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Thursday 26 September 2013

Protein markers as a potential tool for biochemical characterization of silkworm genetic resources

K. Ashok Kumar*, P. Somasundaram, G.K. Rajesh, R. Chandrasekar, N. Balachandran and V. Sivaprasad
The 1st, 2nd, 4th, 5th and 6th authors are with Central Silk Board (CSGRC, Hosur) India. The 3rd author is with Council for Nature Conservation and Environmental Protection (CONCEPT), India


The silkworm Bombyx mori L. is one of the earliest examples of insect domestication and commercial exploitation. The insect is believed to be first identified and put to use by the Chinese nearly 4500 years ago. Since then the species has been subject to severe selective pressure, targeting improved quantitative traits (with reference to silk). As a result the cocoon shell weight has increased manifold while the insect has lost many of its defensive features that once helped it survive in the wild fighting whether, natural enemies and diseases. The fact that one of its closest ancestors namely Bombyx mandarina still lives in the wild enables us comparison which testifies to the above statements. While the progress so far made is quite impressive (in human angle), it is widely perceived that the insect leaves much to be desired in terms of quantitative output and tolerance to adverse environment, pests and diseases. Thus location specific hybridization programs are in vogue towards evolving productive hybrids which are not only tolerant to specific micro climates and pathogenic flora but also adaptable to resource saving techniques such as artificial diet.  The process has lead to creation of silkworm germplasms in many countries offering a wide variety of genetic resources to select from. However, selection of parental breeds has become quite a challenging task, given such a vast and varied collection of geographic races, evolved breeds, transgenic lines, mutant stocks etc. This article sums up a series of studies conducted at the Central Germplasm Resources Centre (CSGRC) Hosur, India and proposes the application of protein markers for characterization of silkworm genetic resources to identify duplicate genotypes and to find out intra and inter racial variations.   

Experimental material

This paper summarises a few laboratory screening studies conducted on 28 silkworm accessions (14 multivoltine races and 14 bivoltine races) maintained at Central Sericultural Germplasm Resource Centre (CSGRC), Hosur Tamil Nadu, India (latitude 12°45’N and longitude 77°5’E, altitude 942M AMSL). The center maintains a total of 421 accessions: 71 multivoltines and 350 bivoltines (Thangavelu et al., 1997 and 2000). The details of the races used for study; such as geographical origin, and cluster groups based on genetic distances are furnished in tables 1-3
Table 1. Details of geographical origin and class of 28 silkworm accessions 
Table 2. Cluster groups based on genetic distances in 14 MV races
Table 3. Cluster groups based on genetic distances in 14 BV races
Biochemical markers for characterization
Identification of suitable biochemical markers is a pre-requisite for the successful implementation of Marker Assisted Selection (MAS), which is being used in the field of animal breeding work (Patnaik and Datta, 995). Allozyme/isozyme markers are the most widely used protein markers and are commonly separated on starch, polyacrylamide or cellulose acetate media and stained using enzyme specific reaction mixtures.
Allozymes of a given enzyme are the products of different alleles at a specific locus. Diploid (2n) organisms can be homozygous or heterozygous having two copies of the same allele or heterozygous having different alleles coding for charge size variants of the same functional enzyme, hence the term Allozymes. Allelic variations through isozyme studies are known to reflect the genetic diversity (Parkash et al., 1992). Recently, haemolymph proteins attracted a great deal of attention as a biochemical model system (Wyatt and Pan,1978; Law and Wells,1989). The main haemolymph proteins, lipophorin(LP), storage protein (SP) and vitellogenin (Vg) are common in insects and have special function for development, metamorphosis and reproduction. Storage proteins are the major reservoirs for amino acids that are utilized for cuticular proteins and for adult development (Levenbook,1985). These biochemical markers are extensively used to describe the genetic constitution of an individual that can also be used to determine the genetic diversity existing in a population. Munn and Greville (1969) used ultra centrifugation data for quantification  of storage protein-1 and Kinnear and Thomson (1975) relied on visual inspection of  stained PAGE bands for gross appraisal of the various storage proteins, while Sekaris and Scheller (1977) estimated storage protein–1 by densitometry of SDS-PAGE gels and storage protein-2 by densitometry in silkworm races( Somasundaram et al.,2004)
Heat shock is known to alter the enzyme activity in different animal systems. A certain fraction of an isozyme may disclose temperature tolerance. Wu et al (1993) reported that the thermo tolerance varies with strains and is positively related to the activity of a heat stable esterase (Hs-EST) analysed in larval body or midgut. This enzyme activity can be adopted as an indicator for the silkworm thermo tolerance.  The stability of an enzyme under higher temperature may have a protective role in the adverse environment.
The information generated from biochemical marker studies on silkworm genetic resources through allozyme/isozyme markers, storage proteins, and heat stable esterase enzymes activity would help in identifying germplasm for hardiness in silkworm, genetic variations among and within races, identification of duplicates and thermo tolerance among silkworm germplasm stocks.

