Recent Achievements in Selection and Breeding of the Silkworm Bombyx mori L in Bulgaria

P. Tzenov

Dr. PANOMIR IVANOV TZENOV was born in Vratza, Bulgaria in 1961. He took his MSc from The University of Zootechnics and Veterinary Medicine – Stara Zagora in Animal breeding engineering – sericulture. He obtained his PhD. in sericulture in 1996. Dr. Tzenov started his career as a technologist in silkworm egg production at the “Silkworm breeding and egg production enterprise”, Vratza, under the Sericulture Experiment Station (SES) Vratza. He became the head of the station in 1987. From 1994 to 2003 he served as The Director of SES, Vratza and as National Director of the project “Rehabilitation of sericulture”, financed by FAO during 2000 – 2002. Presently he holds the position of Executive director of Bulgarian National Center of Agricultural Sciences (NCAS), Sofia. He is member of various reputed national and international forums such as International Working Group on Sericulture Germplasm” under FAO, International Working group on the global silk handcraft cottage industries and silk enterprises development” under FAO, The Publishing council at the National Center of Agricultural Sciences, Bulgaria etc. He is the founder president of Black, Caspian Seas and Central Asia Silk Association (BACSA). He has contributed immensely to general sericulture, silkworm breeding, egg production, rearing technology and cocoon and silk processing. He has 164 published papers to his credit and holds author’s certificates for two commercial silkworm hybrids. Dr. Tzenov is the chairman of the advisory committee of CONCEPT: Council for Nature Conservation and Environmental Protection, India.
Dr. Tzenov can be contacted at: panomir@yahoo.com

ABSTRACT

A method for selection of silkworm breeds and hybrids, tolerant to adverse rearing conditions, based on a previous testing of silkworm breeds/hybrids and choose of the most tolerant was developed. Two new four-way hybrids, manifested the highest tolerance to adverse rearing conditions along with a satisfactory high productivity under optimal rearing were created. By using a polyvoltine strain in the breeding process two new silkworm breeds were created as parents of a F1 silkworm hybrid which is characterized with comparatively good productivity and simultaneously a higher tolerance to adverse rearing conditions. All the 3 new tolerant to adverse rearing conditions silkworm hybrids have been examined in the system of State agency for variety testing and recognized as original. Two new silkworm hybrids for higher silk yield were created. They manifested average fresh cocoon weight 2.674 g, silk shell weight 0.632 g, shell percentage 23.64 %, filament length 1347 m, raw silk percentage 42.00 %, fresh cocoon and raw silk yield by one box of eggs 43.88 kg and 7.57 kg respectively - a productivity, comparable with the best world standard. A method for selection of sex limited for larval markings silkworm breeds, characterized with higher tolerance to adverse rearing conditions was developed. By breeding of 4 new sex – limited for larval markings strains a higher egg production efficiency by means of higher cocoon yield and fecundity at the P1 egg category level was obtained and a F1 four – way hybrid, having high productivity, combined with a higher tolerance to adverse larval rearing conditions was created and approved by the government as original. A method for selection of sex limited for cocoon colour highly productive silkworm breeds was developed. Four new sex – limited for cocoon color silkworm F1 commercial hybrids which manifest a pupation rate and productivity, comparable with those of the control have been created.

Keywords: silkworm, Bombyx mori L., selection, breeding, Bulgaria

INTRODUCTION

The beginning of a purposive silkworm selection and breeding in Bulgaria was dated 114 years ago with the establishment in 1896 of the Sericulture Еxperiment Station (SAES) in Vratza. In the 30th - 40th years of the last century two main strains were maintained and reared at the field level in Bulgaria, namely Yellow local, with yellow cocoons and cream color of raw silk and White Baghdad, having white cocoons. The White Baghdad race is considered as the oldest at the Balkan peninsula and had been selected by the ordinary farmers for many years.
One of the main targets of Sericulture Experiment Station (SES) in Vratza since its establishment and during its more than one century old activity has been the improvement of silkworm races and hybrids in order to increase the cocoon and raw silk yield and their quality.

Up to the beginning of 30’s of 20th century the station had not maintained its own silkworm genetic resources, but tested periodically the strains imported from different countries (mainly France and Italy) before their introduction at the private egg producing companies.

