IJBPAS, June, 2015, 4(6): 3997-4006

ISSN: 2277–4998

EFFECTS OF INDUCED TOLERANCE AGAINST NUCLEAR POLYHEDROSIS ON

ECONOMIC CHARACTERS OF SILKWORM BOMBYX MORI L. AND THEIR
INHERITANCE

MAHESHA HB1* AND THEJASWINI PH2

1: Department of Sericulture, Yuvaraja’s College, University of Mysore, Mysuru-570005,
India
2: Department of Biotechnology, Maharani’s Science College for Women, Mysuru-570005,
India
*
Corresponding Author: E Mail: hbmseri@yahoo.com
ABSTRACT
To study the effects of induced tolerance in silkworm Bombyx mori L. against nuclear
polyhedrosis virus two mulberry silkworm breeds viz., Pure Mysore and NB4D2 were
selected. The breed susceptibility was determined during fifth instar. The silkworms were fed
with known amount of inactivated nuclear polyhedral inclusion bodies (NPIBs) thrice at
various time intervals for induction of tolerance followed by the challenge inoculation with
known amount of live NPIBs. The silkworm larvae of the control, induced, induced followed
by live PIBs inoculated and live NPIBs inoculated batches were allowed to continue
development. The breed susceptibility, effect of polyhedrosis as well as induced tolerance on
economic traits and their inheritance were studied. The induced and induced followed by live
NPIBs inoculated batches exhibited almost similar pattern as in case of control larvae. This
result paves light towards development of a practical control measure to be applied during
mass rearing against the dreaded nuclear polyhedrosis in silkworm Bombyx mori L.
Keywords: Silkworm, Induced Tolerance, Nuclear Polyhedrosis Virus, Economic
Characters, Inheritance
INTRODUCTION
The silkworm, Bombyx mori, lost its natural domestication since time immemorial.
resistance against varieties of disease These pathogens caused an annual crop loss
causing germs because of continuous up to 40%, which runs to the loss of several

3997
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

crores of rupees. Of all silkworm diseases, tolerance against BmNPV on commercial

which cause damage, viral diseases are the characters and their inheritance from
most serious. In India, the silkworm cocoon generation to generation.
crop loss due to nuclear polyhedrosis has MATERIALS AND METHODS
been reported to an extent of 32.9-55.3 % Two mulberry silkworm breeds namely
among the total silkworm diseases [1] and Pure Mysore (multivoltine) and NB4D2
most common in summer season [2]. (tropical bivoltine) were selected for the
Therefore, some attempts have been made present investigation. Disease free layings
to immunize insects with NPV. Carbone and of pure breeds were obtained from the
Fortuna [3] and Gargiulo [4] vaccinated Germplasm, Department of Studies in
silkworms in large scale against jaundice Sericultural Sciences, University of Mysore,
virus. Aizawa [5] observed some degree of Mysuru, India. The silkworm rearing of
protection when silkworm pupae were both parents as well as from F1 progeny was
injected with a vaccine prepared from conducted in the laboratory following the
formalinized infected insect blood. Nataraju method described by Krishnaswami [9]. The
et al. [6] made an effort to develop an oral Nuclear Polyhedral Inclusion Bodies were
vaccine against nuclear polyhedrosis virus collected, confirmed and per orally
in silkworms. Also Mahesha et al. [7] inoculated into the silkworm larvae
reported the induction of tolerance against immediately after fourth moult for the
nuclear polyhedrosis and their effects on multiplication of virus. After the silkworms
some biomolecules. In contrast to pebrine which exhibited milky white haemolymph
(Nosema bombysis Nageli), there is no were collected, the abdominal legs were
clear-cut evidence about the mode of punctured and the haemolymph was
transmission of viruses from generation to collected in a clean, pre cooled 1.5ml micro
generation, or about its phenotypic effects centrifuge tubes. Purification of NPIBs was
on the commercial characters of the carried out by following the method
progeny. However, some scientists reported described by Balakrishnappa and Honnaiah
the effect of BmCPV on commercial [10]. Finally, the stock suspension was
characters and their transmission from prepared which contained 7.125 X 106
generation to generation [8]. Therefore, the polyhedral inclusion bodies ml-1.
present investigation was undertaken in an Enumeration of polyhedral inclusion bodies
attempt to acquire information on the effect was done by following Neuber’s
of nuclear polyhedrosis and induced haemocytometer. For the induction of the

