|Year : 2011 | Volume
| Issue : 1 | Page : 64-68
Modulations in the haemolymph of silkworm [Bombyx mori (l). (Lepidoptera: Bombycidae)] fed with mulberry leaves augmented with cowpeas (Vigna unguiculata)
Manjula Saravanan1, Sabhanayakam Selvi1, Mathivanan Veeranarayanan1, Saravanan Nadanam2
1 Department of Zoology, Faculty of Science, Annamalai University, Annamalai nagar-608 002, Tamil Nadu, India
2 Division of Biochemistry, Rani Meyyammai College of Nursing, Faculty of Medicine, Annamalai University, Annamalai nagar-608 002, Tamil Nadu, India
|Date of Submission||24-Dec-2010|
|Date of Acceptance||02-Jan-2011|
|Date of Web Publication||11-Mar-2011|
Division of Biochemistry, Rani Meyyammai College of Nursing, Faculty of Medicine, Annamalai University, Annamalai nagar - 608 002, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: To evaluate the effect of mulberry leaves supplemented with cowpeas (Vigna unguiculata) on the haemolymph of silkworm. Materials and Method: Finely powdered V. unguiculata was dissolved in distilled water and diluted to 2.5%, 5%, 7.5%, and 10% concentrations. Fresh mulberry leaves (Morus alba L.) were sprayed with each concentration and fed to silkworms, from third to fifth instar, with five feedings per day. Group 1 larvae received mulberry leaves sprayed with distilled water and served as control, group 2 larvae received 2.5% V. unguiculata-sprayed mulberry leaves, group 3 larvae received 5% V. unguiculata-sprayed mulberry leaves, group 4 larvae received 7.5% V. unguiculata-sprayed mulberry leaves, and group 5 larvae received 10% V. unguiculata-sprayed mulberry leaves. Result: Silkworm larvae fed on M. alba L. (mulberry) leaves enriched with 7.5% concentrations of V. unguiculata, significantly gained more cocoon weight, shell weight and shell/cocoon ratio as compared to those fed on normal MR 2 mulberry leaves. Hence, 7.5% dose of V. unguiculata was fixed as an effective dose. Further, same study was conducted to find out the biochemical changes in the haemolymph occurred on the first day of fifth instar larvae. There was significant increase in the haemolymph glucose, cholesterol, urea, total protein, aspartate transaminase, alanine transaminase, and alkaline phosphatase. But haemolymph uric acid decreased significantly. Conclusion: The results suggest that co-administration of V. unguiculata with mulberry leaves at a concentration of 7.5% enhanced the biochemical reaction involved in the silk production in the silkworm.
Keywords: Bombyx mori L ., Haemolymph, Morus alba L., Vigna unguiculata
|How to cite this article:|
Saravanan M, Selvi S, Veeranarayanan M, Nadanam S. Modulations in the haemolymph of silkworm [Bombyx mori (l). (Lepidoptera: Bombycidae)] fed with mulberry leaves augmented with cowpeas (Vigna unguiculata). Int J Nutr Pharmacol Neurol Dis 2011;1:64-8
|How to cite this URL:|
Saravanan M, Selvi S, Veeranarayanan M, Nadanam S. Modulations in the haemolymph of silkworm [Bombyx mori (l). (Lepidoptera: Bombycidae)] fed with mulberry leaves augmented with cowpeas (Vigna unguiculata). Int J Nutr Pharmacol Neurol Dis [serial online] 2011 [cited 2022 May 17];1:64-8. Available from: https://www.ijnpnd.com/text.asp?2011/1/1/64/77534
| Introduction|| |
The rearing of silkworm (Bombyx mori) on commercial scale for the production of cocoons to obtain raw silk is an important cottage industry since the dawn of human civilisation. Silkworm has been used as a sole source of natural silk for producing exquisite textile and dress material. Bombyx mori L. (silkworm) is a phytophagous lepidopteran insect, and a monophagous feeder on Morus alba L. (mulberry leaves). Scientists have tried to use alternative hosts for rearing silkworm, but these were not cost effective. Hence, the scientists used some nutrients, minerals and vitamins as food supplements. Positive impact of supplements was seen on silkworm growth and silk production. Ravikumar  emphasised that the quality and the nutritional status of mulberry has a considerable influence on the silkworm growth, silk yield and disease resistance. Silkworm requires specific essential sugars, amino acids, proteins, and vitamins for its normal growth.  Javed and Gondal  have also reported that silkworm fed on mulberry leaves supplemented with nitrogen and ascorbic acid showed higher growth and lower mortality. The effect of vitamin supplementation on the growth of silkworm has been investigated by many researchers. ,, Different scientists have worked on food supplementation of silkworm larvae to meet the requirements of natural silk production and much information has been compiled on nutritional significance of individual substances in the rearing of silkworm by using the chemically defined diets and their supplementation through mulberry leaves. ,
