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Welcome

Muraghendra P. Ibrahimpur.

PGS12AGR5769

Master’s Seminar – I

onExtra foliation of growth promoting substances to mulberry silk worm for

enhancing cocoon yield

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Contents

Introduction Terminologies Effect of growth promoting substances:

Botanical extractsProtein and carbohydrate supplementsVitamins and minerals Honey by products, milk and others

Summary Conclusion

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Introduction

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Systemic classification of mulberry silkworm

Kingdom- Animalia

Phylum- Arthropoda

Class- Insecta

Sub Class- Pterygota

Order- Lepidoptera

Super Family- Bombycoidea

Family- Bombycidae

Genus- Bombyx

Species- mori

Principle Nutrient Value Energy 43 Kcal Carbohydrates 9.80 g Protein 1.44 g Total Fat 0.39 g

Cholesterol 0 mg Dietary Fiber 1.7 g

Vitamins Folates 6 mcg Niacin 0.620 mg Pyridoxine 0.050 mg Riboflavin 0.101 mg Vitamin A 25 IU Vitamin C 36.4 mg Vitamin E 0.87 mg Vitamin K 7.8 mcg

Principle Nutrient Value

Electrolytes

Sodium 10 mg

Potassium 194 mg

Minerals

Calcium 39 mg

Copper 60 mcg

Iron 1.85 mg

Magnesium 18 mg

Selenium 0.6 mg

Zinc 0.12 mg

Table 1: Nutrient contents in fresh Mulberry leaves (Nutrition Value per 100 g)

Tulasi and Buddolla, 2013 6

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Terminologies

• Extrafoliation :

• Nutrition :

• Growth promoting substances :

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Effect of growth promoting substances

Botanicals • Any deficit in the quality and quantity of mulberry leaves:

• Enrichment of deficit nutrients through botanicals:

• Biochemical constituents - amino acid, proteins, alkaloids, glucoside, phyllanthine, psoralem, betasitosterol - phagostimulants and attractants.

• Administration - plants extracts to larvae increases phagostimulant activity.

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• Botanicals - immense ability to influence the metabolic activities.

• Many attempts - fortify mulberry leaf with botanical extracts to improve mulberry leaf quality and feed efficiency of silkworm - increase cocoon production and silk quality.

• Many studies - plant extracts on various metabolic activities resulting acceleration of most of economical parameters viz., spinning, increase in larval, cocoon & shell weight, silk gland weight, improvement in reeling parameters, increase in silk content, silk filament length and weight.

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Supplementations of botanicals

• Administration of mulberry leaves treated with aqueous leaf extracts of Adhatoda vasica, Phyllanthus niruri and Terminalia arjuna to first instar larvae of PM x CSR2 hybrid resulted in positive response with respect to rearing parameters.

• Highest larval weight in all instars, high silk productivity, increased filament length and reduced denier was noticed in P. niruri followed by A. vesica and T. arjuna.

Saritha Kumari et al., 2011

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Table 2: Effect of botanical extracts on cocoon parameters of PMxCSR2

Botanical extracts Silk productivity(cg/day)

Filamentlength (m) Denier

Adhatoda vasica 3.84 898.93 2.36

Phyllanthus 3.94 912.51 2.31

Terminalia arjuna 3.57 887.97 2.41

Control 3.54 874.95 2.41

F test * * *

SEm± 0.024 3.066 0.012

CD at 5% 0.072 9.193 0.035

* : Significant at 5% Saritha Kumari et al., 2011

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Spirulina- 18 amino acids and vital vitamins - biotin, tocopherol, thiamine, riboflavin, niacin, folic acid, pyrodozoic acid, beta-carotene and vitamin B12.

Supplememtation - Spirulina at 300ppm was found significant increase in all the quantitative cocoon characters- single cocoon weight, single shell weight, pupal weight and silk filament except shell percentage compared to control, 100ppm and 200ppm.

Venkatesh Kumar, 2009

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Table-3: Mean value of quantitative cocoon characters of silkworm under different concentrations of blue green algae (spirulina).

