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Tuesday, 14 February 2012

A New Highly Productive Silkworm Sex-Limited Pure Genetic Line

H.S. Homidy, D. Gahakwa, A.N. Papaskiri and A. Rutayisire

Professor H.S. Homidy

Professor Homidy Khomid Soky (HS. Homidy) is International Expert on program development of sericulture, of the Ministry of Agriculture and Animal Resources, Rwanda (IFAD/PDCRE/RHODA/RAB). His research interest in sericulture are wide and varied such as: breeding of silkworm, biochemistry, physiology, technologies of silkworm hybrid (F1) egg productions, silkworm rearing, productions of cocoons and post-harvest, disease control, maintenance of mulberry plantations, mulberry crop protection etc. During his 23 years of research career he has held important positions as deputy director on research National Research Institute of Sericulture, Uzbekistan, Vice-minister Ministry of Silk Industry republic of Uzbekistan, Director of National Research Institute of Sericulture, Uzbekistan, Manager of scientific technological centre of Uzbek Scientific Research Institute of Sericulture etc. He has to his credits two books, 61 research papers 20 PhD theses and three patented inventions. Professor Homidy is the Vice president of the Black, Caspian Seas and Central Asia Silk Association (BACSA).
He may be contacted at: Email: , Republic of Rwanda Kigali , Tel: +250783280724

Generation of highly productive sex – limited pure genetic silkworm lines remains interesting both in theoretical and in practical aspects using the practice of sericulture to allow simplified production of highly heterocyst silkworm egg hybrids.
During process of selection on preservation of characteristics and adaptation to new environment conditions of silkworm breeds, with the breed named Progress (Uzbek selection - Japanese type), the sixth generation silkworm larvae with different skin color were generated. The new silkworm larvae genetic pure line revealed sex-limited characteristics. Their distinctive feature consist of: silkworm larvae having marking on the first and fourth segment are female and having marking in first to fifth segments (type multi-lunar) are male larvae. ‘Trialson’ the new genetic pure line up to the fifth generation has shown stability of their phenotypic signs and essential differences between females and males. Thus the balance of sex at larvae stage of 1 ♀♀: 1 ♂♂ was achieved. The new genetic pure line is superior in viability of pupation (92,64%), weight of a cocoon (2,48 g) and cocoon shell (0,567 g). Fresh cocoons yield average attained 44,46 kg/box and accordingly with excellent raw silk yield (7,515 kg/box).
Keywords: Sex – limited silkworm pure genetic line, heterosis, hybrids, sexual chromosomes, morphological signs.
Mulberry silkworm (Bombyx mori L.) became the first object on which to practically solve the problem of artificial sex regulation. The main reason for using sex – limited silkworm lines is to make easier and more effective, the process of silkworm egg production especially in terms of sex discrimination. The first attempts to create sex-limited silkworm breeds were made by Tichomirov (1891), followed successfully by other researchers like Tazima (1944), Hasimoto (1948), Astaurov (1972), and Lee at al. (1988). The first silkworm strain, having marked female larvae and plain male was created by Tazima (1944) by treatment of newly laid silkworm eggs with X-rays and translocation of the P+ allele and its connection with the sex chromosome W. Later, using the same method Hasimoto (1948) managed to create a new sex-limited breed having zebra female and plain male larvae. By this method, the initial material used was sex limited races that were crossed with other races having plain larvae or yellow eggs. After that the hybrid population was maintained by batch rearing for four generations and with inside batch mating (inbreeding). Strunnikov (1969) expanded the studies, directed toward the creation of marked differentiation by sex at the egg stage but also with sufficiently good viability. The main distinctive special feature of these lines is that females are developed from the dark eggs while males arise from the bright eggs. On the basis of translocation lines, two breeds, one which is characterized by the oblong form of cocoon, and another with cocoons that are rounded, were created. Concerning the sex-limited character of cocoon color, it is established, that the female cocoon is yellow and the male white. It permits an easy sex-discrimination and the male cocoon can separately be reeled to produce high-grade silk (Chen, 2002).
After discussion of such impressive results on silkworm sex regulation at different stages using X-ray, it was possible to think, that the problem is completely resolved, but however in the literature there is no information on any research or discovery of natural mutation in sex marking of silkworms. In these circumstances, our research was to adapt silkworm breeds delivered from different geographical zones to sub - equatorial climate of Rwanda, to study phenotypical changes including possibilities of occurrence natural mutation in these breeds.
Materials and methods
Study area
This study was conducted at the sericulture unit at the Rubona Research Station, previously under the Rwanda Agricultural Research Institute. Rubona station 2.26 (2° 15' 49 S; 29.81 (29° 48' 27 E) is located in the mid-altitude agro-ecological zone in southern Rwanda, 125 km from Kigali, the capital city. The station has a subequatorial climate and is located at high altitude (1700 m over sea level), with an annual average temperatures around 18-21 °С and rainfall between 1300 -1800 mm. The dry season start from June up to August – September.
Study design
We used a completely randomized design where all the silkworm breeds available were subjected for research of adaptability reaction to new sub - equatorial climate of Rwanda. The study focused on any possible effect of transformation of the silkworm breeds, particularly on revealing mutant genetic lines.
Process that transformed the silkworm
During selection on adaptation of silkworm breeds delivered from different geographical zones to new environment conditions, with breed named Progress (Uzbek selection) Japanese type, in the sixth generation of selection, some silkworm larvae with different skin color were revealed. For improvement and preservation of cleanliness of the silkworm sex-limited breeds, the moderate and remote related breeding – i.e. inbreeding V-VI degree was applied. Selection was carried out based on phenotypical signs of sex-limited silkworms. More than 150 individual families were prepared, but after their detailed analysis, only 105 families were saved. After results on hatchibility of layings, 50 best families were taken for rearing by using Homidy et al., (2001) silkworm rearing procedure.
Data analysis method
Data collected from our work was analysed using common-method of statistical analysis of qualitative and quantitative data (Tabachnick, B.G. & Fidell, L.S. 2007).
Results and discussion 
The idea of Serebrovsky (1935) consisted to use an artificial method, particularly, use of X-ray radiation, to get the silkworms sex-limited. The target was to get male and female silkworms with morphological differences distinguishable at an early stage of development. This phenomenon is due to the genes translocation. The genes responsible for morphological signs are located on sex chromosomes.

