Studies on the protein content in popular bivoltine double hybrid FC1 x FC2 of the silkworm Bombyx mori L. using fortified mulberry leaf

Introduction

The mulberry silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is a commercially important insect owing to its remarkable efficiency in converting mulberry leaf protein into silk fiber. Larval growth, silk gland development and overall silk productivity are directly influenced by the quality and quantity of nutrients available in the diet. Hence, nutritional improvement of mulberry leaves through fortification with proteins, carbohydrates, amino acids, vitamins, hormones and other bioactive compounds has been widely recognized as an effective method for enhancing larval growth and cocoon productivity (Laskar and Datta, 2000; Jeyapaul, et al., 2003) [8,5] . Protein is the most critical nutrient influencing the growth and metabolic efficiency of the silkworm larvae. Among various natural protein supplements, spirulina a cyanobacterial biomass has gained importance due to its exceptionally high protein content and rich profile of essential amino acids such as glycine, lysine, methionine, glutamine and phenylalanine, along with vital vitamins including tocopherol, riboflavin, biotin, niacin, folic acid and βcarotene. Spirulina fortification has been reported to enhance the larval physiology, improve digestion and increase the overall nutritional efficiency of B. mori. Similarly, soya flour also serves as an excellent plant-based protein source enriched with vitamins (A, B and D), minerals such as calcium and magnesium and essential amino acids. Dietary supplementation with soya proteins has been reported to improve larval growth, enhance metabolic activity and significantly elevate the economic traits of Bombyx mori by increasing cocoon weight, shell ratio and filament quality International Journal of Advanced Biochemistry Research https://www.biochemjournal.com (Kamaraj, et al., 2017; Shruti, et al., 2019) [6,9] . Since nearly 70% of the silk filament protein is derived from mulberry leaf protein, efficient digestion and assimilation of dietary proteins are crucial for silk synthesis. The activity of midgut proteases responsible for converting mulberry proteins into silk proteins varies across pure breeds and hybrid combinations (Kumar and Kalpana, 2009) [7] , indicating the importance of evaluating nutritional responses in specific genetic backgrounds. Double hybrids as FC 1 ×FC 2 are widely used in commercial sericulture due to their hybrid vigour, but their physiological response to protein-enriched diets may differ due to genetic and metabolic variations. Therefore, understanding how spirulina and soya flour influence protein accumulation in these hybrids is vital for optimizing feeding strategies and improving silk productivity. Hence, the present study aims to evaluate the impact of spirulina and soya flour supplementation at different concentrations on the protein content of FC 1 ×FC 2 double hybrid of Bombyx mori L., with emphasis on larval physiology and nutritional efficiency to convert the mulberry protein to silk protein when fortified with external protein supplements.

Materials and Methods

The present study was conducted using bivoltine double hybrid namely FC 1 ×FC 2 known for their superior silk yield and hybrid vigour. The disease free layings (DFLs) of the hybrid was procured from the Silkworm Seed Production Centre (SSPC), NSSO, Mysuru, Karnataka. Upon receipt, the DFLs were incubated under controlled conditions (25±1 °C temperature and 80±5 relative humidity) until hatching.

The young larvae were reared on S 36 mulberry variety during the first and second instars (chawki stage), and V 1 variety for the late age larvae (third, fourth and fifth instars). The rearing activities were carried out in the Department of Studies in Sericulture Science, University of Mysore, Manasa Gangothri, Mysuru following the standard protocols suggested by Dandin and Giridhar (2010) [3] . The various treatments were administered after the fourth moult during the fifth instar larval stage on day-2, day-3 and day-4 larvae were divided into subgroups and fed with mulberry leaves treated with the following formulations:  Control batches: Distilled water control and absolute control (no treatment)  Spirulina treatments: 200 ppm, 300 ppm and 400 ppm  Soya flour treatments: 200 ppm, 300 ppm and 400 ppm The powders of the above were dissolved using double distilled water and fresh mulberry leaves were smeared with the solutions prepared. The smeared leaves were air-dried for 15-30 minutes before feeding to the silkworm larvae. The larvae were fed with untreated leaves at 11:00 a.m. and the treated leaves at 4:00 p.m. during 2 nd , 3 rd and 4 th day of the fifth instar as treatments followed by untreated mulberry leaf. The samples such as the silk glands and haemolymph were dissected and collected from the silkworm larvae under sterile conditions. All samples were immediately processed or stored at appropriate temperatures (-20 °C for short-term storage) to preserve their biochemical integrity before analysis. The estimations were carried out following standard biochemical protocols for the silkworm (Chand Asaf and Mahavishnu, 2018) [2] .