3998
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

tolerance, polyhedral bodies were treated in with 70 per cent ethyl alcohol using sterile
2% formaldehyde solution for 24h at 30 cotton wad. Then the leaves were cut to
+1ºC and used [7]. square shape (10 cm2) and 0.1 ml
For breed susceptibility, immediately after inactivated NPIBs suspension (from the
fourth moult, that is, after first two feedings stock of 0.835 x 107ml-1 NPIBs suspension
during fifth instar, mulberry leaves of M5 in sterile distilled water) was uniformly
variety were washed with distilled water and smeared, shade dried and chopped to
surface sterilized with 70 per cent ethyl required size and fed to the silkworms. Such
alcohol using sterile cotton wad. Then the type of inoculation was carried out thrice
2
leaves were cut to square shape (10 cm ) i.e., at the age of third instar second day,
and 0.115 ml NPIBs suspension (from the again at the age of fourth instar second day
stock of different concentrations of BmNPV and also, at the age of fifth instar first day.
viz., 5.44 x 107, 4.76 x 107, 4.08 x 107, 3.4 This was followed by treatment with 0.125
x 107, 2.72 x 107, 2.04 x 107 and 1.36 x ml of live NPIBs (from the stock of 1.36 X
107 polyhedral bodies ml-1 NPIBs 107 ml-1) on second day of fifth instar [7].
suspension in sterile distilled water) was Suitable untreated batches were also
uniformly smeared and fed to the maintained. All experimental batches were
silkworms. Such two leaves (40 cm2) maintained with 100 worms in triplicate.
smeared with NPIBs suspension were made Treated worms were allowed to complete
use for 100 worms. The NPIBs smeared development. Of the cocoons harvested,
leaves were shade dried and chopped to the only uniform and healthy cocoons were
required size and fed to the silkworms. selected, and preserved. The procedure
Suitable untreated batches were also followed for the preparation of layings &
maintained. All experimental batches were incubation, silkworm rearing and
maintained in triplicate. Later, the worms assessment of economic traits are as
were allowed to complete larval stage, described by Narasimhanna [11],
spinning, pupation and moth emergence. Krishnaswamy [9] and Mahesha [12]
Number of cocoons harvested from each respectively. The control batches, induced
batch was considered for calculation of the batches, induced followed by live virus
breed susceptibility. treated and live virus treated batches are
For induction of tolerance, the mulberry mentioned as C, I, IL and L respectively.
leaves of M5 variety were washed with The data obtained from the above
sterile distilled water and surface sterilized experiments were statistically analyzed

3999
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

through SPSS by one way ANOVA, [13], viability of 96% (from treatment to cocoon
Scheffe’s post hoc test [14] and probit spinning), followed by 68% in N1, 58% in
analysis [15] wherever they were applicable. N2, 48% in N3, 42% in N4, 34% in N5, 11%
RESULTS AND DISCUSSION in N6 and 12% in N7 batches. In case of
In Pure Mysore breed, the viability rate was single cocoon weight, N0 (control) batches
higher in the control batches and gradual revealed a highest mean weight of 1.895 g
reduction was noticed as the dose of followed by 1.715, 1.695, 1.665, 1.573,
BmNPV increased. The control (NO) worms 1.480, 1.448 and 1.380 g in N1, N2, N3, N4,
recorded a mean viability of 100% followed N5, N6 and N7 batches respectively. In case
by N1 of 72%, N2 of 64%, N3 of 52%, N4 of of shell weight, again, the control (N0) batch
41%, N5 of 32%, N6 of 22% and N7 of 16% registered a highest weight of 0.366 g
(Table 1). The N1, N2, N3, N4, N5, N6 and followed by 0.286, 0.267, 0.251, 0.236,
N7 are the silkworm batches inoculated with 0.221, 0.214 and 0.198 g in N1, N2, N3, N4,
BmNPV doses of 5.44 x 107, 4.76 x 107, N5, N6 and N7 sets respectively. The shell
4.08 x 107, 3.4 x 107, 2.72 x 107, 2.04 x 107 ratio also showed gradual reduction from
7 -1
and 1.36 x 10 NPIBs ml respectively. In 19.32% in the control to 14.39% in N7
case of single cocoon weight also, same batch.
pattern was noticed. The gradual reduction The F1 progeny obtained by selfing the
in the cocoon weight from 1.062 g in the moths emerged from the silkworms
control to 0.755 g in N7 (heavily inoculated inoculated with BmNPV, exhibited
batch) was observed. In case of shell inferiority as compared to control as well as
weight, gradual reduction from 0.133 g in induced, induced followed by live PIBs
the control to 0.071 g in N7 batch was inoculated batches in almost all the
noticed. There was sharp reduction in shell economic characters analyzed. The size of
weight from N0 to N7 batches. In the shell the eggs in the silkworm batches treated
ratio, the same pattern was noticed. A with NPIBs was small in both the strains.
gradual reduction from 12.52% in N0 batch Almost all commercial characters like
was reduced to 9.41% in N7 batches. fecundity, hatchability, cocoon yield,
In the NB4D2 breed, again, the larval cocoon weight, shell weight, shell ratio,
mortality was less in the control batches and filament length and effective rate of rearing
gradual reduction was observed as the dose showed reduction when compared to the
of BmNPV increased (Table 2). Silkworms control, induced and induced followed by
of control (N0) batch recorded a mean