S. No Treatments

Concentration (ppm)

Cocoon weight (gm)

Shell weight (cgm)

Pupal weight (gm)

Shell percentage

Silk filament

length (m)

1 100 0.905* 22.250* 0.645* 25.250 664.575

2 200 0.935* 22.250* 0.675* 24.615 682.203*

3 300 1.083** 24.500** 0.828** 22.640 866.605**

4 Control 0.805 20.500 0.590 25.975 661.943

F Test HS HS HS NS HS

CD @ 5%

0.080 1.585 0.081 - 14.192

CD @ 1%

0.112 2.208 0.113 - 19.770

Venkatesh Kumar, 2009

Note: HS-Highly Significant (p = 001); NS-Not Significant **-Significant at 1% level *-Significant at 5% level

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The administration of mulberry leaves fortified with medicinal botanical extracts of Withania somnifera, Terminalia arjuna and Tinospora cordifolia to silkworm (CSR2 x CSR4) and (PM X CSR2) recorded highest cocoon weight, shell weight, shell ratio, silk filament length, silk filament weight and finer denier compared to control.

Sridevi, 2003

16Sridevi, 2003

Table 4: Effect of meditional extract treated mulberry leaves on 5 th instar larval duration, mature larval weight and effective rate of rearing (%) of silkworm, Bombyx mori L.(CSR2 XCSR4)

Treatments 5th instar larval duration(hrs) Mature larval weight Effective rate of rearing

Concentration (%) Mean Concentration (%) Mean Concentration (%) Mean

0.1 0.5 0.1 0.5 0.1 0.5

T1: Sauropus androgynus

144.00 144.00 144.00 2.07 1.98 2.02 82.55 76.77 79.66

T2 : Phyllanthus niruri 141.66 143.00 142.33 2.15 2.04 2.09 82.33 79.33 80.83

T3: Withania somnifera 140.00 140.00 140.00 2.48 2.34 2.41 92.66 90.99 91.82

T4 :Tagetes errecta 143.00 143.00 143.00 2.25 2.11 2.18 82.00 80.00 81.00

T5 : Tinospora cordifolia

141.00 141.00 141.00 2.39 2.24 2.31 87.00 83.67 85.34

T6: Leptadenia reticulata

142.00 142.00 142.00 2.38 2.18 2.28 84.67 82.00 83.34

T7: Terminalia arjuna 141.00 141.00 141.00 2.43 2.21 2.35 90.34 88.00 89.16

T8: Adathoda viscica 143.00 143.00 143.00 2.34 2.13 2.23 83.34 81.67 82.50

T9: Control 144.00 144.00 144.00 1.96 1.89 1.92 68.54 64.78 66.66

Mean 142.18 142.33 142.33 2.27 2.13 84.03 80.69

F-Test SE± CD(0.05%)

F-Test SE± CD(0.05%)

F-Test SE± CD(0.05%)

Concentrations NS - - * 0.0051 0.0137 * 0.845 1.730

Treatments * 0.761 2.0594 * 0.0108 0.0291 * 1.580 3.237

Interaction NS - - * 0.0152 0.412 NS - -

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Table 5: Overall performance of the silkworm B. mori in response to the influence of Aloe vera tonic during final instar

Manimuthu, et al., 2010

Experimental Rearing (%)

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• The A. vera @ 2.0 % - higher larval growth and increased the weight of cocoon.

• The larval weight, relative growth rate, effective rearing rate, larval consumption index of the final instar larvae of silk worm, increased with the supplementation of A. vera tonic. Maximum shell weight and shell ratio - noticed.

Rajkumar and Vitthalrao , 2013

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• Ethanol extract of 20 botanicals in 3, 2.5%, 5% and 10% concentrations were evaluated for their efficacy to improve performance of mulberry silkworm through feeding botanical enriched mulberry leaves.

