marker assisted selection employed in the breeding
programme that reduced the breeding cycles, for improving the efficiency of selection
particularly at the early stage.
CDFD, Hyderabad and APSSRDI, Hindupur in collaboration with University of
Claude Bernard, Lyon, France have shown successfully germline transfer of anti NPV genes
(single and multi-genes) in silkworm which have shown high tolerance to NPV. Large
number of transgenic silkworm strains were developed through biotechnological approaches
to meet the requirement of present day silk industry.
Nineteen polyvoltine novel transgenic Nistari silkworm lines (donor stock) and were
utilized for transfer of BmNPV resistance genes into the productive breed CSR2 (bivoltine).
This has resulted in 10 bivoltine CSR2 transgenic lines. Among them the transgenic 727 line
used as a donor parent to introgress baculovirus gene to other bivoltine breeds namely CSR4,
APDR115 and APS12.
During maintenance the standard rearing technique and operational procedures
followed. Besides, the wild type BmNPV which is prevalent in farmer’s silkworm rearing
house was used for infection studies (BmNPV 40000 OBs per larva) and reared in separate
rearing house with respective control. Mortality was recorded up to moth emergence. The
batch selection of lines was effected based on visual observation and cocoon traits. The inter-
batch cross system meticulously followed in each cycle is maintained the cocoon traits and
vigor of the lines. Currently large scale exploitation of these transgenic lines is under
progress to bring about economic transformation of sericulture.

19. Table 2: Infectious diseases of the silkworm Bombyx mori, L .
Disease Causative agent Pathogenicity Symptoms
Viruses
Nuclear polyhedrosisBombyx mori nuclear
polyhedrosis virus (BmNPV)
Chronic Swell on inter segmental region shinning and fragile skin, milky
white fluid.
Cytoplasmic
polyhedrosis
Bombyx mori cytoplasmic
polyhedrosis virus
(BmCPV)
Chronic Translucent cephalothoraxic region; diarrhea; retarded growth;
milky white midgut; whitish feaces.
Infectious flacherieBombyx mori infectious
flacherie virus (BmIFV)
Chronic Translucent cephalothoraxes; retarded growth; vomiting and
diarrhea.
Densonucleosis Bombyx mori densonucleosis
virus (BmDNV)
Chronic Translucent cephalothoraxes; retarded growth; vomiting and
diarrhoea.
Mycosis
White muscardine Beauveria bassiana Chronic Oily specks on the body surface; larva on death softens, turns hard
and latter mummifies; mummified larvae appear white.
Green muscardine Spicaria prasina (Nomuraea
riyali), Metarhizium
anisopliae
Chronic Large specks with black periphery; mummified larvae green in
color
Yellow muscardinePacilomyces farinosus Acute/
Chronic
Large diseases specks around stigma and small on skin ,
mummified larvae yellow.
Red muscardine Sporosporella urella Acute/
Chronic
Develop red patches few hours before death; no external growth.
Bacterimia
Bacterial diseases of
digestive organ
Streptococci / Stephlococci spChronic Sluggish movement; retarded growth; transparent cephalothoracic
region
Septicemia Bacillus sp., Streptococci
sp., Stephylococci sp.,
Serratia marcescens
Acute Sluggish movement low appetite; swollen thorax; shrinkage;
vomiting softening and discolored body.
Toxicosis Bacillus thringiensis var.sottoAcute/
Chronic
Sluggish movement; retarded growth; caesation of feeding ;
vomiting ; paralysis and death; corpse stretch and cephalothoracic
region bend like hook.
Microsporidiosis Nosema bombycis Chronic Flaccid larvae; retarded growth; white pustules all along the
length of the silk gland

20. Table 3: Non-infectious diseases of silkworm Bombyx mori
Diseases Causative agent Symptoms
Arthopod diseases Exorista sorbillans
Euproctis similes
Setora postomata
Black scar on the body
Poisoning Poisonous chemicals Vomiting gut juice; shrinkage
Physiological ailments Poor nutrition, high & low temperature
and humidity
Flaccidity
Table 4: Viruses pathogenic to the silkworm, Bombyx mori L
Pathogen Family Insect host order
Particle
symmetry
Nucleic acid
Inclusion
body
Bombyx mori nuclear polyhedrosis
(BmNPV)
Baculoviridae Lepidoptera, Diptera
Hymenoptera,
Helical dsDNA +
Bombyx mori cytoplasmic
polyhedrosis virus (BmCPV)
Reoviridae Hymenoptera, Diptera,
Lepidoptera
Isometric dsDNA +
Densonucleosis viruses Bm(DNV)Parvoviridae Lepidoptera Isometric SsDNA -
Flacherie viruses (BmIFV) PicornaviridaeLepidoptera, Orthoptera
Hymenoptera, Coleoptera,
Isometric Ss RNA -