2.1 Procedure: In the Lowry's et al., (1951) method for protein estimation, 1 ml of the sample, 1 ml of BSA standard and 1 ml of distilled water (blank) are taken in separate test tubes. To each tube, 5 ml of freshly prepared alkaline copper reagent is added, mixed thoroughly, and kept undisturbed for 10 minutes at room temperature. Following this, 0.5 ml of diluted (1:1) Folin-Ciocalteu reagent is added to each tube, mixed immediately and incubated for 30 minutes at room temperature for color development. After the incubation period, the optical density (OD) is measured at 660 nm using a spectrophotometer against the blank, and the protein concentration in the sample is determined by comparing the optical density (OD) with that of the standard values. During the rearing of silkworm larvae, data collected pertaining to selected economic traits namely fecundity, hatching percentage, larval duration, matured larval weight, cocoon weight, shell weight, shell ratio, filament length, denier and renditta were collected to correlate the same with the protein estimation.

Statistical Analysis

The data collected were subjected to statistical analysis, including the calculation of mean and standard error. A ONE-WAY analysis of variance (ANOVA) was performed and the data were further analyzed using Factorial Analysis of Variance (p≤0.05, p≤0.01) (Sundarraj et al., 1972) through SPSS for Windows, version 21.0 to assess the significance of treatments, seasons, and day-wise effects on the economic and biochemical parameters. Graphs were plotted using the MS-Excel application (version-2013).

Results

Protein content in silk glands

The protein content in the silk glands of FC 1 ×FC 2 double hybrid under various treatments (Fig. 1) was estimated across different developmental days (day-3, day-4 and day-5) in the 5th instar larval stage during pre-monsoon, monsoon and post-monsoon seasons. The results are presented in figure 1, the protein content is expressed in µg/mg and µg/ml depending on the tissue taken. During the pre-monsoon season, protein content in silk glands increased progressively from day-3 to day-5 across spirulina and soya flour treatment batches. On day-3, the highest protein content among spirulina treatments was observed in SP-400 (117.6 µg/mg), while the lowest was in SP-200 (97.45 µg/mg). Among the soya flour treatments, the highest was recorded in SF-400 (116.2 µg/mg) and the lowest in SF-200 (95.51 µg/mg). On day-4, SP-400 treatment observed the highest protein level (142.3 µg/mg), whereas the lowest was in SP-200 (111.5 µg/mg); for soya flour treatments, the highest was SF-400 (140.7 µg/mg) and the lowest was SF-200 (110.8 µg/mg). On day-5, the highest protein content among spirulina treatments was noted in SP-400 (154.6 µg/mg), and the lowest in SP-200 (122.2 µg/mg); similarly, in soya flour treatments, SF-400 had the highest (151.2 µg/mg) and SF-200 the lowest (121.9 µg/mg). In the monsoon season, a consistent rise in protein content was observed. On day-3, the highest protein level among spirulina treatments was found in SP-400 (120.7 µg/mg), while the lowest was in SP-200 (99.74 µg/mg); for soya flour treatments, the highest was in SF-400 (119.2 µg/mg) and the lowest in SF-200 (98.71 µg/mg). On day-4, SP-400 recorded the highest protein content (147.3 µg/mg), and the lowest was in SP-200 (115.3 µg/mg); within soya flour treatments, the highest was observed in SF-400 (146.3 µg/mg), and the lowest in SF-200 (114.3 µg/mg). On day-5, the highest protein content among spirulina treatments was again found in SP-400 (158.7 µg/mg), while the lowest was in SP-200 (127.6 µg/mg); similarly, in soya flour batches, SF-400 had the highest value (156.3 µg/mg) and SF-200 the lowest (126.6 µg/mg). In the post-monsoon season, protein content followed the same increasing trend. On day-3, the highest protein content among spirulina treatments was recorded in SP-400 (119.5 µg/mg), and the lowest in SP-200 (99.52 µg/mg); for soya flour treatments, the highest was in SF-400 (118.7 µg/mg), and the lowest in SF-200 (97.05 µg/mg). On day-4, SP-400 exhibited the highest protein level (144.7 µg/mg), while the lowest was observed in SP-200 (113.7 µg/mg); in soya flour treatments, SF-400 had the highest (142.3 µg/mg), and SF-200 the lowest (112.7 µg/mg). On day-5, the highest protein content among spirulina treatments was found in SP-400 (156.7 µg/mg), with the lowest in SP-200 (125.5 µg/mg); in soya flour treatments, the highest was SF-400 (154.2 µg/mg) and the lowest was SF-200 (124.4 µg/mg). Among all three seasons, the highest protein content was recorded during the monsoon season, followed by post-monsoon and pre-monsoon seasons. The result revealed analysis of variance (one-way ANOVA) conducted on the protein (silk gland) estimation parameter revealed highly significant differences (p<0.01) among seasons, day wise treatments and treatment batches.

