J. Silk Sci. Tech. Jpn. 27, 15-22（2019） Treatise Study of breeding large Antheraea yamamai for greater silk productivity by backcross Hiroaki Ishikawa1), Tomohiro Hirano2), Chikahisa Kiriyama2), Takao Okuno2), Mitsuharu Jounai3), Suguru Takeuchi2), Yuta Sobue2), Mika Jitsukawa4), Kiyoko Sakurai4), and Zenta Kajiura2) * 1) Department of Bioscience and Textile Technology Doctoral Program, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokita, Ueda, Nagano 386-8567, Japan 2) Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokita, Ueda, Nagano 386-8567, Japan 3) Department of Textile Science and Technology, Applied Biology, Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokita, Ueda, Nagano 386-8567, Japan 4) Faculty of Textile Science and Technology, Applied Biology, Shinshu University, 3-15-1 Tokita, Ueda, Nagano 386-8567, Japan (Received : September 30, 2018, Accepted : December 6, 2018) Backcross of Japanese oak silkmoth Antheraea yamamai (A. yamamai) was undertaken to breed a strain with greater silk production capacity by hybridizing a strain showing a high fertilization rate (SUB-52) and a strain showing heavy cocoon weight (SUB-11). By repeating backcross from 2010 to 2018, we obtained and stored backcross strain (Bn; n represents the number of generations) from third generation (B3) to seventh generation (B7). Comparing SUB-52, SUB-11 and each Bn by statistic method showed that B6 was superior to other strains in terms of silk productivity. Although cocoon weight and cocoon shell weight in the latest backcross strain B7 was significantly higher than that of SUB-52, there was no significant difference between 7B and their recurrent parent SUB-11. We consider that B7 was getting closer to SUB-11 and appropriate backcross should be limited until B6. As for future research, we plan to improve the fertilization rate by using Bn for hybridization. (*: To whom correspondence should be addressed, E mail: [email protected]) Key Words: Antheraea yamamai (A. yamamai), back cross, cocoon shell weight 1. Introduction is known to make cocoon that contain blue and yellow pigments that result in bright green cocoon Silks produced by non-mulberry silkmoths shell.4,5) were regarded to use as biomaterials because of Silk from A. yamamai has been used in Ja- their properties such as color, strength and bio- pan as a fabric material for a long time however, logical compatibility are different from the silk it has also been investigated for use as biomate- produced by domestic silkmoth Bombyx mori (B. rials because of its characteristic amino acid mo- mori). Japanese oak silkmoth Antheraea yamamai tifs.1) Therefore, we consider that demand for silk (A. yamamai) is one of the non-mulberry, satur- from A. yamamai would be preferably increased niid silkmoth native to east Asia.1-3) A. yamamai to enhance using and researching. However, even 15 日本シルク学会誌 第 27 巻 though the body of A. yamamai is much bigger which inherit objective characteristics were used than B. mori, silk productivity of A. yamamai is for the next hybridization to obtain the next gen- lower than that of B. mori. erations Bn+1. By repeating backcross from 2010 to We consider that cross breeding is one of the 2018, we stored backcross strains from B3 to B7. methods for improving the efficiency of silk pro- Theoretically, the more backcrosses are per- ductivity per cocoon weight or increasing the total formed, the closer the latest backcross strain is to amount of silk product by enhancing the cocoon the recurrent parent6). Therefore, we investigated weight itself or increasing the number of harvest- the appropriate number of backcrosses to breed a able cocoons. For cross breeding, we collected backcross strain which has high productivity of A. yamamai from various areas and reared them silk by comparing SUB-52, SUB-11 and B3-7. We to investigate each characteristic as a strain. As a also reeled cocoon filaments from each strain and result, we selected two strains for cross breeding, measure their fiber properties to investigate the strain showing a high fertilization rate (SUB-52) differences between the strains. which