In the 40’s the station introduced several yellow cocoon races like Askoli, Var, Abrucio and Almeria from France and Italy and using this material some local yellow cocoon lines were evolved.

During the period 1944 – 1952 hybrids between the Yellow local and Golden Chinese race had been tested in Bulgaria and introduced to the field. In order to improve the lines of the Yellow local race, European yellow cocoon breeds Askoli, Var and Alpen were imported during the period 1949-1950. In the end of this period white cocoon silkworm breeds and hybrids became more popular in the world due mainly to their better silk dyeing quality. From 1965 to 1970 many new silkworm lines have been selected in Bulgaria by segregation from Italian and Japanese white cocoon F1 hybrids.

In 1969 the government approved the first white cocoon Bulgarian F1 commercial hybrids.

After 1970 the silkworm breeding work in Bulgaria was expanded and many new breeds and hybrids had been created and introduced until nowadays.

Taking into account the very big importance of the genetic resources for a successful breeding work the number of breeds, maintained at SAES - Vratza and the Agricultural universities was increased from 20-25 at the beginning of 70’s to over 300 in 2010.

The research work in the field of silkworm genetics and breeding was directed to studies on the interactions between the genotype and environment, heterosis expression in F1, positive transgressions expression in F2, combining ability, heritability, correlations, regressions, inheritance of the main qualitative and quantitative characters, implementation of the intensive inbred-lines breeding, methods for creation of initial populations for silkworm breeding, breeding of sex-limited breeds, use of partheno and androgenesis in the silkworm breeding, breeding of silkworm lines and hybrids for summer-autumn rearing, and having higher tolerance to adverse rearing conditions etc..

In the recent years the silkworm breeding in Bulgaria was directed to three main aims: viz. Breeding strains and hybrids tolerant to adverse rearing conditions; Breeding strains and hybrids for higher silk yield and Breeding sex-limited for larval markings and cocoon color strains as parents of commercial F1 hybrids, tolerant to adverse rearing conditions and hybrids, with high cocoon and silk productivity.

Breeding strains and hybrids tolerant to adverse rearing conditions.

In this work we used two methods. They were 1. Method of breeding tolerant to adverse rearing conditions silkworm breeds and hybrids by a pre-testing some accessions from the genetic resources and 2. Method of breeding tolerant to adverse rearing conditions silkworm breeds by using polyvoltine strain.

Method of breeding tolerant to adverse rearing conditions silkworm breeds and hybrids by a pre-testing some accessions from the genetic resources.

For the purpose 15 silkworm breeds of the Japanese type, 13 breeds of the Chinese type and 16 F1 hybrids between some of the breeds were used. All the breeds/hybrids were with white cocoon color and uni and bivoltine. The breeds were chosen from the silkworm germplasm, maintained at SAES-Vratza for having comparatively high values of the main quantitative characters (Tzenov and Grekov, 2007). Then the breeds were tested for three times regarding their tolerance to adverse rearing conditions during the 4th and 5th instars (t 28-31 oC, RH 75-80%, feeding amount and rearing space – reduced by 50 %, very reduced ventilation). The breeds who manifested the highest pupation rate under adverse larval rearing conditions were crossed between some of them using the scheme Japanese type x Chinese type and the opposite and the F1 hybrids were further tested also under both standard and adverse rearing regime. Then the hybrids performed the best under the adverse rearing conditions and manifested comparable to the control productivity under standard rearing conditions were chosen and further tested. As a result two new four-way hybrids, namely (KK x Hesa 1) x (Vesletz 2 x Gergana 2), (KK x AS) x (Vesletz 2 x Gergana 2) and the reciprocal crosses, manifested the highest tolerance to adverse rearing conditions along with a satisfactory high productivity under optimal rearing were created (Tzenov and Grekov, 2008, 2009). Both the hybrids have been examined under the system of State variety testing commission and recognized as original.

Simultaneously, each silkworm breed selected by the adverse rearing test was mass reared for 6 generations under adverse conditions and only the individuals survived and having the highest cocoon weight, shell weight and shell percentage were selected for further reproduction. After the 6th generation the breeds have been maintained by batch rearing, following the standard methodology for pure lines.

