Effect of nutritional fortification of mulberry leaf powder and extracts on Bombyx mori L

Introduction

Nutrition plays a fundamental role in the growth and productivity of organisms, including silkworms. The mulberry (Morus spp.) leaf, being the exclusive food source for the silkworm Bombyx mori L., significantly influences the growth, development, health, feed consumption, and feed conversion efficiency of silkworms. Approximately 70% of the silk protein synthesized by silkworms is directly derived from the proteins present in mulberry leaves, making larval nutrition and leaf nutritive value crucial for silk production. Fortifying mulberry leaves with supplementary compounds to enhance cocoon production has gained significant attention. Previous studies have demonstrated that enriching mulberry leaves with various nutrients can improve larval feeding and cocoon production (Etaberi, 2002; Etaberi et al., 2004; Islam et al., 2004; Rajabi et al., 2007) [6,7 10, 21] . Although mulberry leaves naturally contain balanced nutrients for silkworm growth, their quality often falls short due to varying growing conditions and management practices (Luskar & Datta, 2000) [13] . Recent advancements in sericulture research have focused on fortifying mulberry leaves with botanical extracts and nutrients to enhance leaf quality and feed efficiency, thereby improving cocoon production and silk quality (Murugan et al., 1998; Masthan et al., 2011) [16,15] . The addition of amino acids, vitamins, glucose, and other supplements has shown positive effects on larval growth, cocoon characteristics, and silk production (Sarkar et al., 1995) [24] . Various plant extracts, chemical nutrients, and natural products, such as Lantana camara, Tridax procumbens, Withania somnifera, Aloe vera, algal extracts, honey, and Moringa oleifera, have been explored for their potential to enhance mulberry leaf quality and silkworm performance (Rajashekar et al., 1997; Bhaskar et al., 2015; Raja & Kumar, 2016) [ 20,9,27] . Additionally, commercial nutrient supplements like 'Serifeed' have demonstrated promising results in improving silkworm growth, cocoon parameters, and yield (Kumar & Michael, 2006, 2011) [2,3] . Despite these advancements, most studies have focused on fortifying normal-quality mulberry leaves, often overlooking the impact of leaf quality on fortification outcomes. An exception is the work of Bongale and Krishna (1996) [4] , who fortified chlorotic mulberry leaves with sucrose. There is limited research on the effect of fortifying poor-quality mulberry leaves with extracts or dust derived from superior-quality leaves. To address this gap, the present study was undertaken to evaluate the impact of fortifying poor-and good-quality mulberry leaves with leaf powder extracts and dust prepared from superior-quality leaves on silkworm performance. This approach aims to provide an innovative solution for overcoming the challenges associated with poor-quality mulberry leaves in silkworm rearing.

Materials and Methodology

The study was conducted to evaluate the impact of fortified mulberry leaves on silkworm performance under various treatments. The treatments included poor-quality mulberry leaves fortified with 5% aqueous extract of superior quality leaf powder (T 1 ), water-ethanol (1:1) extract of superior quality leaf powder (T 2 ), superior quality leaf powder dust (T 3 ), distilled water spray (control, T 4 ), and no spray (control, T 5 ). Similar treatments were applied to goodquality mulberry leaves, denoted as T 6 through T 10 . The mulberry leaves used were of cultivars S36 for young silkworms and V1 for adult silkworms. Superior-quality leaves were harvested from well-maintained gardens of the V1 cultivar, while good-quality leaves came from regularly maintained gardens of S36 and V1 cultivars. Poor-quality leaves were obtained from V1 gardens maintained with limited irrigation, organic manure, and without chemical fertilizers. The bivoltine hybrid FC1 × FC2 was selected for the study, and standard rearing techniques (Dandin & Giridhar, 2010) [5] were followed. Superior-quality leaves were powdered, dried in the shade, and sieved to produce fine dust. Extracts were prepared using 5 g of leaf powder soaked in 100 ml of either distilled water (aqueous extract) or a 1:1 waterethanol mixture (overnight), followed by filtration through a muslin cloth. Ethanol was evaporated from the extract before use. For uniform application, fortified leaves were sprayed with 300 ml of solution per kg of leaf, and the treatments were imposed from the 3rd to 5th instar. Experimental design included 10 treatments with 4 replications and 40 randomized batches, each containing 100 silkworms. Parameters studied included larval weight, cocoon weight, sex-wise pupal and shell weights, filament length, denier, renditta, and raw silk percentage. Leaf quality parameters such as carbohydrate content (Anthrone method; Plummer, 1971) [19] , protein content (Lowry's method; Lowry et al., 1951) [14] , and chlorophyll content (Arnon, 1949) [1] were analyzed. Statistical analysis was performed using SPSS (version 10.0), employing ANOVA and Tukey's HSD test at p = 0.01 and p = 0.05.