Cowpeas are a common food item in the southern United States, where they are often called field peas. Two subcategories of field peas are crowder peas, so called because they are crowded together in their pods, causing them to have square ends, and cream peas. The beans are called thatta kaai, and are an integral part of the cuisine in southern region of India. According to the USDA food database, cowpeas have the highest percentage of calories from protein among vegetarian foods.  Although the effects of nitrogen, vitamin, and salt supplementation on the growth of silkworm have been investigated by many researchers, the effect of mulberry leaves enriched with Vigna unguiculata was not investigated. Thus, the present study aimed to find out the effective dose of V. unguiculata application to mulberry leaves on cocoon weight, shell weight, and shell/cocoon ratio of silkworm. Till date, no authors have focussed on biochemical changes in silkworm fed with food supplements. By using the effective dose, further biochemical studies were done in the haemolymph of first day of fifth instar larvae of B. mori. The ultimate aim was to find whether the change in concentration of biochemicals have an impact on the growth and silk production of silkworm.
| Materials and Methods|| |
Eggs of silkworm L NB4, D2 (local Bivoltine) race were collected from farmers' training centre at Jayankodapattiam, Tamil Nadu, India. The eggs were placed in an incubator for hatching, at an ambient temperature of 25±2ΊC and relative humidity of 70-80%. After hatching, larvae were isolated from stock culture. The larvae were divided into five experimental groups, including the control group (distilled water control), with each group consisting of 10 larvae. The larvae were reared in cardboard boxes measuring 22 Χ 15 Χ 5 cm 3 covered with polythene sheet and placed in an iron stand with ant wells. V. unguiculata was purchased from the local market surrounding Chidambaram, Tamil Nadu, India, and was identified and authenticated by the Department of Botany, Annamalai University. It was then shade dried and powdered using mortar. Finely powdered V. unguiculata was dissolved in distilled water and diluted to 2.5%, 5%, 7.5%, and 10% concentrations. Fresh mulberry leaves were sprayed with each concentration and then dried in air for 10 min. Supplemented leaves were fed to silkworms, five feedings per day. Group 1 larvae received mulberry leaves sprayed with distilled water and served as control, group 2 larvae received mulberry leaves sprayed with 2.5% V. unguiculata, group 3 larvae received mulberry leaves sprayed with 5% V. unguiculata, group 4 larvae received mulberry leaves sprayed with 7.5% V. unguiculata, and group 5 larvae received mulberry leaves sprayed with 10% V. unguiculata. The feedings were maintained up to the cocoon stage of the silkworm. Cocoon weight, shell weight, pupa weight, and shell cocoon weight ratio were determined for all groups.
Same protocol was repeated only with mulberry leaves sprayed with 7.5% V. unguiculata and control larvae received mulberry leaves sprayed with distilled water for estimation of biochemical parameters. On the first day of fifth instar, 10 larvae were selected randomly from each group. The larval haemolymph was taken with a cut through one of the prolegs. From each larva, 0.5 ml of haemolymph was extracted, and then pooled haemolymph for each treatment was used for biochemical measurement. To avoid the activity of prophenol oxidase followed by melanisation of haemolymph, 1 mg of phenylthiourea was added to the samples.  The samples were then centrifuged for 10 min at 10,000 rpm.  The supernatant was transferred to new tubes and kept at -20°C until the beginning of experiments.
