Effect of Use of Lactobacillus plantarum as a Probiotics to improve Cocoon
Production of Mulberry Silkworm, Bombyx mori (L.)”
K.K.Singh, R.M.Chauhan, A.B.Pande, S.B.Gokhale and N.G.Hegde
Journal of Basic and Applied Sciences Vol. (1), June–Nov. pp 1-8.
Abstract
The silkworm larvae were fed on mulberry leaves treated with a probiotic bacteria
namely Lactobacillus plantarum during different instars and its effect on larval body
weight, mature body weight, cocoon weight, pupa weight, shell weight, shell ratio and
pupation percentage were studied. Approximately all the treatment with L. plantarum
a probiotics was significantly superior to that of control. L. plantarum treated groups
recorded significantly higher values for larval body weight, cocoon weight, shell
weight, pupation percentage at all the instars namely 1st, 2nd, 3rd, 4th and 5th instar
respectively as compared to control. The study indicated that the probiotic bacteria
exhibit inducement of certain growth factors leading to increase in silk yields and
pupation % in silkworm rearing however the mechanism of interaction of host native
microbiota with the probiotic bacteria influencing physiology and innate immunity of
silkworm require further investigation.
Keywords:
production
Probiotics, Lactobacillus plantarum, Bombyx mori (L.) and Cocoon
Introduction
Probiotics are the live microbial food supplements beneficially affecting the host by
improving the microbial balance (Fuller, 1991and Austin et al., 1995).Several
researchers have reported about beneficial role played by use of probiotics in Humans
(Chan et al., 1985; Brigidi et al., 2000 & 2001), ruminants ( ), aquaculture (Douillet
and Langdon, 1994 and Gildberg et al., 1997) and insects (Dillon and Dillon,
2004).Therefore, products containing probiotic bacteria are gaining popularity,
increasing the importance of their accurate speciation(Yeung et al ,2002 ) .Similarly,
in mulberry silkworm (Bombyx mori ) presence of different types of bacteria in the
gut have been reported (Roy et al, 2000 and Kodama,2001).Most of the species
belonging to Genus Streptococcus are found to be pathogenic to Bombyx mori larvae
while bacteria from Genus Pediococccus, Leuconostoc and Lactobacillus didn’t
produce any infected silkworm. The precise mechanism of beneficial effect on host or
interaction among the different bacterial strains present as micro-flora is not known
(Steinhaus, 1949 and Kodama, 2001).
Different species of lactic acid bacteria have been extensively studied
(Bruno et al .,1993; Bernet-Camard et al.,1997 ;Gibson et al 1998; Kodama,2001)
and found to be beneficial as probiotics (Fuller, 1991;Bernet –Camard,
1997;Sakamoto et al ; 2001).Therefore it was planned to study the effect of a lactic
acid bacteria namely Lactobacillus plantarum a known probiotics on economic
parameters of Bombyx mori (L..)
Materials and Methods
Standard culture of Lactobacillus plantarum procured from National Collection of
Industrial Micro-organisms (NCIM 2083), National Chemical Laboratory, Pune was
cultured in MRS broth medium. The broth culture of L. plantarum (106 cfu/ml) was
used for the experiment. The leaves of mulberry S-1635 variety and an indigenous
multivoltine silkworm race namely BAIF-1 were used known to be susceptible to
nuclear polyhedrosis virus. Experiment was carried out under controlled laboratory
conditions (temperature 28±30C and humidity of 70 to 85%).
Mulberry leaves were fed to Bombyx mori (L.) larvae as per rearing methods
suggested by Krishnaswami et. al. ., 1978 .Experimental as well control groups had
3 replications consisting of 100 larvae each . Mulberry leaves of equal weight and
approximately same number were used for feeding B. mori larvae in control and
experimental groups. Experimental groups were fed with mulberry leaves treated by
dipping the leaves in the suspension of L .plantarum (106 cfu / ml) and partially
drying at room temperature, only once soon after hatching and first feed after 1 st, 2nd,
3rd and 4th moult pass larvae. The control group of B. mori (L.) larvae (100 larvae in 3
replications) were fed with mulberry leaves without treatment of the L. plantarum
culture. Parameters like single larva weight (6th day of 5th instar) and mature larva
weight ( just before mounting ) , total cocoon weight (gm), shell weight (gm), pupa
weight (gm), shell ratio (%) and effective pupa formation (%) were recorded and data
was analysed statistically to study the effect.
Results and discussion
The results on effect of probiotic bacteria L.plantarum on economical parameters of
silkworm are presented in Table 1. Results showed that the larvae of treatment groups
had significantly higher larval weight, cocoon wt. pupal wt. shell ratio and pupation
rate as compared to control group. Also overall analysis (Table 2) of interaction
between different treatment groups and control group showed significant higher
values for larval weight, cocoon, pupa, and shell weight and ratio and pupation %.