Protein content in haemolymph

The protein content in the haemolymph of FC 1 ×FC 2 double hybrid under various treatments (Fig. 2) was estimated across different developmental days (day-3, day-4 and day-5) in the 5th instar larval stage during pre-monsoon, monsoon and post-monsoon seasons. The results are presented in figure 2. During the pre-monsoon season, protein content in haemolymph increased progressively from day-3 to day-5 across spirulina and soya flour treatment batches. On day-3, the highest protein content among spirulina treatments was observed in SP-400 (110.2 µg/ml), while the lowest was in SP-200 (90.41 µg/ml). Among soya flour treatments, the highest was recorded in SF-400 (109.6 µg/ml) and the lowest in SF-200 (89.85 µg/ml). On day-4, SP-400 treatment observed the highest protein level (122.6 µg/ml), whereas the lowest was in SP-200 (102.3 µg/ml); for soya flour treatments, the highest was SF-400 (121.3 µg/ml) and the lowest was SF-200 (101.4 µg/ml). On day-5, the highest protein content among spirulina treatments was noted in SP-400 (129.7 µg/ml), and the lowest in SP-200 (110.1 µg/ml); similarly, in soya flour treatments, SF-400 had the highest (128.8 µg/ml) and SF-200 the lowest (109.4 µg/ml). In the monsoon season, a consistent rise in protein content was observed. On day-3, the highest protein level among spirulina treatments was found in SP-400 (113.8 µg/ml), while the lowest was in SP-200 (93.21 µg/ml); for soya flour treatments, the highest was in SF-400 (113.2 µg/ml) and the lowest in SF-200 (93.06 µg/ml). On day-4, SP-400 recorded the highest protein content (126.7 µg/ml), and the lowest was in SP-200 (106.8 µg/ml); within soya flour treatments, the highest was observed in SF-400 (125.5 µg/ml), and the lowest in SF-200 (105.5 µg/ml). On day-5, the highest protein content among spirulina treatments was again found in SP-400 (134.8 µg/ml), while the lowest was in SP-200 (114.3 µg/ml); similarly, in soya flour batches, SF-400 had the highest value (132.3 µg/ml) and SF-200 the lowest (113.2 µg/ml). In the post-monsoon season, protein content followed the similar increasing trend. On day-3, the highest protein content among spirulina treatments was recorded in SP-400 (112.8 µg/ml), and the lowest in SP-200 (92.25 µg/ml); for soya flour treatments, the highest was in SF-400 (111.5 µg/mg), and the lowest in SF-200 (91.32 µg/ml). On day-4, SP-400 exhibited the highest protein level (124.4 µg/ml), while the lowest was observed in SP-200 (104.3 µg/ml); in soya flour treatments, SF-400 had the highest (123.6 µg/ml), and SF-200 the lowest (103.3 µg/ml). On day-5, the highest protein content among spirulina treatments was found in SP-400 (131.7 µg/ml), with the lowest in SP-200 (112.2 µg/ml); in soya flour treatments, the highest was SF-400 (130.3 µg/ml) and the lowest was SF-200 (111.2 µg/ml). Among all three seasons, the highest protein content was recorded during the monsoon season, followed by post-monsoon and pre-monsoon seasons. The result revealed analysis of variance (one-way ANOVA) conducted on the protein (haemolymph) estimation parameter revealed highly significant differences (p<0.01) among seasons, day wise treatments and treatment batches.