4000
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

live NPIBs inoculated sets of both the as well as the pathogen for their mutual
strains (Tables 3 and 4). defense mechanism.
In the present investigation, breed The F1 progeny obtained by selfing the
susceptibility was carried out by smearing moths emerged from the silkworms
the mulberry leaves with PIBs. Of the inoculated with BmNPV, exhibited
tested breeds, Pure Mysore exhibited more inferiority as compared to their respective
susceptibility when compared to NB4D2 control batches in all the economic traits
breed. In both the breeds, the viability rate analyzed. Similar observations made by
was higher in control sets; but, the treated Vail et al.[16] and, Vail and Gouch, [17] in
with BmNPV PIBs showed gradual Trichoplusia ni; Bullock et al. [18] in
reduction as the dose of the pathogen Pectinophora gossipiella; Simmons and
increased. However, contrary to the general Sikorowski [19] and, Sikorowski and
trend, the multivoltine, Pure Mysore was Thomson [20] in Heliothis virescens;
found to be more susceptible to BmNPV as Mahesha and Honnaiah [8] in Bombyx mori.
compared to a bivoltine. Such a However, Sikorowski et al. [21] observed
contradiction might have direct relationship that the virus particles are present in the
to the body weight that is a lighter race is haemolymph of Heliothis virescens larvae
more susceptible than the heavier races. and in the adult. This result suggested that
Similar observation was recorded by the developing eggs might be under the
Mahesha [12] during cytoplasmic influence of haemolymph, which contains
polyhedrosis. In addition to the mortality of virus particles. In addition, there was
silkworms there was a significant reduction variation protein content [22] and [23],
in the cocoon weight, shell weight and shell reduced enzyme activities [24] and
ratio in the surviving silkworms of all the haemolymph glucose level [25] were also
three silkworm varieties when compared to reported. Even in the case of nuclear
their respective control batches. The polyhedrosis also, the eggs produced by the
reduction in cocoon and shell weight might live BmNPV infected mother moth were less
be due the reduced feeding since pathogen in number as well as size; altered enzyme
attack. Moreover, the food material activities [26] and. [7] were reported. Thus,
absorbed by the pathogen might have been inferior commercial characters might be due
utilized for its multiplication. In addition, to weak larvae hatched out from the
the energy derived from the ingested food, undersized eggs produced under the
must have been distributed between the host influence of virus. On the other hand the