Pratheesh Kumar et al., 2007

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Five botanicals viz., Asparagus recemosus, Achyranthes aspera, Tribulus terrestris, Withania somnifera, and Parthenium histerophorus ranked first were evaluated at 8%, 5% and 3% concentrations to confirm their effectiveness and standardize effective concentration.

Among the rearing parameters, mature larval weight, shell ratio and number of cocoons/kg were not significantly influenced by the treatments.

• Fifth instar larvae of mulberry silkworm (PM x CSR2) fed by mulberry leaves smeared with aqueous extracts Azolla, significantly increased the mature larval weight, cocoon weight, shell weight, and pupal weight but non significant in shell per cent over the control.

Patil , 2011

The silkworm (PM x CSR2) fed by mulberry leaves smeared with extracts of Azolla

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Patil et al., 2005

• Supplementation of mango leaf water extract ( 30-40%) improved most of the economic traits especially larval weight, pupal weight, shell weight, shell ratio, filament length and filament weight by improving ERR.

• The extract of Azadirachta indica and Vitex negundo supplemented to mulberry feeding of silkworm there was significant improvement in shell ratio and filament length at 2.5 % of neem extract and 5.0% concentration of Vitex negundo leaf extract.

Sujatha et al., 2003

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• Silk worm supplemented - potato leaf extract recorded the highest larval weight and lower larval duration.

Krishnaprasad et al. , 2001

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• Improvement in cocoon weight, shell weight, shell ratio, silk productivity, filament length, filament weight, denier due to spraying of Tridax procumbens, Tribulus terrestris and Parthenium hysterophorus at 0.4 % concentration.

• Silkworm fed on mulberry leaf supplemented with Coffee arabica leaf extract at 1:25 concentration resulted significantly higher cocoon weight, pupal weight and shell %.

Murugesh, 2002

Jeypul et al. 2003

• The Pure mysore - most responsive to 5% dust formulation of Lantana camara and Clerodendron inermae.

• Larvae - PM, NB18 and PM x NB18 grew vigorously and had maximum larval weight, cocoon weight as compared to control

Santhosh Kumar, 199726

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Proteins and carbohydrate supplements

The fifth instar larvae of mulberry silkworm hybrids PM x CSR2 and

CSR2 x CSR4 fed on mulberry leaves supplemented with flours (cereals

and pulses) at two ratios (1:10) (1:20) (Flour: leaf) shows significantly

higher filament length, finer denier, more fibroin percentage and lower

sericin content of cocoons - reared on flour supplemented leaf over the

control

Rekha and Neelu, 201028

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Treatments Feed additive Feed additive mean

Hybrid X Feed additive

Ratio X Feed additive

Hybrid X Feed additive X Ratio

H1 H2

H1 H2 D1 D2 D1 D2 D1 D2

T1 Ragi 929 851 1007 932 927 852 849 1011 1044T2 Rice 921 849 994 923 919 850 848 997 991T3 Wheat 911 842 980 913 908 84 807 983 977T4 Sorghum 857 809 905 854 859 810 807 898 911T5 Soybean 981 890 1072 984 978 892 888 1077 1067T6 Horsegram 965 875 1055 96 964 877 874 1055 1054T7 Greengram 945 856 1035 947 943 858 854 1037 1033T8 Cowpea 903 841 966 907 900 842 840 971 961T9 Bengalgram 879 819 940 822 877 820 817 943 936

T10 control 836 786 886 841 831 788 785 894 878Mean 842 984 915 911 843 840 987 981

F-Test, * * NS NSS.Em±, 2.260 3.196 1.429 3.169

CD @ 5% 6.357 8.990 - -

Table 7: Influence of feed additives on filament length (m) of mulberry silkworm hybrids

Hybrid RatioH1 - PM X CSR2 D1-1:10 (Flour: Leaf)

H2 -CSR2 X CSR4 D2 – 1:20 (Flour: Leaf)Rekha and Neelu, 2010

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• Cocoon weight - maximum - larval feeding was supplemented with soya flour 10g / kg of leaves among all the hybrids as compare to control.

