Research Paper Probiotics

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: Probiotics, Lactobacillus plantarum, Bombyx mori (L.) and Cocoon production

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 1st, 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 phago- stimulants 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 Single larva Mature larva Cocoon wt. Pupae wt. Shell wt. (gm) Shell ratio Pupation group wt. (gm) wt. (gm) (gm) (gm) % %

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 Mean of square value variation Single Matur Cocoon Pupae Shell Shell Pupation larva e larva wt. wt. wt. ratio wt. wt. Interactio 5 0.091 0.0636 0.1361 0.1000 0.0099 19.5531 1531.800 n between control and treatment groups * NS ** ** ** ** **

*Significant at 5%, ** Highly significant at 1%, NS: Non -significant

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CRS/SERI/2005/

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