MATERIALS AND METHODS

The samples were collected form Silkworm Physiology Laboratory, Central Sericultural Research and Training Institute Mysore, India during the months of January-February 2006. Cocoon filament and degummed fiber of pure bivoltine breeds (CSR₂ and CSR₄), bivoltine hybrid (CSR₂ x CSR₄), cross breed of multivoltine x bivoltine hybrids (PM x CSR₂) and pure multivoltine (PM) were taken for the present study. The samples were cross sectioned by slicing them into small pieces of about 20 μm thickness with the help of a sharp blade. Three samples were taken for each breed from the middle of the cocoon for cocoon filament study. However, for the degummed fiber, the adhesive protein i.e., sericin was removed from cocoon filament by standard methods (Gulrajani, 1988) and the blocks were prepared using dental wax and then the blocks were sliced into cross sections. The sections of both cocoon filament and degummed fiber of both the sexes were mounted onto copper stubs using double side sticking tape. The mounted specimens were immediately coated with 20 nm thickness of gold on their cut surface, in a Sputter coater (EMS-550) to minimize charging under the electron beam. The gold coated samples were observed using scanning electron microscope (SEM) (JEOL 100 CX II ASID 4D, Tokyo Ltd., Japan) under an accelerating voltage of 20 kV with a beam current around 0.1 nA. Low magnification was used to show the cross sections and longitudinal sections of the cocoon shell however the higher magnification was selected to observe the number of voids/striae present in the cocoon filament and degummed fiber. The photographs were taken at different magnifications for observations.

RESULTS AND DISCUSSION

The cocoon filament and degummed fiber of different breeds/hybrids of Bombyx mori were examined under a scanning electron microscope in order to clarify the characteristics of the cocoon filaments and degummed fiber and to confirm the presence of voids or any other similar structures. Low magnification photomicrographs of the cross section of female cocoon of CSR₄ revealed that the cocoon filament had cut transversely and longitudinally (Fig. 1a). The cocoon fibers were tightly packed in the cocoons of pure bivoltine breeds (Fig. 1a and b) whereas the same filament observed loosely packed in the cocoons of pure multivoltine breed i.e., in pure Mysore (Fig. 1f).

The morphological studies on the cross sections of cocoon filaments in different pure breeds and hybrids reveals the presence of number of voids and their shape, size varies at ultrastructure level. The number of voids in pure multivoltine breed i.e., Pure Mysore, is much higher than those found in pure breeds and hybrids of bivoltine.

Fig. 1a:	Cross section of the cocoon shell (Ccs) reveals the cross section (Cs) and longitudinal strands (Ls) of cocoon filament of female CSR4 breed. Note cocoon filaments are closely packed. (x 500)

Fig. 1b:	A magnified view of the cross section (Cs) of cocoon shell indicated the presence of voids (arrow) in male CSR4 breed (x 1700)

Fig. 1c:	Cross section of fibroin strand (Fs) with a number of voids in the central part of filament (arrow heads) in female CSR2 breed. (Sl = Sericin layer). (x 12800)

Fig. 1d:	Two fibroin strands (Fs) coated with a sericin layer (Sl). Arrow heads indicated the voids in the cross section of fibroin strands of male CSR2 x CSR4 . (x 6400)

Fig. 1e:	Long channel shaped voids (arrow heads) in the cross section cocoon filament of PM x CSR2. (x 5000)

Fig. 1f:	Cross section of the cocoon shell showing the cross section of cocoon filament (Cs) and longitudinal strands (Ls) of Pure Mysore. The cocoon filaments are loosely packed (ooo) in multivoltine. (x 500)

Fig. 1g:	Two fibroin strands (Fs) coated with sericin layer (SI) revealing a large number of voids (arrows) in the female cocoon filament of Pure Mysore (x 5000)

Fig. 1h:	A large number of channel shaped voids (Vs) in the central part of the cross section of fibrion strand in female Pure Mysore (x 12800)