Biochemical markers for hardiness 
Amylase enzyme as a biochemical marker was studied in silkworm genetic resources for its effective usage as surrogate breeding parameter to correlate amylase activity with economic traits. This study was aimed at identification of suitable biochemical markers for the successful implementation of Marker Assisted Selection (MAS) which being used in other fields of breeding (Datta, 1998, Jayaswal et al., 2000). Out of a number of biochemical parameters viz., digestive amylase, invertase, protease, alkaline phosphatase and haemolymph trehalose, only the digestive amylase has a significant positive correlation with survival on one hand and negative correlation with weight of larvae, cocoon and shell on the other (Chatterjee et al., 1992). The genetic divergence in term of activity as well as isozyme, polymorphism coupled with its role in better digestibility and survivability indicates the prospect of using amylase as a marker in silkworm breeding. It is reported that multivoltine races contain dominant amylase gene (Ae+) with four to five band polymorphism responsible for inducing hardiness in multitovltine races. This gene (Ae+) recessive in temperate races with no amylase isozyme band (Null type) may be responsible for less tolerance to climatic fluctuations.  Thus the dominant (Ae+) gene can be transferred to temperate races through a suitable vector for developing hardy bivoltine races. Similarly haemolymph amylase isozyme studies conducted so far showed variability in its activity in different silkworm stocks. Most of the hibernating high yielding races of Japanese and European races are homozygous for Amy dn Detailed characterizations of silkworm germplasm stocks are vital for the effective use in potential breeding resources. Hierarchical agglomerative clustering by UPGMA method using Euclidean distance of 54 stocks on the basis of yield attributes alone resulted in 7 clusters indicating the significance of such data in the characterization and classification of silkworm germplasm stocks(Chatterjee and Datta, 1992). The categorization of silkworm strains into five yielding groups on the basis of discrimination function scores of economic characters and esterase isozyme activity at different developmental stages was done on twenty silkworm genotypes (Ram and Lal, 2002).  

Biochemical marker for genetic variations 
Studies carried out on biochemical characterization of silkworm races of different origins from germplasm stocks with four metabolic enzymes viz.,  a esterase, b esterase , acid phosphatase, and alkaline phosphatase were able to compare and relate the genetic relationship among 14 MV races viz.,   Pure Mysore, Sarupat, Moria, Tamil Nadu white, C.Nichi, Hosa Mysore, Mysore Princes, Kolar Gold, Kollegal Jawan, MY-1, P2D1, Rong Daizo, Guangnang and OS-616  and 14 BV races viz., Alps jaunne, Alps yellow, Cevenesse yellow, Ascoli yellow, Meigtsu, B-36, B-37, B-39, B-40, J-112, J-122, Yakwei, Changnaung and C-122. The results on electrophoretic variation on isozyme profiles are seen in the band mobility (Fig. 1a,b).

Fig.1a. Genetic polymorphism on esterase isozyme among 14 MV races. Various bands designated as A,B,C & D.

Fig.1b. Genetic polymorphism on esterase isozyme  among 14 BV races. Various bands designated as A,B,C & D.  
A total of 4 bands was observed in multivoltine races and 7 bands in bivoltine races. All the observed 4 bands in multivoltine and 7 bands in bivoltine were not present uniformly in all 28 races studied. These variable bands present in them were identified as race specific markers and analysed through pop-gene software package for genetic analysis (Yeh et al., 1999).  Thus 9 clustering groups were identified among 14 multivoltine races and 8 clustering groups among 14 bivoltine races.  
The clustering helps us to relate their origin, racial characters and the parentage of the evolved breeds. Among 14 MV silkworm races studied, higher genetic distance was seen between C.Nichi (Original race of Japan origin) and Rong Daizo (Evolved breed of China origin). Similarly in BV silkworm races, such higher genetic distance was observed between B-36 (Japan origin) and C-122 (China origin).  A detailed study on biochemical characterization in these line and proper classification of germplasm stocks is vital for the effective use in potential breeding resources. The genetic diversity and population genetic structure of twelve silkworm races revealed in the present study would be utilized for an effective conservation plan and breeding strategies. Based on the results observed in the present study, it is inferred that populations of silkworm races J-112 and NB4D2 would be very useful in a breeding programme, because their higher genetic diversity and alleles. 
Storage protein profiles for genetic variations.  
Studies on storage protein expression in the final instar larvae of silkworm races help to identify and cluster them based on the genetic variability in storage protein profiles. Storage protein variation was studied in eleven popular multivoltine silkworm (Bombyx mori) breeds encompassing different parentage and origin, as furnished in table 4. The main feature of storage protein variation in the multivoltines is found to be inter-origin variability in unit area expression of storage protein. The storage protein levels (SP-2) among these popular breeds as seen in SDS-PAGE also differed as evident from the SDS PAGE profiles in Fig. 2. 
Table 4:Variability in protein expression of storage protein (SP-2) level in  
            multivoltine Silkworm breeds of Bombyx mori
Fig.2. Genetic polymorphism on storage proteins in 11 MV silkworm races
Based on densitometry scanning of SDS-PAGE bands of storage protein levels, six clustering of eleven races was done using Ward’s Minimum Variance clustering analysis. Storage protein has an important role to play on metamorphic features of larval weight and pupation rate of B.mori as evident from correlation factors. Thus study on genetic variability existing among storage protein level in silkworm breeds may be useful to cluster the silkworm races into different clusters having similarity in the storage proteins and their effect on growth traits.