Method of breeding tolerant to adverse rearing conditions silkworm breeds by using polyvoltine strain.

In this breeding process two uni-bivoltine white cocoon pure lines Super 1 and Hesa 2 and the polyvoltine strain Bonde 517, characterized with yellow-green cocoon color were used.

he breeds Super 1 and Hesa 2 are highly productive with fresh cocoon weight of 2.100 – 2.400 g, shell weight 0.460 – 0.520 g and shell percentage 21 – 22 %. Bonde 517 strain is a typical polivoltine, the cocoons are spindle shaped and flossy, the fresh cocoon weight is 1.000 – 1.100 g, shell weight 0.115 – 0.130 g and the shell percentage is 11 – 12 %. On the other hand this strain has a very high tolerance to adverse rearing conditions, especially high temperature and humidity and it is also comparatively resistant to NPV.

The first step of the breeding process was making F1 and F2 crosses of Super 1 x Bonde 517 and Hesa 2 x Bonde 517. In F2 there was segregation in colored and white cocoons with different shape and texture (Tzenov at al., 1997, 1998, 1999, 2000). Seven male cocoons out of 32 kg cocoons obtained per each cross, having white color, elongated (oval) shape, and similar to those of the uni-bivoltine line texture were selected and mated with selected females of the lines Super 1 and Hesa 2 respectively. Then the two populations were bred by inbreeding for 6 generations, batch rearing, selection for cocoon color, shape, texture and the main quantitative characters. After that the selection process continued with outbreeding for 8 generations, batch rearing, selection for cocoon color, shape, texture and the main quantitative characters. Finally the new silkworm breeds SB 1 and HB 2 were created. The breed SB 1 is of the typical Japanese type, namely larvae with markings, white and elongated with constriction cocoons. The breed HB 2 is of the Chinese type with plain larvae, white and oval cocoons. The main characteristics of the newly evolved breeds are presented in Table 1to3.

Then we tested the F1 hybrids between the two new breeds under optimal larval rearing conditions (Table 4 and 5.). The results manifested that the two hybrids had significantly lower cocoon and silk shell weight than the control, the hybrid Super 1 x Hesa 2. However we also tried the newly evolved breeds as components of a four – way hybrid SB1xSuper 1 x HB2xHesa 2 and the reciprocal. The four – way hybrids manifested a productivity, comparable to the control (Table 4 and 5.). Both the single and four – way hybrids were also tested under adverse rearing conditions during the last two instars (Table 6) and showed remarkably higher pupation rate than the control. As a result the new silkworm hybrid SB1xSuper 1 x HB2xHesa 2 and the reciprocal cross which is characterized with comparatively good productivity and simultaneously a higher tolerance to adverse rearing conditions was created, tested in the system of State agency for variety testing and recognized as original.

Breeding strains and hybrids for higher silk yield.

For the aims of breeding two new silkworm strains, named B1 and Svila 2 have been created for a period of 7 years selection work. The breed B1 is of the Japanese type, having larvae with markings and elongated cocoons with constriction. The breed Svila 2 is of the Chinese type, with plain larvae and oval cocoons. Both the strains are characterized with cocoon weight 2.100 – 2.300 g, shell weight 0.500 – 0.600 g and shell percentage 24 – 26 %. Then from the silkworm germplasm, maintained at SAES – Vratza four other silkworm breeds have been chosen (Hesa 1 and Vratza 35 of Japanese type and Hesa 2 and Merefa 2 of Chinese type) which performed comparatively the highest silk productivity. Using the 6 strains, 10 new F1 silkworm hybrids were created and tested while the Bulgarian hybrid Super 1 x Hesa 2 and the Japanese hybrid Shunrei x Shogetsu had been used as controls (Table 7 and 8).

The results obtained manifested that some of the new hybrids perform higher values than the Bulgarian control and weree close to the Japanese control as regards the fresh cocoon weight, silk shell weight, shell percentage, fresh cocoon and raw silk yield by one box of eggs and filament weight characters values.