L. plantarum treated mulberry leaves fed to larvae at 1st ,2nd ,3rd ,4th and 5th instar
recorded maximum larval weights (2.90, 2.86, 2.90, 2.86 and 2.93gm respectively ),
cocoon weight (1.09,1.27,1.30,1.29,1.22 and 1.33gm), pupal weight (1.07,1.10,1.08
and
1.02gm)
shell
wt.(0.20,0.20,0.21,0.20,and
0.21gm),
shell
ratio
(15.98,15.33,16.21,16.32 and 15.65%) respectively as compare to control group. The
higher value of larval weights and cocoon parameters indicated the greater suitability
/acceptability of probiotic L. planterum along with food plants. The amount of food
consumed by a larva influences its growth rate, development, final body weight and
probability of survival (Slansky and Scriber, 1985).These results show that a
fundamental shift in the microbial profile in silkworm larval gut is beneficial to the
host which in turn may significantly contribute to increase silk production. Food
intake is also regulated by the physical nature of food and also presence of phagostimulants in the food (Dadd, 1970). Silkworm B. mori (L) reared on mulberry leaves
supplemented with minerals, oral protein supplementation, cereal flours, medicinal
extracts, plant growth hormones ( Sunder Raj et al.,2000 Singh ,1997) are reported to
have beneficial effects on economic parameters. Sukumar (1983) has reported
enhancing effect on silk yield using mulberry phylloplane yeast Sporobolomyces
roseus .In present study similar observation may be attributed to increased efficiency
of digestion and assimilation of food materials leading to increased protein synthesis
and subsequent accumulation of storage proteins in the body on account of activity of
a probiotic microbial flora in gut of host. Comparatively, the food consumption in the
control category with indigenous micro flora reflects (with respect to treatment
groups) the low silk production ability as feeding quality leaves influences the
synthesis of total RNA and translation of fibroin messenger RNA and DNA synthesis
(Chavancy and Flournier, 1979). Statistically no significance (table 2) was observed
with respect to mature larva weights in treatment and control groups which may due
to common timing of picking the ripe larvae in treated and control groups.
Significantly higher pupation % (Table1 and 2) was observed in all treatment groups
except 2nd instar treatment group. L.plantarum treated larvae recorded maximum
pupation % in 4th instar (83%) followed by 5th instar (82%), 1st instar (78%) and 3rd
(73%) respectively as compare to control group. This may be partly due to higher
food digestibility leading to higher survival rate (Slansky and Scriber, 1985) and due
to increase in activity of red florescent protein (RFP) a protein responsible for
providing innate immunity by inactivating virus among the silkworm ( Kodama,
2001). It is well substantiated by Hayashiya,et al .,1971 , the concentration of RFP is
maximum during the moulting period (when the larvae do not feed) especially during
4th and 5th instar while it is low in earlier instar. Since in silkworm more than 90% of
total larval feed is ingested and also RFP is maximum during 4th and 5th instar of
B.mori, (Kodama, 2001) the supplementation of L.plantarum soon after the moult
may have influenced RFP as well along with feeding. This definitely have resulted in
higher pupation % as compare to control group in present study during 4th instar
followed by 5th instar 3rd instar and 1st instar. Though 2nd instar showed enhancement
in cocoon character but pupation % was similar to control group may be due to less
influence of L.plantarum on activity of RFP during this stage of B.mori.
Beneficial effects by modulation of gut micro-flora and influence on mucosal
immunity or through altering enzymatic activities has been extensively studied in
human ,animals and many insects (Yeung et al ,2002 ) leading to commercialization
of products as probiotics. Present preliminary study indicate the beneficial effects of a
probiotic L. plantarum on economic traits of silkworm B.mori, however the
mechanism of interaction of host native microbiota with the probiotic bacteria
influencing directly and indirectly on food assimilation , physiology and innate
immunity of silkworm require further investigation.
Table 1: Effect of L.plantarum on economic parameters of a multivoltine B.mori
Parameters
Treatment
group
Single larva
wt. (gm)
Mature larva
wt. (gm)
Cocoon wt.
(gm)
Pupae wt.
(gm)
Shell wt. (gm)
Shell ratio
%
Pupation
%
Control
2.73±0.039
2.37±0.060
1.09±0.033
0.91±0.030
0.15±0.005
13.51±0.462
62±0.524
1st instar
2.90±0.044
2.52±0.035
1.27±0.032
1.07±0.032
0.20±0.005
15.98±.540
78±1.572
2nd instar
2.86±0.047
2.52±0.056
1.30±0.048
1.10±0.043
0.20±0.005
15.33±0.333
63±1.428
3rd instar
2.90±0.044
2.52±0.035
1.29±0.045
1.08±0.041
0.21±0.005
16.21±0.477
73±1.103
4th instar
2.86±0.047
2.52±0.056
1.22±0.039
1.02±0.036
0.20±0.006
16.32±0.449
83±0.524
5th instar
2.93±0.045
2.52±0.053
1.33±0.052
1.12±0.047
0.21±0.006
15.65±0.391
82±1.299
SE(m) ±
0.019
0.021
0.018
0.017
0.003
0.200
0.935
CD at 5%
0.094
0.094
0.080
0.076
0.013
0.894
4.179
CV %
6.80
8.60
15.33
16.63
16.07
13.44
13.19
Table 2. Analysis of variance on effect of L. planatrum treatment on economic
parameters of a multivoltine B.mori (L.)
Source of Df
variation
Single
larva
wt.
Interaction 5 0.091
between
control
and
treatment
groups
*
Mean of square value
Mature Cocoon Pupae Shell
Shell
Pupation
larva
wt.
wt.
wt.
ratio
wt.
0.0636 0.1361 0.1000 0.0099 19.5531 1531.800
NS
**
**
**
**
**
*Significant at 5%, ** Highly significant at 1%, NS: Non -significant
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CRS/SERI/2005/
To,
Sub: Regarding submission of a research paper entitled “Effect of use of
Lactobacillus plantarum as a probiotics to improve cocoon production in the
mulberry silkworm Bombyx mori (L.)”
Dear Sir,
We are submitting herewith a research paper entitled “Effect of use of Lactobacillus
plantarum as a probiotics to improve cocoon production in the mulberry silkworm
Bombyx mori (L.)”
May we request you to please inform the about acceptance of the same.
Thank you for your cooperation.
With kind regards
Yours sincerely,
(Dr.S.B.Gokhale)
Vice President & Head
CRS,BAIF,Uruli-kanchan
Enclosures 1. Hard copy of the research paper