New pure silkworm sex-limited pure line GH-02
During our research work on sex-limited silkworm, mainly using the selection technique, we produced a new genetic pure line that has code named GH-02. This new genetic pure line
revealed sex-limited traits in the larvae stage as well. The distinctive feature of this line consist of the silkworm larvae having carpet type markings on segments, typical characteristics of females; and silkworms having usual type marking on their segments, characterizing male larvae . Breeding and selection studies on the new genetic pure line GH-02 up to the fifth generation showed stability on the phenotypic signs, which are essential differences between females and males. Thus, the balance of sex at larvae has now been stabilized at the ratio of 1 ♀♀: 1 ♂♂ (Table 1).
Cocoons of New pure silkworm sex-limited breed (GH-02)
The analysis of the obtained indicators on phenotypical signs of new genetic pure line GH-02, shows that viability of pupation rate of 90,24±0,3 - 95,04±0,2 %, weight of a cocoon-2,80±0,02-2,16±0,01g, cocoon shell -0,582±0,1-0,553±0,2 g and percent cocoon shell-21,0±0,01 - 25,6±0,02%. Fresh cocoons yield by 1 box silkworm eggs an average makes 44,46 kg, thus cocoons yield at females reaches 49,49±3,2 kg/box. The essential difference was not observed in color of female and male cocoons, thus essential differences were revealed in their technological parameters, like filament length-1242±6,7 - 1287±5,3 m, good cocoons reelability (average) 87,5 % and excellent raw silk yield 7,32±1,2 - 7,71±1,5 kg/box.
Table 1. Testing new silkworm six-limited pure line GH-02

It is important to understand that sericulture is focusing on deducing new highly productive breeds with improved raw silk yield and disease resistance. During the process of research on adaptability response of silkworm breeds delivered from different geographical origins to local condition, for the first time in Rwanda, at larval stage it was discovered a new sex limited genetic line of silkworm. This new genetic pure line (code named GH – 02) had showed to be disease resistant and highly yielding in terms of raw silk with good technological parameters of filament. The difference was significantly observed in body coloration during 4th and 5th instars larval stage, in female carpet coloration appeared while in male usual coloration appeared and thus it was easy to make separation according to sex, they can be used for production of 100% silkworm hybrid eggs. The reason of occurrence of this sexual segregation could be natural mutation, attributable to the geographical location of our scientific laboratory (1700m over sea level).

Tazima Y. (1944) Studies on chromosome aberrations in the silkworm. II Translocation involving second and W-chromosomes //Ibid. N12. Р.109-181.

Hasimoto H. (1948) Sex-limited zebra in X-ray mutation in the silkworm. //J.Sericulture Japan. 1948, Р.16.

Astaurov B.L. (1972) Prospect of animal sex regulation //J. Nature.№2. P.48-57.

Lee S., Kim S., Kim K., Lee S., Lee H., Lee Y. (1988) Induction of sex-limited cocoon color character by translocation of yellow blood gene Y(TT -25.6) on to W chromosome by gamma irradiation in silkworm. Bombyx mori. //Korean J. of Breeding, 21(3), P.219-223.

Strunnikov V. (1969) Greeting of man's posterity at silkworm (Bombix mori L). //Academic scientific Reports. V.189. №5. P. 1155-1158.

Chen Y. (2002) Conservation status of silkworm genetic resources in China. Expert consultation on promotion of global exchange of sericulture germplasm resources", Satellite session of XIXth ISC Congress, 21st -25th, September 2002, Bangkok, Thailand.

Homidy H., Ahmadaliev A. and Papaskiri A. (2001) Technology of silkworm rearing. Patent № 055075 Uz.

Serebrovsky A. (1935) Hybridization at animals. M.: Biomedgis. P.290.

Tabachnick, B.G. & Fidell, L.S. (2007). Using Multivariate Statistics, Fifth Edition. Boston: Pearson Education, Inc. / Allyn and Bacon, ISBN 978-0205459384

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