It may be concluded that the best hybrids for higher cocoon and raw silk yield are Hesa 1 x Svila 2 and the reciprocal, B1 x Merefa 2, Vratza 35 x Svila 2, B1 x Svila 2 and the reciprocal cross. Among them the best one are Hesa 1 x Svila 2 and B1 x Svila 2 and their reciprocal crosses having average fresh cocoon weight 2.674 g, silk shell weight 0.632 g, shell percentage 23.64 %, filament length 1347 m, raw silk percentage 42.00 %, fresh cocoon and raw silk yield by one box of eggs 43.88 kg and 7.57 kg respectively.

It may be concluded that the two new Bulgarian hybrids have a productivity, comparable with those of the Japanese hybrid Shunrei x Shogetsu, recognized as the best world standard.

Breeding sex-limited for larval markings and cocoon color strains as parents of commercial F1 hybrids, tolerant to adverse rearing conditions and hybrids, with high cocoon and silk productivity.

Breeding sex-limited for larval markings strains as parents of commercial F1 hybrids, tolerant to adverse rearing conditions.

The aims of the breeding process were three fold, viz. To create 4 new silkworm breeds, sex – limited for larval markings as analogues of ordinary breeds, characterized with higher tolerance to adverse rearing conditions; To use the newly bred strains as parents of four – way hybrid in order to obtain higher cocoon yield and fecundity at the P1 egg category level and The parents of the F1 hybrid (P1) to be sex – limited for zebra larval markings in order to make easier the sex discrimination.

The method of creation the 4 new sex – limited for larval markings breeds was as follows (Tzenov & Guzman, 2004, Tzenov, 2005):

1. Initial population: cross between females of sex limited for larval markings strain, received from Japan and males of strain, tolerant to adverse rearing conditions;

2. Backcrosses for 9 generations, using as male parent the tolerant to adverse rearing conditions strain, mass rearing;

3. Backcrosses for 3 generations, batch rearing;

4. New sex – limited breed.

The newly created sex – limited breeds have the following morphological characteristics:

SN1: the females are zebra, the males are with normal markings. The cocoons are white, elongated with constriction.

Iva 1: the females are with normal markings, the males are plain. The cocoons are white, dumbbell with high constriction.

Magi 2: the females are zebra, the males are plain. The cocoons are white in color, oval.

Nova 2: the females are with normal markings, the males are plain. The cocoons are white in color, oval.

In order always in the P1 generation to have females with zebra markings we performed only the crosses SN1 x Iva 1 and Magi 2 x Nova 2 (Tzenov, 2005, 2008). The average number of normal eggs in a laying in the 4 pure breeds was 618, while in the P1 crosses it’s 664. The performance of the pure breeds, their P1 crosses and the F1 commercial hybrid are given in tables 9 and 10

It is evident that both P1 and F1 hybrids manifest a comparatively high productivity. The F1 hybrid SN1 x Iva 1 x Magi 2 x Nova 2 was also tested under adverse rearing conditions (table 6) and showed a comparatively high pupation rate, compared with the control. The hybrid has been tested in the system of the State variety testing commission and approved as original.

It may be concluded that by breeding of 4 new sex – limited for larval markings strains a higher egg production efficiency by means of higher cocoon yield and fecundity at the P1 egg category level was obtained and a F1 four – way hybrid, having a high productivity, combined with a higher tolerance to adverse larval rearing conditions was created.

Breeding sex-limited for cocoon color strains as parents of commercial F1 hybrids, with high cocoon and silk productivity.

Three silkworm breeds were used, namely the Japanese breed D 50 and the Bulgarian breeds Super 1 and Hesa 2. The Japanese breed D 50 was received from the Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan. The breed D 50 is genetically sex limited for cocoon color – the female cocoons are yellow and the male cocoons are white. The breed has larvae with ordinary markings and elongated cocoons with constriction. On the other hand the breed performs a very low productivity and the population contains the so called gene „translucent skin larva”. It’s known that this character is controlled by over 23 alleles, located at different chromosome loci. The „translucent skin larva” gene is also lethal or it determines sterility in the female moth. In the case of the breed D 50 the females with translucent skin lay eggs which embryo dies later on. In the D 50 breed population about 50 % of the larvae are with translucent skin and the rest have normal skin.