The number of the voids in the cross sections of pure breeds of bivoltine i.e., CSR₂ and CSR₄ were observed relatively very less in both the sexes. Further, the shape and size of these voids also differ from that of the voids noticed in cross section of cross breed of multivoltine and pure multivoltine. The shape of the voids in pure bivoltine and their hybrids were observed to be round or elliptical and sometime two or three small voids joined to form a small channel shaped void in CSR₂ and CSR₂ x CSR₄ breeds (Fig. 1c-e). However, a large number of bigger voids were noticed in the central part of cocoon filament of Pure Mysore and its hybrid (Fig. 1g and h). Narumi et al. (1992, 1993) and Lin et al. (1993) stated that the voids were distributed in the central part of the filament cross section and were typically circular or elliptical in the cocoon filament of wild silkworms. The voids studied in all the breeds of B. mori were elongated in the direction of the fiber axis. It is also observed that the diameter of the voids distributed in the central part of the filament was larger than that of the voids near the surface in the cocoon filament. Large sized and more number of voids in male cocoon filaments was observed than that of female cocoon filament in all the breeds/hybrids studied. Narumi et al. (1994) reported that the number of voids in male filament was larger than that of female ones and there were many large voids in the central part of male cocoon filaments of wild silkworms.

Fig. 2a-c:	Cross sections of degummed fiber (Dfs) in the dental wax media (Dw) reveals the striae(St) in CSR2 female (2a), CSR2 male (2b) and male CSR4 (2c). (x 3300 for Fig. 2a; x 6660 for Fig 2b and x 4270 for Fig. 2c)

Fig. 2d-e:	Magnified views of cross sections showing the striae (St) which were formed after cooking and degumming of cocoon filament in male CSR4 (2d) and in the female of CSR2 x CSR4 (2e) (x 8530 for Fig. 2d and x 8530 for Fig. 2e)

The cross sectional areas of filament after cooking showed about 6% reduction compared to the raw cocoon filaments. Cocoon cooking removes sericin from the peripheral areas of filaments, resulting in a decrease in the cross sectional area of the filament. Following cocoon cooking, the number of voids was found to have reduced to about 30% in the cocoon filament and about 20% after degummed cocoon filament in all the breeds/hybrids. Some voids were gradually flattened during the cocoon cooking and degumming process and finally closed and were transformed into striae in all the breeds/hybrids studied, except in the females of Pure Mysore (Fig. 2a-h). Assuming that striae is a closed void, we found that the total number of voids and striae observed after cooking and degumming was smaller than the number of voids in the cocoon filament. This suggests that the closer of voids has progressed further and they have fused into fibroin to such an extent that they were not detected in the SEM. After degumming, the number of voids in the filament was further reduced, while the number of extremely flat voids and streak-like striae increased in all the breeds/hybrids except in Pure Mysore. The number of the striae, their shape and size were found almost similar in CSR₂, CSR₄ and CSR₂ x CSR₄ and PM x CSR₂ (Fig. 2a-f). However, more number of striae were found in PM x CSR₂ male degummed fiber (Fig. 2g). Narumi and Kobayashi (1997b) have studied the morphological charactertics of voids in wild silks used in woven fabrics and mentioned that the larger diameter voids found in cocoon filaments of wild silkworms are flattened at a significant degree in woven fabrics structure and transformed into striae.

Fig. 2f and g:	Cross sections of the degummed fibers of male CSR2 x CSR4 (2f) and female PM x CSR2 (2g) reveals the striae (St) in the central part of degummed fiber. (x 4270 for Fig. 2f and x 6660 for Fig. 2g)

Fig. 2h:	Magnified view of a cross section of degummed fiber of female Pure Mysore showing a large number of voids (Vs) in the peripheral area of cross section. (x 8530)

The present findings clearly indicate that the large sized and more number of voids present in male cocoon filaments than in female in all the breeds/hybrids of B. mori studied. Further, there are considerable differences in shape, size, number and distribution pattern of the voids in the cocoon filaments of different breeds/hybrids of mulberry silkworm. However, the number of voids/striae was higher in wild silkworms than that of the breeds/hybrids of B. mori. Further, the voids gradually flattened during the cocoon cooking and degumming processes and finally closed and transformed into striae resulting in an increased compactness of degummed fiber. In conclusion, the results of the present study confirmed that the presence of more number of voids/striae in multivoltine and less number in bivoltine cocoon filament and degummed fiber. More number of voids/striae found in inferior silk hence bivoltine silk is superior over the multivoltine silk. Therefore, the results of the present study will find use in the selection of the breeds/hybrids of superior quality silk for breeding programmes in addition to other reeling parameters.

ACKNOWLEDGMENTS

The authors are thankful to Dr. S.B. Dandin, Director, Central Sericultural Research and Training Institute, Mysore, India for encouragement.