• The highest shell weight of 0.513g and 0.383g was recorded in (CSR6 x CSR26) x (CSR2 x CSR27) and CSR2 x CSR4 on soya flour 10g / kg of leaves followed by PM x CSR2, APM2 x APS12 and APM1 x APS8 (0.332, 0.278g and 0.274 g).

• It also improved the shell ratio in all hybrids.

Pallavi and Muthuswami, 2012

Pallavi and Muthuswami, 2012

Table 8: Cocoon weight (g) of silkworm B. mori reared on feed additives

Treatments Cocoon weight (g) in silkworm hybrids

PM x CSR2CSR2 x CSR4 APM1 x APS8 APM2 x APS12 (CSR6 x CSR26)

x(CSR2 x CSR27)

Soya flour 10g / kg of leaves

1.770a 1.800a 1.395b 1.293b 2.053a

Soya flour 5g /kg of leaves

1.730b 1.743b 1.372c 1.278c 1.977c

Corn flour 10g / kg of leaves

1.703c 1.690c 1.331d 1.266d 1.903d

Corn flour 5g /kg of leaves

1.670d 1.660d 1.327e 1.252e 1.880d

Horse gram flour 10g / kg ofleaves

1.620e 1.591e 1.301f 1.245f 1.840e

Horse gram flour 5g /kg of leaves

1.530f 1.583e 1.288g 1.239f 1.810f

Control 1.527f 1.579e 1.283h 1.157g 1.777g

SEd ± 0.0080 0.0063 0.0017 0.0080 0.0114

CD (0.05) 0.0169 0.0133 0.0036 0.0170 0.0242

Figures followed by similar letters are not different statistically at 5% level 31

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• The highest protein content - recorded on supplementation of soya flour - 2nd instar @ 10g/kg of leaves on 5 hybrids [PM x CSR2, CSR2 xCSR4, APM1 x APS8, APM2 x APS12 and (CSR6 x CSR26) x (CSR2 x CSR27)].

• Highest carbohydrate content - body of young silkworm was recorded on supplementation of corn flour @ 10g / kg of leaves followed by corn flour 5g / kg of leaves in all the silkworm breeds.

Pallavi et al., 2011

Table 9: Protein content (mg) in young age silkworm B. mori reared on feed additives

Figures followed by similar letters are not different statistically at 5% level Pallavi et al., 2011 33

Treatments Protein content (mg) in silkwormPM x CSR2 CSR2 x CSR4 APM1 x APS8 APM2 x APS12 (CSR6 x CSR26)

x(CSR2 x CSR27)

Soya flour10g / kg of leaves

19.91a 21.44a 19.83a 19.71a 24.03a

Soya flour 5g /kg of leaves

19.61c 21.19c 19.53b 19.43b 23.75c

Corn flour10g / kg of leaves

19.19d 19.18d 19.08c 19.00c 23.45d

Corn flour 5g /kg of leaves

19.01e 19.03e 18.96d 18.87d 23.09e

Horse gram flour10g / kg ofleaves

18.07f 18.57f 18.05e 17.92e 22.86f

Horse gram flour 5g /kg of leaves

17.92g 18.25g 17.89f 17.81f 22.76g

Artificial diet 19.86b 21.36b 19.83a 19.72a 23.86b

Control 17.63h 18.02h 17.58g 17.51g 22.72hSEd 0.0258 0.0280 0.0331 0.0149 0.0142CD (0.05) 0.0547 0.0593 0.0702 0.0316 0.0302

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Fig: 1 Protein content (mg) in young age silkworm B. mori reared on feed additives

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• Supplementation - 50, 100 and 150 μg/ml arginine - silkworm larvae - significant increase - larval weight, cocooning percentage, cocoon weight, cocoon shell weight, cocoon shell ratio, filament length.

• Silk gland weight and denier in only 150 μg/ml

• The dietary supplementation - 50, 100 and 150 μg/ml histidine - significant increase- larval weight, silk gland weight survival rate, cocoon weight, shell weight and shell ratio, filament length and weight and denier in all the treated groups but cocoon shell weight only in 150 μg/ml fed groups.