Results and Discussion

The study systematically examined the effects of fortifying poor and good-quality mulberry leaves with aqueous extract, water-ethanol extract, and leaf powder dust on the rearing performance of silkworms and silk production parameters. The findings, presented in the table, demonstrated that fortifications significantly improved various rearing parameters and silk-related traits in silkworms fed with poor-quality leaves, whereas the effects were minimal or negligible for silkworms fed with good-quality leaves.

The larval weight increased significantly in all fortified treatments of poor-quality leaves, highlighting the positive effect of fortification. Among the treatments, the waterethanol extract recorded the highest larval weight (4.51±0.14 g), followed by leaf powder dust (4.49±0.11 g) and aqueous extract (4.44±0.11 g). These values were significantly higher than the positive control (4.32±0.10 g) and absolute control (4.21±0.03 g). On the other hand, in silkworms reared on good-quality leaves, fortifications did not lead to statistically significant differences. The larval weight remained consistent across treatments, with values such as 4.58±0.08 g for the water-ethanol extract and 4.55±0.16 g for the aqueous extract, indicating that the inherent nutritional quality of the good-quality leaves rendered fortifications unnecessary.

Cocoon Weight

Fortifications also had a substantial impact on the cocoon weight of silkworms fed with poor-quality leaves. The water-ethanol extract treatment resulted in the highest cocoon weight (2.21±1.02 g), followed closely by the aqueous extract (2.20±0.68 g) and leaf powder dust (2.01±0.67 g). These treatments significantly outperformed the positive control (2.09±0.30 g) and the absolute control (2.22±0.52 g). In contrast, no significant improvement was observed in cocoon weight for good-quality leaves, as values like 2.21±1.23 g (water-ethanol extract) and 2.20±0.14 g (aqueous extract) were comparable to the controls. This result further supports the finding that fortification primarily benefits poor-quality leaves.

The female pupa weight followed a similar trend, with significant improvements observed only in treatments with poor-quality leaves. The water-ethanol extract produced the highest female pupa weight (1.88±0.04 g), followed by leaf powder dust (1.903±0.06 g) and aqueous extract (1.83±0.31 g). These values were significantly higher than those in the positive control (1.80±0.06 g) and absolute control (1.76±0.08 g). For male pupa weight, the leaf powder dust treatment recorded the highest weight (1.85±0.80 g) in poorquality leaves, outperforming the water-ethanol extract (1.83±0.46 g) and aqueous extract (1.81±0.91 g). Notably, in good-quality leaves, fortifications did not significantly impact female or male pupa weights, with values remaining consistent across treatments.

The shell weight of both female and male cocoons showed marginal improvements across treatments in both leaf qualities. For female cocoons, the water-ethanol extract treatment resulted in the highest shell weight (0.35±0.00 g) in poor-quality leaves, while for male cocoons, the same treatment recorded the highest weight (0.31±0.05 g). However, these changes were not statistically significant, particularly in good-quality leaves, where fortifications yielded similar values across treatments.

The filament length, a critical parameter for silk production, showed notable improvements in silkworms reared on fortified poor-quality leaves. The water-ethanol extract resulted in the longest filament length (12090±0.05 m), followed by leaf powder dust (1264±0.00 m) and aqueous extract (1126±0.03 m). These were significant improvements over the positive control (1099±0.00 m) and absolute control (1021±0.05 m). In good-quality leaves, filament length was slightly longer in fortified treatments, with the highest value recorded in the water-ethanol extract treatment (1286±0.00 m). However, these improvements were not statistically significant.