4001
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

induced and induced followed by live virus Authors wish to thank University Grants
treated exhibited almost similarity as in case Commission, New Delhi, India, for financial
of control silkworms in the expression of assistance and University of Mysore for
commercial characters. Hence, the present extending the facilities to carry out this
investigation clearly indicated that the work.
progeny obtained by selfing the moths REFERENCES
emerged from live BmNPV inoculated [1] Nataraju B, Datta RK, Baig M,
silkworms, exhibited inferiority in almost all Balavenkatasubbaiah M, Samson
characters analyzed, and this knowledge can MV and Shivaprasad V, Studies on
be used in the sericulture industry during the the prevalence of NPV in
selection of parent seed cocoons for the sericultural areas of Karnataka,
preparation of disease free layings either for Indian J. Seric., 37 (2), 1998, 154-
commercial or academic purposes, which 158.
have a vital role in the improvement of [2] Christi MZ and Sohaf KA, Studies
sericulture industry. The information on the polyhedral diseases of
gathered in this research work may be silkworm, Bombyx mori L. in
applied in grainage activities during the Jammu and Kashmir state. Indian J.
selection of seed cocoons for the preparation Seric., 29, 1990, 155-157
of DFLs. In addition, the progeny obtained [3] Carbone D and Fortuna E, La
by selfing the moths emerged from vaccinazione dei bachi da seta, Terza
inactivated and inactivated followed by the Nota Preventiva. 11, 1932, 204-210.
live NPIBs inoculated silkworms, exhibited [4] Gargiulo F, Ulteriori ricerche sul
insignificant variation from the control sets giallume del baco da seta.
of almost all characters analyzed. Hence, Treatmenti immunizanti, Bioll. Lab.
the immunization techniques used in this Zool. Agr. Bachicol Milano., 4,
work may be used to develop a practical 1932, 103-112 .
control measure to be applied at rearers’ [5] Aizawa K, Sedimentation of the
level to avoid crop loss during required silkworm jaundice virus by the ultra
season. In addition, the information centrifuge. I. Effect of the vaccine
obtained from this research work made from the sediment. Japan. J.
contributes to basic virology in general. Appl. Zool. 18, 1953, 141-142.
ACKNOWLEDGMENTS [6] Nataraju B, Shivaprasad V and Datta
RK, Studies on development of an

4002
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

oral vaccine against nuclear of Bombyx mori L., treated with

polyhedrosis in silkworm Bombyx cytoplasmic polyhedrosis virus and
mori L., Sericologia, 40 (3), 2000, a mutagen. Ph. D. Thesis,
421-427. University of Mysore, Mysore,
[7] Mahesha HB, Rahamathulla G and India, 1997.
Thejaswini PH, Studies on the [13] Fisher AR, and Yates F., Statistical
tolerance against nuclear tables for biological, agricultural
polyhedrosis in silkworm Bombyx and medical research, 6th Ed. Pub.
mori L and its biochemical aspects. by Longman Group Ltd., England,
IJBPAS, 2(7), 2013, 1501-1512. 1953.
[8] Mahesha HB and Honnaiah S, [14] Scheffe HA, The Analysis of
Effects of cytoplasmic polyhedrosis Variance, Wiley Publications, New
on silkworm (Bombyx mori L) and York, (1959)
its transmission from generation to [15] Fenny D. J., Probit analysis, III
generation. J. Seric., 8-10 (1&2), Edn., 1971, 90-233.
2002, 38-43. [16] Vail PV, Hall, IM and Gouch D,
[9] Krishnaswami S, New technology of Influence of a cytoplasmic
silkworm rearing. Central polyhedrosis on various
Sericultural Research &Training developmental stages of the
Institute Bulletin No.2, Pub. by cabbage looper. J. Invertebrate
Central Silk Board, India, 1978, 1- Pathol. 14, 1969, 237-244.
23. [17] Vail PA and Gouch D, Effects of
[10] Balakrishnappa YK and Honnaiah cytoplasmic polyhedrosis virus on
S, Isolation and purification of adult cabbage looper and their
cytoplasmic polyhedrosis virus progeny. J. Invertebrate Pathol.14,
from Bombyx mori L. J. Mysore 1970, 237-244.
University, Sect. B XXXII, 1992, [18] Bullock HR, Martiner E and
331-333. Stuermer CW, Cytoplasmic
[11] Narasimhanna MN, Manual on polyhedrosis virus and the
silkworm egg production. Pub. by development and fecundity of pink
Central Silk Board, India, 1988. bollworm. J. Invertebrate Pathol.,
[12] Mahesha HB, Cytogenetic and 15, 1970, 109-12.
biochemical studies in a few races