Chakrabarty and Kaliwal 2012

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• The oral supplementation - 100 and 150 μg/ml mixture of arginine and histidine to the silkworm larvae resulted in a significant increase in the larval weight, silk gland weight, survival rate, cocoon weight, shell weight and it’s ratio, male cocoon weight, cocoon shell weight and shell ratio and denier were significantly increased in all the treated groups, but filament length was increased only in 100 and 150 μg/ml treated groups.

Chakrabarty and Kaliwal 2012

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Vitamins and minerals

• Vitamins - required - their function - cofactors of enzymes and needed -appropriate catalytic activity.

• B- complex vitamins - biotin, cholin, incitol, niacin, pyridoxine, riboflavin, and thiamine.

• Mulberry leaves - rich in vitamin B complex, except for vitamin B12. Vitamin B-12 does not occur in in mulberry leaves, but a considerable amount of this vitamin is present in the silkworm larva and pupa.

• Ascorbic acid - posses gustatory stimulating activity. β- carotene - gustatory stimulating activity.

Vitamins Minimum amount required mg/g of dry

diet

Amount in mulberry leaves mg/g of dry

matterBiotin(B8) 1 0.2-0.8

Choline 750 930-1550

Inositol 1000 4000

Nicotinic acid(B3) 20 69-99

Pantothenic acid(B5) 20 16-35

Pyridoxine(B6) 5 43-50

Riboflavin(B2) 5 13-21

Thiamine(B1) 0.5 6.7

Ito, 1978

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Table 10: Amount of minerals present in mulberry leaves and requirement by silkworms

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Table 11: Amount of minerals present in mulberry leaves and requirement by silkworms

Inorganic elements Minimum amount required mg/g of dry

diet

Amount in mulberry leaves mg/g of dry

matter

Potassium 9.0 25-33

Phosphorous 2-3 1.6-3.4

Magnesium 1.0 2.0-4.9

zinc 0.02 0.021

Horie et al., 1967

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The feed efficacy and growth rate of silkworm larvae (V instar), enhanced by 0.2% Vitamin C treated group than control and other Vitamin C treated groups (0.1%, 0.4% and 0.8%)

Balasundaram, et al., 2013

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Table. 12: Morphometric growth rate of V instar larvae of Bombyx mori fed with control and different concentrations of Vitamin C treated MR2 mulberry leaves

Table. 13: Morphometric data of control and different concentrations of Vitamin C treated MR2 mulberry leaves fed Bombyx mori larvae produced cocoon

Values are Mean ± Standard Deviation of six observations. Values in the same column with different superscript letters (a, b & c) differs significantly at P<0.05 (DMRT).

Balasundaram, et al., 2013

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Supplementation of vitamin C at 0.5% and 1% in the last larval stages in the bivoltine hybrid NB4D2 X SH6 of silkworm exerted significant improvement in the larval weight, cocoon weight, shell weight, shell % over the control.

Amardev and Shamim, 2012

d) On single cocoon weight (g).

b) On single shell weight (g).

c) On shell %.

a) On 10 larval weight (g).

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Fig 2: Supplementation effect of synthetic Vit C on larval weight, single cocoon weight, shell % and single shell weight

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Spraying of 100ppm Folic acid solution on mulberry leaf and feeding to silkworm significantly improved larval weight, silk gland weight and growth rate, cocoon weight, shell weight and shell ratio.

Rahmathulla, 2007

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• Zinc chloride, pyridoxine, methoprene at 2 μg/ml and with mixed dose (Zn+B6+H) 2 μg/ml each on alternate days in 4th and 5th instar significantly elevated economic parameters of the cocoons.

• The supplementation of 0.01 mg/lit FeCl3 significantly increases larval weight and economic parameters such as cocoon weight, shell weight and shell ratio.