The breed Super 1 is of the Japanese type, having larvae with markings and spinning white cocoons with elongated shape. The breed Hesa 2 is of the Chinese type, having plain larvae and spinning white cocoons with oval shape. Both the breeds are highly productive and used as parental lines of several Bulgarian F1 commercial silkworm hybrids.

The breeding target was to create two new breeds having the same morphological characters and similar productivity as the original Bulgarian breeds Super 1 and Hesa 2, but sex limited for the cocoon color. For the purpose female individuals of D 50 strain were crossed with male individuals of Super 1 and Hesa 2. The breeding scheme is given in Fig. 1. The first generation (G1) was mass reared as mixture of 60 laying (one laying are the eggs laid by one mother moth) separated in 10 replications. In G1 all the female individuals spun only yellow cocoons and all the males – white cocoons which proved the stable inheritance of the linked with the W chromosome „y” allele, responsible for yellow haemolymph and yellow cocoon color. From the cocoons obtained, females with golden yellow cocoon color were selected and then measured individually to detect their fresh cocoon and silk shell weights and the shell ratio. The individuals having the highest parameters were selected and then mated with selected male individuals from the breeds Super 1 and Hesa 2. A random cocoon sample had been taken in order to check the silk reeling parameters of the population. The second generation (G2) obtained was reared and maintained by the same method and then crossed again with selected males of Super 1 and Hesa 2. During the fifth instar the larvae of the population D 50 x Hesa 2 G2 were separated into plain and with markings while only females from the plain larvae group were used for further breeding. Then in the 3rd generation (G3) a separate batch rearing was performed where the eggs of each silkworm moth were grown separately. Each population was reared in a volume of 32 laying each one having 200 larvae counted after the 2nd molt and then a selection of the 10 best laying regarding the pupation rate, cocoon, silk shell weights and shell ratio was implemented. The cocoons of the chosen 10 laying were measured individually and then only 20 female and 20 male cocoons from each laying, having the highest parameters were selected.

The selected cocoons from each laying were sib - mated in order to obtain the 4th generation (G4) which was inbreed. The purposes of this inbreeding were first to check the population uniformity reached at this stage through detecting the segregation after the inbreeding and secondly to reject the laying which manifest the „translucent skin larva” character. The fourth generation was reared in a volume of 32 separate batches, each 4 originated from one laying of G3 after inbreeding. After rejection of several 4 – laying batches manifesting the „translucent skin larva” character the rest laying/individuals were object of the same selection procedure like in G3. The selected female individuals of G4 were again crossed with males of the breeds Super 1 and Hesa 2 respectively in order to obtain the 5th generation (G5). Then the 5th generation was further reared and all the morphological and economically important parameters were detected.

During the larval rearing of each selection generation, separate batches of the breeds Super 1 and Hesa 2 have been also reared in parallel in order to make comparison between the productivity of the newly selected populations and the original breeds. The data obtained were processed statistically using the well recognized methods. Then in G 6 generation we repeated the inbreeding in order to obtain G 7, which generation was again crossed with males of Super 1 and Hesa 2 respectively. The 8th generation we already considered as a new breed and gave names of the newly bred strains as L 1 (D 50 x Super 1) and L 2 (D 50 x Hesa 2).

Hybrids between the new breeds L 1 and L2 as well as four – way hybrids L1 x Iva 1 x L2 x Nova 2 and the reciprocal crosses have also been produced and tested.

It is evident from the data shown in Table 11 that the breeds L 1 and L 2 still manifest lower values of the basic productive characters than those in the strains Super 1 and Hesa 2. May be an other reason is the depression which causes the translocation of the Y allele on the W chromosome (Kimura at al., 1971; Yamamoto, 1989; Datta at al., 2001). On the other hand the productivity of the newly selected breeds is much higher if compared with the Japanese sex limited strain D 50. This picture is however different in the new hybrids, compared with the control.

The data presented in Table 12 show that all the four sex limited for cocoon color silkworm F1 hybrids display a higher pupation rate than the control. The hybrid L1 x L2 and the reciprocal has fresh cocoon weight, silk shell weight and shell ratio very close to the control. Both new four way hybrids manifest lower than the control values of the three characters studied, which we explain by the participation in them as mail parents of the sex limited for larval markings breeds Iva 1 and Nova 2, having a higher tolerance to adverse rearing conditions, but a little lower productivity.