All biochemical estimations were done in ERBA Smartlab, fully auto analyser. Glucose estimations in the haemolymph were done by glucose oxidase-peroxidase (GOD-POD) based end-point method using the kit from Span Diagnostics Ltd, Surat. The total cholesterol in the haemolymph was estimated based on the method of Richmond.  The principles of this method are based on hydrolysis of cholesterol esters by cholesterol oxidase, cholesterol esterase, and peroxidase. Urea concentration was determined by measuring ammonia produced from urea, using a commercial urea assay kit (Span Diagnostics Ltd, Surat). Uric acid contents in the haemolymph were determined using uricase as described by Valovage and Brooks.  Serum total protein was estimated by Biuret's method.  Aspartate aminotransferase (AST, EC 126.96.36.199) and alanine aminotransferase (ALT, EC 188.8.131.52) were assayed using the diagnostic kit based on the method of Reitman and Frankel.  Alkaline phosphatase (ALP, EC 184.108.40.206.1) was estimated using the diagnostic kit based on Kind and King's method. ,
| Satistical Analysis|| |
Data were analysed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) using a commercially available statistics software package (SPSS® for Windows, V. 16.0, Chicago, USA). Results were presented as means ± SD. P values <0.05 were regarded as statistically significant.
| Results and Discussion|| |
[Table 1] shows the effects of supplementation of mulberry with various concentrations of V. unguiculata on cocoon weight, shell weight, and shell/cocoon ratio of silkworm. There is a significant raise in the cocoon weight, shell weight, and shell/cocoon ratio of the larvae fed with V. unguiculata-supplemented mulberry when compared with control groups. This may be due to the increased protein content of the mulberry supplemented with V. unguiculata. This is in agreement with the work done by Javed  and Islam et al,  regarding the increased cocoon weight, shell weight, and shell/cocoon ratio when silkworm were treated with various combinations of urea, ascorbic acid, and nickel chloride. There was no significance between the 7.5% and 10% dose of V. unguiculata with respect to cocoon weight and shell weight, but significant difference was noted with respect to shell/cocoon ratio. This may be due to the slight decreased in cocoon weight in the 10% dose. Therefore, the effective dose can be fixed at 7.5%.
|Table 1: Effects of various concentrations of V. unguiculata-supplemented mulberry on the cocoon weight, shell weight, and shell/cocoon ratio of B. mori.|
Click here to view
[Table 2] shows the effects of 7.5% V. unguiculata-supplemented mulberry on the haemolymph glucose and cholesterol levels of silkworm. A significant increase was noted in the haemolymph glucose in the V. unguiculata-supplemented group when compared with control group supplemented with distilled water. Increase in glucose levels indicates full fed state and stress-free condition, as decrease in glucose levels in the haemolymph is seen in starvation and external stress or a response to suppress the stress. V. unguiculata could enhance the feeding of larvae by favourable effects on leaves or decrease the anti-feedant characteristics. Etebari and Matindoost , have demonstrated that even starvation could have caused the reduction in levels of many biochemical compounds of haemolymph, such as glucose. Haemolymph cholesterol level in V. unguiculata groups showed a significant increase when compared with control groups. Needless to say, lipids are an important source of energy for insects. Decrease in levels of some compounds, including glucose and cholesterol, to a physiological stress could be attributed to the interruption in absorption system. Normal cholesterol in haemolymph indicates that there is no interruption in absorption system when mulberry leaves were enriched with V. unguiculata. Furthermore, increased cholesterol level may indicate the effective activation of absorption system, which, in turn, enhanced growth of silkworm and silk production.
|Table 2: Effects of 7.5% concentrations of V. unguiculata-supplemented mulberry on the haemolymph glucose and cholesterol of B. mori.|
Click here to view
[Table 3] shows the effects of mulberry supplemented with 7.5% V. unguiculata on the haemolymph urea, uric acid, and total protein levels of silkworm. There is a significant elevation in the haemolymph urea in V. unguiculata-supplemented group when compared with control group. From the standpoint of urea formation in the insects, enzymes such as arginase in several tissues might be induced by a diet with higher protein content such as an artificial diet. V. unguiculata also has high protein content. Changes in liver arginase activity according to dietary protein intake are well documented in mammals.  Preliminary studies on fat body arginase in silkworm larvae indicate that mitochondrial arginase activity per individual insect is higher in the larvae reared on an artificial diet than in those reared on fresh mulberry leaves (unpublished data). There is significant decrease in the haemolymph uric acid of the larvae fed with V. unguiculata-supplemented mulberry when compared with control groups. Generally, there is an adverse correlation between the amount of protein and uric acid in the haemolymph of silkworm and larvae with lowered protein have elevated uric acid. One of the important factors for measuring uric acid and urea was to know the nitrogen catabolism procedure in silkworm because V. unguiculata has high nitrogen content as well. Decrease in uric acid level in this group of larvae represents a decrease in some metabolisms, especially protein catabolism activity in them. There is significant elevation in the haemolymph total protein in the V. unguiculata-supplemented group when compared with control group. Studies on protein metabolism are considered most important in silkworm physiology because of its vital role in determination of chemical characteristics of silk proteins.  In general, the breakdown of proteins dominates over their synthesis due to enhanced proteolytic activity.  Maintaining protein levels in a highly organised state requires an active and continuous supply of energy. In the present study, V. unguiculata has high protein content, so the larvae groups may be supplied with uninterrupted energy.