Economic Traits

The mean values of the selected economic traits of bivoltine double hybrid FC 1 xFC 2 of the silkworm, Bombyx mori are presented in Table 1. Among the bivoltine hybrids selected for the study, SP400 on day-5, day-4, day-3 in fifth instar showed higher matured larval weight, cocoon weight and shell weight. During the pre-monsoon season, the highest matured larval weight (48.00 g) was recorded in SP400 treatment in day-5 compared to day-4 and day-3, whereas the lowest value (42.79 g) was observed in SP200 treatment in day-3 compared with day-4 and day-5 of 5 th instar larvae. The larval weight was found to be higher (42.21 g) in SP400 in day-5 compared to day-4 and day-3; minimum of (35.30 g) in SP200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The cocoon weight was highest (20.60 g) treated with SP400 on day-5 and lowest (16.74 g) in SP200 on day-3, while shell weight showed a maximum (4.200 g) for SP400 treatment in day-5 and a minimum (3.300 g.) in SP200 treatment in day-3 compared to day-4 of 5 th instar larvae. The filament length ranged from (1405 m) in SP400 in day-5 and (1188 m) SP200 of day-3; while in soya flour the highest matured larval weight (47.12 g) was recorded in SF400 treatment in day-5 compared to day-4 and day-3, whereas the lowest (42.02 g) was observed in SF200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The larval weight was found to be maximum (41.05 g) in SF400 in day-5 compared to day-4 and day-3, and a minimum of (34.17 g) in SF200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The cocoon weight was highest (20.10 g) in SF400 on day-5 and lowest (16.69 g) in SP200 on day-3; the shell weight was found to be maximum (4.247 g) in SF400 on day-5 and a minimum (3.060 g) in SF200 treatment in day-3 compared to day-4 and day-5. The filament length ranged from (1283 m) in SF400 in day-5 and (1176 m) SP200 of day-3 of 5 th instar. During the monsoon season, the highest matured larval weight (51.12 g) was recorded in SP 400 treatment in day-5 compared to day-4 and day-3, whereas the lowest (44.35 g) was observed in SP200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The larval weight was found maximum (42.44 g) in SP400 in day-5 compared to day-4 and day-3 of 5 th instar; minimum of (35.85 g) in SP200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The cocoon weight was highest (21.82 g) in SP400 on day-5 and lowest (17.17 g) in SP200 on day-3, while shell weight showed a maximum (5.100 g) for SP400 treatment in day-5 and a minimum (3.693 g) in SP200 treatment in day-3 compared to day-4 of 5 th instar. The filament length ranged from (1473 m) in SP400 in day-5 and (1197 m) SP200 of day-3; while in Soya flour the highest matured larval weight (49.67 g) was recorded in SF 400 treatment in day-5 compared to day-4 and day-3, whereas the lowest (44.67 g) was observed in SF200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The larval weight was found to be maximum of (41.53 g) in SF400 in day-5 compared to day-4 and day-3, and a minimum of (34.98 g) in SF200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The cocoon weight was highest (21.58 g) in SF400 on day-5 and lowest (17.10 g) in SP200 on day-3; shell weight was found to be maximum (4.727 g) in SF400 on day-5 and a minimum (3.353 g) in SF200 treatment in day-3 compared to day-4 and day-5. The filament length ranged from (1411 m) in SF400 in day-5 and (1212 m) SP200 of day-3 of 5 th instar. During the post-monsoon season, the highest matured larval weight (49.03 g) was recorded in SP 400 treatment in day-5 compared to day-4 and day-3, whereas the lowest value (44.76 g) was observed in SP200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The larval weight was found to be higher (42.12 g) in SP400 in day-5 compared to day-4 and day-3; minimum of (35.78 g) in SP200 treatment in day-3 compared with day-4 and day-5 of 5 th instar larvae. The cocoon weight was highest (21.20 g) in SP400 on day-5 and lowest (16.99 g) in SP200 on day-3, while shell weight showed a maximum value (4.96 g) for SP400 on day-5 and a minimum (3.56 g) in SP200 treatment in day-3 compared to day-4 of 5 th instar larvae. The filament length ranged from (1453 m) in SP400 on day-5 and (1189 m) SP200 of day-3; while in soya flour treatment the highest matured larval weight (48.96 g) was recorded in SF 400 in day-5 compared to day-4 and day-3, whereas the lowest (43.65 g) was observed in SF200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The larval weight was found to be maximum of (41.29 g) in SF400 in day-5 compared to day-4 and day-3 and was minimum (34.40 g) in SF200 treatment in day-3 compared with day-4 and day-5 of 5 th instar. The cocoon weight was highest (21.10 g) in SF400 on day-5 and lowest (16.72 g) in SP200 on day-3, the shell weight was found to be maximum (4.42 g) in SF400, day-5 and a minimum (3.19 g) in SF200 treatment in day-3 compared to day-4 and day-5. The filament length ranged from (1383 m) in SF400 in day-5 and (1194 m) SP200 of day-3 of 5 th instar larvae.