4003
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

[19] Simmons CL and Sikorowski PP, A proteins during cypovirus infection

laboratory study of the effects of and in mutants raised from EMS
cytoplasmic polyhedrosis virus on treated silkworm Bombyx mori L.
Heliothis virescens (Lepidoptera: Int. J. Pure App. Biosci. 3(2), 2015,
Noctuidae) J. Invertebrate Pathol., In Press.
22, 1973, 237-244. [24] Mahesha HB Thejaswini PH and
[20] Sikorowski PP and Thomson AC, Honnaiah S, Effect of cytoplasmic
Effects of cytoplasmic polyhedrosis polyhedrosis on amylase and
virus on diapausing Heliothis succinate dehydrogenase activity
virescens. J. Invertebrate Pathol., levels in silkworm Bombyx mori L.
33: 1979, 66-70. Entomon, 27(3), 2002, 269-279.
[21] Sikorowski PP, Andrews GL and [25] Mahesha HB and Thejaswini PH,
Broome JR, Presence of Studies on the haemolymph glucose
cytoplasmic polyhedrosis virus in level in silkworm Bombyx mori
the haemolymph of Heliothis during cytoplasmic polyhedrosis.
virescence larvae and adults. J. Int. J. Pure App. Biosci. 1(3), 2013,
Invertebrate pathol. 18, 1971, 167- 11-16.
168. [26] Mahesha HB and Thejaswini PH,
[22] Mahesha HB, Thejaswini PH and Studies on Isozymes of amylase,
Honnaiah S, Effect of cytoplasmic superoxide dismutase and esterase
polyhedrosis on haemolymph during induction of tolerance
proteins of silkworm Bombyx mori against nuclear polyhedrosis in
L. Annals of Entomology, 18(1), silkworm Bombyx mori L. Int. J.
2000, 27-32. Pure App. Biosci. 2(2), 2014, 48-
[23] Mahesha HB and Thejaswini PH, 55.
Comparative studies on the midgut

4004
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

Table 1: Effects of Nuclear Polyhedrosis on Larval Mortality

and Cocoon Characters of Pure Mysore strain
Dose of BmNPV No. of cocoons Cocoon Shell weight Shell ratio
 harvested weight (g) (g) (%)
No 100 1.062 0.133 12.52
N1 72 0.955 0.116 12.14
N2 64 0.950 0.098 10.36
N3 52 0.909 0.089 9.87
N4 42 0.878 0.083 9.51
N5 32 0.851 0.079 9.33
N6 22 0.842 0.079 9.41
N7 16 0.755 0.071 9.41
The variations between control and experimental sets and among different doses are all significant at 5%
level; Fifty worms in triplicate for each dose were used for inoculation.

Table 2: Effects of Nuclear Polyhedrosis on Larval Mortality

and Cocoon Characters of NB4D2 strain
Dose of BmNPV No. of cocoons Cocoon Shell weight Shell ratio
 harvested weight (g) (g) (%)
No 96 1.895 0.366 19.32
N1 68 1.715 0.286 16.68
N2 58 1.695 0.267 15.79
N3 48 1.665 0.251 15.11
N4 42 1.573 0.236 15.04
N5 34 1.480 0.221 14.96
N6 22 1.448 0.214 14.82
N7 12 1.380 0.198 14.39
The variations between control and experimental sets and among different doses are all significant at 5%
level; Fifty worms in triplicate for each dose were used for inoculation

4005
IJBPAS, June, 2015, 4(6)
Mahesha HB* and Thejaswini PH Research Article

Table 3: Effects of Nuclear Polyhedrosis on Commercial Characters of F1 Progeny. Race: Pure Mysore

Dose of Fecundity Hatching Yield per 10000 larvae brushed Cocoon wt. (g) Shell wt. (g) Shell ratio Filament ERR
BmNPV (%) (%) Length
Number Weight (kg) (mt)

C 514 98.49 9460 10.141 1.072 0.144 13.43 455 94.60

I 504 98.21 9458 10.120 1.070 0.141 13.17 445 94.58

IL 489 98.11 9458 10.120 1.071 0.141 13.16 445 94.58

L 401 85.41 9309 9.039 0.971 0.120 12.36 379 93.09

The variation between control and experimental sets and among different treatments is all significant at 5% level.

Table 4: Effects of Nuclear Polyhedrosis on Commercial Characters of F1 Progeny. Race: NB4D2

Dose of BmNPV Fecundity Hatching Yield per 10000 larvae brushed Cocoon wt. (g) Shell wt. (g) Shell ratio Filament ERR
(%) (%) Length
(mt)
Number Weight (kg)

C 580 98.11 9449 18.425 1.950 0.376 19.28 985 94.49

I 575 98.16 9421 18.333 1.946 0.375 19.27 982 94.21

IL 576 98.02 9411 18.136 1.921 0.368 19.15 964 94.11

L 466 90.01 9311 15.955 1.774 0.284 16.00 743 93.11

The variations between control and experimental sets and among different treatments are all significant at 5% level.

4006
IJBPAS, June, 2015, 4(6)