Heat stable esterase isozyme profiles for thermo tolerance

The climatic condition of tropics, particularly in summer is not conducive to rear high yielding bivoltine hybrids. Keeping this in view, characterization of silkworm breeds for thermo tolerance is imperative in order to utilize such those identified thermo tolerant breeds in the breeding plan to evolve productive bivoltine /multivoltine breeds/hybrids. Genetic dissection of silkworm races carried out by several Japanese scientists (Sarkar, 1998) has indicated that certain enzymes are responsible for hardiness and related quantitative characters. Wu et al (1993) reported that the thermo tolerance varies with strains and is positively relative to the activity of a heat stable esterase (HsEST) analysed in larval body or midgut. This enzyme activity can be used as an indicator for the silkworm thermo tolerance. Further, there is always a heritable linkage between characteristics of high temperature tolerance and disease resistance (Samson and Chandrashekaraiah, 1998). In view of this, breeds with such trait should be characterized in the germplasm as resource materials for developing thermo tolerant breeds/hybrids. Non specific b-esterase band (Est 3) in haemolymph of CB5 (GP) and its syngenic lines among the silkworm stocks withstood a temperature up to 80 ±° C for 10 min indicating naturally available thermo stable esterase protein in the haemolymph of thermo tolerant races (Chattopadhyay et al., 2001).   With this main objective, the presence of heat stable esterase isozyme band activity was studied in selected silkworm races along with thermo tolerant races  CSR-18 and CSR-19 to identify such those breeds which can withstand higher temperature prevailing in some of the areas where during summer the temperature may shoot up to 40 °C and above. A total 10 MV races viz. Pure Mysore, Nistari, A25, A4e, PMX, MU-1, MU-11,MW-13, LMP and LMO and 12 BV races viz., Meigitsu, J-112, Chukwei, Sanish-17, Sanish-18 (P), JD6, JAM-125, PAM-105, BL-1, NB4D2, CSR-18 and CSR-19 has been studied for heat stable esterase isozyme activity to relate thermal tolerance in the above silkworm races. Heat stable esterase isozyme study showed the presence of active b-esterase band in multivoltine silkworm races of Pure Mysore, Nistari, A25, A4e, MW-13,LMP and LMO and in Chaukwei, PAM-105,BL-1,CSR-18 and CSR-19 of bivoltine  silkworm races. Such active b-esterase band was absent in multivoltine races of MU-1, MU-11 and PMX and in Meigtsu, J-112, Sanish-17, Sanish (18), JD-6, JAM-125, NB4D2 in bivoltine silkworm races. The presence of active b-esterase band in the haemolymph of silkworm races may be responsible for thermo tolerance. The differential Esterase isozyme band activity observed in 11 multivoltine and bvoltine breeds is furnished in table 5. 

Table 5: Esterase isozyme band activity in the haemolymph of MV & BV races
Biochemical markers on higher amylase activity and the presence of Amy dn allele in homozygous conditions are useful to identify multivoltine and bivoltine silkworm races with strong amylase activity. 54 silkworm germplasm stocks were grouped into 7 clusters on the basis of yield attributes and amylase activity. Allozyme techniques were used on twelve silkworm races with different geographic origin to identify the genetic similarity and genetic diversity among them. The results showed six clusters and among them NB4D2 and NB18 are found to be genetically similar while BL-24 and Nistari are found to be genetically distant races. Silkworm races J-112 and NB4D2 showed the highest genetic diversity, per cent polymorphism and more alleles respectively. Storage protein expression level in the haemolymph proteins analyzed in different silkworm races is found to be useful in identifying robustness of the race/breed having inherent genetic potential for growth and cocoon yield. Presence of mid-gut esterase band 5 in thermo tolerant silkworm breeds and the presence of active b-esterase band in the haemolymph of silkworm races could be used as a biochemical marker to relate thermo tolerance in silkworm genetic resources. From the findings of the series of studies we make a strong argument in favor of using protein markers as a reliable tool for screening silkworm germplasms resources in commercial hybridization programmes.

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