Discussion

The silkworm (Bombyx mori) growth and silk productivity are largely determined by nutritional status, enzymatic efficiency, and environmental conditions. Biochemical components such as proteins, carbohydrates, and enzymes like amylase and phosphatases play crucial roles in larval metabolism and silk synthesis. Dietary supplementation is widely recognized as an effective approach to enhance larval physiological performance and cocoon quality, and its impact may vary with tissue type, developmental stage, and seasonal conditions. The protein content in FC 1 ×FC 2 is a vital indicator of metabolic and synthetic potential in silkworm larvae. In the present study, the protein content of FC 1 xFC 2 were significantly influenced by both the type of dietary supplement and tissue type studied. In the silk glands, spirulina fortification at SP400 produced the highest protein content, followed by SP300 and SP200, indicating that enriched protein sources directly enhance silk protein synthesis. In contrast, soya flour treatments (SF400, SF300, and SF200) showed moderate increases, while control batches (DWC and AC) exhibited the lowest protein levels. Similar trends were observed in the haemolymph, reflecting systemic metabolic enhancement, where protein accumulation suggests improved storage and mobilization for biosynthetic purposes. Seasonal variation revealed that the monsoon season supported higher protein levels across tissues, likely due to optimal temperature and humidity for metabolic activity. Among the seasons, larvae reared during the monsoon season exhibited higher protein accumulation than the larvae reared during pre-monsoon and post-monsoon seasons, possibly due to optimal temperature and humidity favoring digestion and metabolism. Across all treatment batches, a steady increase in protein content was noted from day-3 to day-5 of 5 th instar larva. Among all three seasons, the highest protein content is found in silk glands when compared with haemolymph was recorded during the monsoon season, followed by postmonsoon and pre-monsoon seasons.

Conclusion

The present investigation clearly establishes that fortification of mulberry leaves with spirulina and soya flour markedly influences protein metabolism in FC 1 ×FC 2 double hybrid of Bombyx mori. A consistent and significant increase in protein content was observed in silk glands and haemolymph from day-3 to day-5 of the 5th instar larvae, indicating enhanced metabolic activity and improved assimilation efficiency for conversion of leaf protein to silk protein.

Acknowledgement

AcknowledgementsWe thank The Chairman, Department of Studies in Sericulture Science, University of Mysore, Mysuru for providing the necessary facilities to carry out this research work.

References

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