For poor-quality leaves, the water-ethanol extract showed the most consistent improvements in silk quality parameters, producing the finest denier (2.64±0.02), the best renditta (5.04±0.03), and the highest raw silk percentage (4.86±0.18%). These values were significantly better than those of the positive and absolute controls. Fortifications with leaf powder dust and aqueous extract also improved these parameters but to a slightly lesser extent. In goodquality leaves, however, the fortifications had little impact, as denier, renditta, and raw silk percentage remained comparable to controls, reflecting the limited need for additional supplementation in nutritionally rich leaves. Bongale and Krishna (1996) [4] have reported the improvement in larval weight, cocoon weight and shell weight in the chloratic leaf fortified with sucrose. Similarly, extracts of Parthenium hysterophorus (Rajashekar et al., 1997) [20] , Phylanthus niruri (Shubha, 2007) [25] , Tribulus terrestris (Murugesh and Mahalingam, 2005) [30] , Aloe vera oil (Singh et al., 2014) [18] , etc., have shown to improve the larval weight, cocoon weight, pupal weight, shell weight etc., however, in these studies the fortification was made to the good quality leaves. The data on filament length, denier, renditta and raw silk percentage of silkworm reared on various treatments shown marginal improvement indicating the beneficial effect of fortification of mulberry leaf with its powder extracts. The present study clearly indicated that the various rearing parameters of silkworm hybrid are improved substantially through fortification of poor quality leaf. Among the three fortifications, poor quality mulberry leaves fortified with extracts of water-ethanol gave best results followed by the leaves fortified with leaf powder dust and aqueous extracts.

Conclusion

The present experiment was conducted to understand the effect of fortification of poor and good quality mulberry leaves with 5% aqueous extract, 5% extract in water-ethanol (1:1) mixture and powder dust of leaf powder prepared out of superior quality leaf. Three qualities of mulberry leaf viz., poor, good and superior, of V1 and S36 cultivars were used with the silkworm hybrid FC1 x FC2 in this comparative study. Improvements ranging from marginal to highly significant, in silkworm rearing parameters viz., larval weight, cocoon weight, male and female pupal weights and also male and female shell weights are recorded in three fortification treatments with only poor quality leaves. Among the three fortification treatments studied, mulberry leaves fortified with the extract of water-ethanol was found most effective followed by fortification with leaf powder dust and fortification with aqueous extract. From this investigation, it may be concluded that mulberry leaves if they are found not upto the mark while rearing silkworm due to various reasons or inadequacies in the management of mulberry garden, such leaves could be fortified with mulberry leaf powder extracts to overcome the problem to some extent.