|Table 3: Effects of 7.5% concentrations of V. unguiculata-supplemented mulberry on the haemolymph urea, uric acid, and total protein of B. mori.|
Click here to view
[Table 4] shows the effects of 7.5% V. unguiculata supplementation with mulberry on the haemolymph aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels of silkworm. There is a significant increase in the AST, ALT, and ALP activities in the haemolymph of V. unguiculata-supplemented group when compared with control group. The transaminases are important components of amino acid catabolism, which is mainly involved in transferring an amino group from one amino acid to another keto acid, thus forming another amino acid. The AST and ALT serve as a strategic link between carbohydrate and protein metabolism.  Elevation in activities of both AST and ALT enzymes in the haemolymph of silkworm supplemented with V. unguiculata indicated an active transportation of amino acids, which provided keto acid to serve as precursors in the synthesis of essential constituents for synthesis of silk fibres. Alkaline phosphatase is a group of hydrolytic enzymes that hydrolyse phosphomonoesters under alkaline condition. The activity of these enzymes is related to the physiological situation of silkworms and reflects absorption, digestion, and positive transportation of nutrients in the midgut. Different stress and disease result in considerable decrease in the ALP activity.  In the present study, the increase in ALP activity significantly indicates a better physiological situation and reflects the absorption, digestion, and positive transportation of nutrients in the midgut. It also indicates stress-free and disease-free condition of silkworm, which indicates that silkworm are healthy for the synthesis of silk. It is also reported that treatment with 20-hydroxyecdysone increased ALP activity in all tissues, except in the midgut. 
|Table 4: Effects of 7.5% concentrations of V. unguiculata-supplemented mulberry on the haemolymph AST, ALT, and ALP of B. mori.|
Click here to view
| Conclusion|| |
It may be concluded that supplementing V. unguiculata in mulberry as food for silkworm has different effects on its level of biochemicals. V. unguiculata showed considerable change in all biomolecules, which indicates more utilisation and turnover of carbohydrates, lipids, and proteins. This may be responsible for healthy, disease-free, and stress-free growth of the silkworm. Therefore, this supplementation can be incorporated to sericulture farms.
| References|| |
|1.||Ravikumar C. Western ghat as a bivoltine region prospects, challenges and strategies for its development. Indian Silk 1988;26:39-54. |
|2.||Sengupta K, Singh BD, Mustafij C. Nutrition of silkworm. Bombyx mori L. 1: Studies on the enrichment of mulberry leaf with various sugars, proteins, aminoacids and vitamins for vigorous growth of the worm and increased cocoon crop production. Indian J Sci 1972;11:11-27. |
|3.||JavedH, Godal HG. Effect of food supplementation by N and ascorbic acid on larval mortality of silkworm (Bombyx mori L.). Asian J Plant Sci 2002;5:556-7. |
|4.||Kanafi RR, Ebadi R, Mirhosseini SZ, Seidavi AR, Zolfaghari M, Etebari K. A review on nutritive effect of mulberry leaves enrichment with vitamins on economic traits and biological parameters of silkworm Bombyx mori L. Inv Sur J 2007;4:86-91. |
|5.||Kanafi RR, Ebadi R, Fazilati M, Mirhoseini SZ. Nutritive effects of mulberry leaves enrichment with riboflavin vitamin on bio-economic characters of silkworm, Bombyx mori L. 9th Arab Congress of Plant Protection. 19-23 November, 2006; Damascus, Syria. |