It may be concluded that we created 4 new sex – limited for cocoon color silkworm F1 commercial hybrids which manifest a pupation rate and productivity, comparable with those of the control, the hybrid Super 1 x Hesa 2.

REFERENCES

Datta, R. K., Basavaraja, H.K., Mal Reddy, N., Kalpana, G.V., Joge, P.G., Palit, A.K., Jayaswal, K.P. (2001)„CSR8” Sex limited bivoltine breed. A boon for graineurs. Indian silk 39 (11): 5-7.

Kimura, K., Harada, C., Akai, H. (1971) Studies on „W” chromosome translocated in yellow blood gene in silkworm, Jap. J. Breed. 21: 199 – 203.

Tzenov, P., Y.Natcheva, N.Petkov (1997) Study of on the cocoon coulor and cocoon shape characters inheritance in F1 crosses between in bivoltine and multivoltine races of the silkworm Bombyx mori L., Bulg. J. of Agric. Sci., 2, 181-185.

Tzenov, P., Y. Natcheva, N. Petkov (1998) Inheritance of the larval marking character in hybrids between multivoltine and unibivoltine silkworm, Bombyx mori L., races, Bulg. J. of Agric. Sci., 4, 3, 373-378.

Tzenov, P., Y.Natcheva, N.Petkov (1998) Cocoon shape inheritance in hybrids between multivoltine and uni-bivoltine silkworm, Bombyx mori L. races. Bull. Ind. Acad. Sericultural, vol. 2(2), 8-15, India.

Tzenov, P., N.Petkov, Y.Natceva (1999) Study on the inheritance of food ingestion and digestion in hybrids between univoline and multivoltine silkworm, Bombyx mori L., races, Sericologia, 39 (2), France.

Tzenov, P., Y.Natcheva, N.Petkov (1999) Study of the cocoon color segregation manifested in crosses between uni-bivoltine and multivoltine silkworm, Bombyx mori L. rаces, Sericologia, 39 (3), France.

Tzenov, P., Y.Natcheva, N.Petkov (2000) Study on the cocoon color segregation manifested in hybrids between uni-bivоltine and multivoltine silkworm, Bombyx mori L. races, Sericologia, 40 (1), 67-73 (France).

Tzenov P. & Z.I. De Guzman (2004) Breeding the new Bulgarian sex-limited for larval markings silkworm Bombyx mori L. commercial hybrid Ze/4 and study on its performance in the Philippines and Bulgaria, Sericologia 44(3), 297-312. (France)

Tzenov P. (2005) Study on the larval markings inheritance in some four-way hybrids between sex-limited for larval markings silkworm, Bombyx mori L. breeds. In: “International Workshop on Revival and Promotion of Sericultural Industries and Small Silk Enterprise Development in the Black & Caspian Seas Region”, Tashkent, Uzbekistan; 415-419.

Tzenov P. (2005) Breeding of new silkworm, Bombyx mori L. sex-limited for larval markings analogues of parental pure lines by the method of back crosses. In: “International Workshop on Revival and Promotion of Sericultural Industries and Small Silk Enterprise Development in the Black & Caspian Seas Region”, Tashkent, Uzbekistan; 419-424.

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Tzenov P. D. Grekov (2008) Development of methods for breeding new silkworm, Bombyx mori L. commercial hybrids tolerant to adversе rearing conditions. Annals of agrarian science, vol. 6, № 2, 71 – 76.Georgia.

Tzenov P., D. Grekov (2009) Breeding of new tolerant to adverse rearing conditions silkworm, Bombyx mori L. four – way uni – bivoltine commercial hybrids. In: Application of Biotechnology in Sericulture, 243 – 252. Bangalore, India

Tzenov P. (2008) Heterosis Expression in Some Main Quantitative Breeding Characters in Four – Way Sex-Limited for Larval Markings Silkworm, Bomby mori L. F1 Hybrids, 21st Congress of the International Sericultural Commission, 3 – 6 November 2008, Athens, Greece.

Yamamoto, T. 1989. Breeding of sex limited yellow cocoon races of silkworms by chromosome manipulation. Farming Japan 23 (5): 42 – 48.

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