References

1. Arnon DI. Copper enzymes in isolated chloroplasts, polyphenoxidase in Beta vulgaris. Plant Physiol. 1949;24:1-15.
2. Anandakumar MD, Michael AS. Influence of nutritive additive on digestive physiology of silkworm Bombyx mori L. Department of Life Science, Bangalore University, Jnana Bharathi, Bangalore, Karnataka, India. 2006;1(11):9-14.
3. Anandakumar MD, Michael AS. Effect of Serifeed, a feed supplement enriched feed of silkworm Bombyx mori L., on its nutritional and economic parameters. Int J Sci Eng Res. 2011;1(11):9-14.
4. Bongale UD, Krishna M. Fortification of chlorotic leaf of mulberry (Morus indica L. var. M5) with sucrose for improvement of feed value to the silkworm, Bombyx mori L. Indian J Seric. 1996;35(1):64-66.
5. Dandin SB, Giridhar K. Hand Book of Sericulture Technologies. Central Silk Board, Bangalore, Karnataka, India. 2010. 427 p.
6. Etebari K. Effect of enrichment of mulberry leaves (Morus alba) with some vitamins and nitrogenous compounds on some economic traits and physiological characters of silkworm Bombyx mori (Lepidoptera: Bombycidae). Isfahan University of Technology, Iran. 2002.
7. Etebari K, Ebadi R, Mantindoost L. Effect of vitamin C on biological, biochemical and economical characteristics of the silkworm Bombyx mori L. Int J Indust Entomol. 2004;8:81-87.
8. Etebari K, Ebadi R, Matindoost L. Physiological changes of silkworm (Bombyx mori L.) larvae fed on mulberry leaves supplemented with nitrogenous compounds. J Ent Res Soc. 2007;9(2):1-15.
9. Gobena, Bhaskar RN. Fortification of mulberry leaves with medicinal botanical plant extracts effect on silkworm Bombyx mori L. (PM x CSR2) larval growth and cocoon traits. J Biol Sci. 2015;15(4):199-206.
10. Islam MR, Ali MAO, Paul DK, Sultana S, Banu NA, Islam MR. Effect of salt, nickel chloride supplementation on the growth of silkworm Bombyx mori L. (Lepidoptera: Bombycidae). J Biol Sci. 2004;4:170-172.
11. Jolly MS. Appropriate Sericulture Techniques. CSRTI, Mysore. 1987;1-16.
12. Khedr MMA, EI-Shafiey SN, Mead HMI. Influence of fortification of mulberry leaves with natural and synthetic multivitamins on growth and development of Bombyx mori L. J Plant Prot Path. Mansoura Univ. 2013;4(1):111-123.
13. Laskar N, Datta M. Effect of alfalfa tonic and its organic ingredients on growth and development of silkworm Bombyx mori L. race instar. Environ Ecol. 2000;18:591-596.
14. Lowry OH, Rosenbourgh NJ, Farr AL, Randal RJ. Protein measurement with the Folin-phenol reagent. J Biol Chem. 1951;193:265-275.
15. Masthan K, Kumar TR, Murthy CN. Beneficial effects of blue-green algae Spirulina and yeast Saccharomyces cerevisiae on cocoon quantitative parameters of silkworm Bombyx mori L. Asian J Microbiol Biotechnol. 2011;13(1):205-208.
16. Murugan K, Jayabalan D, Senthilkumar N, Senthil Nathan S, Sivaprakasam N. Growth-promoting effect of plant products on silkworm. J Sci Ind Res. 1998;57:740-745.
17. Prajapathi ND, Purohit SS, Sharma AK, Kumar T. A Hand Book of Medicinal Plants. Agrobios, India. 2003. 554 p.
18. Singh P, Surendra, Upadhyay VB. Effect of Aloe vera oil on hatchability of multivoltine mulberry silkworm Bombyx mori L. Pinnacle J Pub. 2014.
19. Plummer DT. An Introduction to Practical Biochemistry. Tata McGraw Hill Publishing Company Ltd., Bombay and New Delhi, India. 1971.
20. Rajasekaragouda R, Gopalan M, Jeyaraj N, Natarajan N. Field performance of plant extracts on mulberry silkworm Bombyx mori L. Entomon. 1997;22(3-4):235238.
21. Rajabi RK, Ebadi R, Mirhosseini SA, Zolfaghari A, Etebari K. A review on nutritive effect of mulberry leaves enriched with vitamins on economic traits and biological parameters of Bombyx mori L. ISJ. 2007;4:86-91.
22. Rajeshwari K, Isaiarasu L. Influence of leaf, flower, and pod extracts of Moringa oleifera on the growth and reproductive parameters of Bombyx mori L. J Entomol. 2004;29(4):331-338.
23. Radjabi R. Effect of mulberry leaves enrichment with amino acids supplementary nutrient on silkworm Bombyx mori L. North of Iran. Acad J Entomol. 2010;3(1):45-51.
24. Sarkar AA, Haque MR, Rab, Absar MN. Effect of feeding mulberry (Morus sp) leaves supplemented with different nutrients to silkworm (Bombyx mori L.). Curr Sci. 1995;69:185-188.
25. Shubha K, Bhaskar RN, Chinnaswamy KP. Influence of medicinal plant extracts on rearing parameters of PM x CSR2. Bull Indian Acad Seric. 2007;11(2):1-3.
26. Selvi S, Selvisabhanayakam E, Mathivanan V. Effect of Spinacia oleracea (spinach) on morphometric parameters of silkworm Bombyx mori L. Int J Recent Sci Res. 2014;2:544-547.
27. Raja VS, Muthu Kumar SA. Influence of Chlorella and Scenedesmus algal extracts on the economic traits of silkworm Bombyx mori L. Int J Adv Res. 2016;4(9):1521-1525.
28. Thulasi N, Sivaprasad S. Synergetic effect of ascorbic acid and lemon juice on the growth and protein synthesis in the silkworm Bombyx mori L. and its influence on economic traits of sericulture. J Bio Innov. 2013;2(4):168-183.
29. Thulasi N, Sivaprasad S. Larval growth, silk production and economic traits of Bombyx mori L. under the influence of honey-enriched mulberry diet. J Appl Nat Sci. 2015;7(1):286-292.
30. Murugesh K, Mahalingam I. Influence of Tribulous terrestris L. on the growth of silkworm, Bombyx mori L. and its impact on economic traits. Progress of research in organic sericulture and Seri-by products utilization, University of Madras, Vellore; 2005. p. 151155.