|6.||Kanafi RR, Ebadi R, Fazilati M, Mirhoseini SZ. The effects of mulberry leaves enrichment with pyridoxine-HCl on economic traits and biological parameters of silkworm Bombyx mori L. 17th Iranian Plant Protection Congress, Teheran, 2006. p. 391. |
|7.||Chenthilnayaki N, Selvisabanayakam V, Mathivanan, Rajathi V. Studies on the comparative efficacy of two varieties of mulberry leaves (Morus Sp) MR2 and V1 on Bombyx mori L. (Lepidoptera: Bombycidae) in relation to larval and pupal parameters. J Curr Sci 2004;5:49-54. |
|8.||Chenthilnayaki N, Selvisabhanyakam, Mathivanan V. Development of issue on mulberry cultivation in relation to silk production. Indian J Environ Ecoplan 2004;8:111-7. |
|9.||Shaw M. 100 most protein rich vegetarian foods. SmarterFitter Blog. Available from: http://smarterfitter.com/blog/2007/10/28/100-most-protein-rich-vegetarian-foods/. [cited in 2007 Oct 28]. |
|10.||Etebari K, Mirohoesieni SZ, Matindoost L. Study on interaspecific biodiversity of eight groups of silkworm (Bombyx mori) by biochemical markers. Insect Sci 2005;12:87-94. |
|11.||Nath BS, Suresh A, Varma M, Kumar BR. Changes in protein metabolism in haemolymph and fat body of the silkworm, Bombyx mori L., in response to organophosphorus insecticides toxicity. Ecotoxicol Environ Saf 1997;36:169-73. |
|12.||Richmond W. Preparation and properties of cholesterol oxidase from Nocardia sp. and its application to enzymatic assay of total cholesterol in serum. Clin Chem 1973;19:1350-6. |
|13.||Valovage WD, Brooks MA. Uric acid quantities in the fat body of normal and aposymbiotic german cockroaches Blattella germanica. Ann Entomol Soc Am 1979;72:687-89. |
|14.||Reinhold JG. Standard methods in clinical chemistry. In: Reiner M, editor. New York: Academic Press; 1953. p. 88. |
|15.||Reitman S, Frankel S. A colorimetric method for the determination of serum glutamate oxaloacetic and glutamate pyruvic transaminases. Am J Clin Pathol 1957;28:56-63. |
|16.||Kind PR, King EJ. Estimation of plasma phosphatases by determination of hydrolyzed phenol with amino-antipyrine. J Clin Pathol 1954;7:330- 32. |
|17.||King J. Practical clinical enzymology. In: Van D, editor. London: Nastrand. Co; 1965. p. 83-93. |
|18.||Islam MR, Ali MA, 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;4:170-2. |
|19.||Etebari K, Matindoost L. The study on effects of larval age and starvation stress on biochemical macromolecules abundance of haemolymph in silkworm Bombyx mori, In: Proceedings of the Sixteenth Iranian Plant Protection Congress, General Entomology Symposium, August 28-September 1, University of Tabriz, Iran, 2004. p. 435. |
|20.||Etebari K, Matindoost L. Effects of hypervitaminosis of vitamin B3 on silkworm biology. J Biosci 2004;29:417-22. |
|21.||Morris SM Jr. Regulation of enzymes of urea and arginine synthesis. Annu Rev Nutr 1992;12:81-101. |
|22.||Shigematsu H. Protein metabolism in the fat body of the silkworm, Bombyx mori L. Bull Seric Exp Stn 1960;16:165-70. |
|23.||Harper HA, Rodwell VM, Mayer PA. In review of physiological chemistry, 17th ed. California: Longe Medical Publications, Maruzer Company Limited; 1979. |
|24.||Martin MK, Osborn E, Billing P, Glickstein N. Bombyx mori supplemented with Vigna unguiculata Marine Pollution Bulletin 1981;12:305-8. |
|25.||Miao Y. Studies on the activity of the alkaline phosphatase in the midgut of infected silkworm, Bombyx mori L. J Appl Entomol 2002;126:38-42. |
|26.||Yi SX, Adams T. Age- and diapause-related acid and alkaline phosphatase activities in the intestine and malpighian tubules of the Colorado potato beetle, Leptinotarsa decemlineata (Say). Arch Insect Biochem Physiol 2001;46:152-63. |
[Table 1], [Table 2], [Table 3], [Table 4]