Shrivastava Sudha, 2013

Lakshmi Devi and Yellamma, 2013

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Honey bee by products, milk and others

• Some honey bee products and their mixtures as food additives to 5th instar larvae increased weights of mature larvae, pupae, fresh cocoons and cocoon shells in two strains Chinese F1 (SN 1 x Iva 1) and (SA105)

• Larvae fed on mulberry leaves treated with royal jelly (RJ) (10mg/ml) - significant increase- weights of mature larvae, pupae, fresh cocoons and cocoon shells followed by honey (H) (1g/100ml) and pollen (P) in the broad strain. While in the local strain, RJ gave the maximum weights followed by P (50mg/100ml) and then H.

Abir , 201347

Table14: Effect of honey bee products on some biological parameters of B. mori Chinese F1 (SN 1 x Iva 1)

Abir , 2013 48

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The larvae gained 82.5% more weight by the end of fifth instar larval of CSR2 xCSR4 when fed with mulberry leaves dipped in milk than when fed with fresh mulberry leaves without milk.

Bovine milk

Konala et al., 2013

Figure 1. Relationship between weight of the larvae during different days of the fifth instar when fed with fresh mulberry (M) and mulberry leaves dipped in milk.

Figure 2. The larvae fed with milk treated leaves gained 310% weight from day 1 to day 7 of the fifth instar, while the larvae fed with fresh leaves (M) gained 153% weight during th same period.

Figure 3. The relationship between the weights of the cocoons at the end of the fifth instar when larvae were fed with fresh mulberry (M) and mulberry leaves dipped in milk.

Konala et al., 2013 50

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Table 15: The effects of various nitrogenous compounds on cocoon parameters of silkworm.

ns: Not Significant

Kayvan Etebari et al., 2007

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• Nutritive additive of 1% “greenleaf” as a foliar applicant on V1 mulberry resulted a maximum increase in the macromolecule content during rainy season.

• The bivoltine hybrid CSR2 xCSR4 and poly - bivoltine crossbreed

PM x CSR2 fed on 1% treated leaves showed an increased enzyme

activity and nutritive components in their midgut and haemolymph tissues.

• The enzyme activity and conversion efficiency of mulberry nutrients was found to be significantly higher in CSR2 xCSR4, (bivoltine

hybrid) than the PM x CSR2. Which intern showed significant

increase in economic parameters except shell percentage.

Anandakumar and Sandhya, 2012

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The fortification of mulberry leaves with alfalfa tonic and its inorganic ingradients results in increased 5th instar larval weight.

Topical application of indol 3- acetic acid to the bivoltine breed (NB18) of B. mori resulted in significant increase in silk gland, cocoon and shell weight, filament length and egg productivity.

Hugar and kaliwal, 1997

Laskar and Dutta, 2000

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Summary

Mulbery silkworm is a monophagous insect it is neessary to supply nutrients through fortification on mulberry leaves.

Administration - plants extracts to larvae increases phagostimulant activity.

Supplementation of locally available botanicles like Adhatoda vasica, Phyllanthus niruri, Terminalia arjuna, Withania somnifera, Terminalia arjuna and Tinospora cordifolia increases economic parameters.

Supplementation of protein rich sources like soybean enhance cocoon yield.

Supplementation of arginine and histidine @ 100 and 150 μg/ml increases economic parameters of silkworm.

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Summary

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Supplementation of vitamin C at 0.5% to the bivoltine hybrid NB4D2 X SH6 in 5th instar of silkworm gives higher larval weight, cocoon weight, shell weight, shell % over the control.

Zinc chloride, pyridoxine, methoprene at 2 μg/ml and 0.01 mg/lit FeCl3 on alternate days in 4th and 5th instar significantly elevated economic parameters of the cocoons.

Some honey bee products and their mixtures as food additives at 5th instar increased weights of mature larvae, pupae, fresh cocoons and cocoon shells.

The larvae gained 82.5% more weight by the end of fifth instar larval when fed with mulberry leaves dipped in milk than when fed with fresh mulberry leaves without milk.

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Conclusion

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