was expected to increase the number of harvestable cocoon and strain showing a heavy 2. Materials and Methods cocoon weight (SUB-11) which was expected to contribute to increase the production of silk. 2.1 Insects and reagents In this study, backcross was used as the meth- A. yamamai were captured from various ar- od of cross breeding. The biggest SUB-11 females eas in Japan and reared at the Shinshu University were used for hybridized with SUB-52 males to Faculty of Textile Science and Technology. Two breed first generation hybrids. Male hybrids were strains were used for backcross, one showing a then hybridized with the biggest SUB-11 females high fertilization rate (SUB-52) (Table 1) (Fig. as recurrent parents to breed backcross strains, 1) and another showing a heavy cocoon weight Bn (n represents the number of generations). Bn (SUB-11) (Table 2) (Fig. 2). Table 1. Number of hybridized pairs, fertilization pairs and fertilization rate of SUB-52, SUB-11 and other strains in 2013 Strain Number of hybridized pairs Number of fertilized pairs Fertilization rate a) [%] others 1 21 3 14.3 SUB-11 71 15 21.1 others 2 38 11 28.9 others 3 38 22 57.9 SUB-52 49 38 77.6 a): Fertilization rate = (Number of fertilized pairs) x100 / (Number of hybridized pairs) [%]. Table 2. Cocoon weight of SUB-52, SUB-11 and other strains in the female group in 2013 Strain Number of samples Cocoon weight ± SD a) [g] others 1 9 9.021 ± 0.910 SUB-11 24 12.513 ± 1.004 others 2 5 10.517 ± 0.787 others 3 15 8.039 ± 0.650 SUB-52 13 9.348 ± 1.238 a): Value of cocoon weight shown the average (left) and standard deviation (right). 16 Breeding large A. yamamai Chlorinated lime granules (with the main managed by the Shinshu University Faculty of component of calcium hypochlorite trihydrate) and Textile Science and Technology. Rubber boots NaOH were used to disinfect or sterilize eggs, the were worn by all staff working on the oak field oak field and other instruments. Proteinase K, an and were disinfected by spraying NaOH solution enzyme that degrades protein, disodium hydrogen (0.001% v/v) after use to diminish the risk of death phosphate 12-water (Na2HPO412H2O) and sodium by diseases. dihydrogen phosphate dihydrate (NaH2PO42H2O) were used to make a solution for cocoon cooking 2.2 Disinfection (Wako Pure Chemical Industries, Ltd., Japan). Chlorinated lime granules were dissolved by Oak branches, Quercus acutissima (Q. acu- tap water to prepare an antiseptic solution (3 l) at tissima) were harvested as food from the oak field a concentration of 1% (w/v). Eggs of A. yamamai were put in the empty tea bag in group sorted by their mother moth. Eggs in the tea bags were and 100.0 90.0 soaked in the antiseptic solution and stirred with 77.6 80.0 a magnet stirrer (15 min) to bleach and disinfect ] 70.0 % [ e 57.9 t 60.0 the egg surface. Eggs were rinsed under runnning a r n o 50.0 i tap water until the smell of chloride was removed t a z i l 40.0 i t r 28.9 from the egg bags. Eggs were air dried at room e F 30.0 21.1 20.0 14.3 temperature (RT) and stored in petri dishes sepa- 10.0 rated by group until hatching. 0.0 others 1 SUB-11 others 2 others 3 SUB-52 To diminish the risk of diseases such as nu- clear polyhedrosis virus, soil in the oak field was Fig. 1. Ranking of fertilization rate in 2013, red bar: SUB-52 group records the highest fertilization rate, scorched by fire throwers for disinfection in win- green bar: SUB-11 group records the forth place in ter. Oak trees were also disinfected by spraying fertilization rate, blue bar: groups of other strains with NaOH solution (0.001% v/v). Slaked lime that not referred to the paper. was spread on the oak field and covered by agri- cultural mulch to protect the trees and A. yamamai 14.000 larvae from natural enemies and to prevent weed 12.000 growth on the field before spring. ] 10.000 g [ 7) t h 2.3 Rearing g i 8.000 e w n Hatched larvae were moved from the petri o 6.000 o c o C dishes to fresh Q. acutissima branches in the rear- 4.000 ing cases. Branches were wrapped in wet paper 2.000 at the cut portion to retain moisture and fresh- 0.000 others 3 others 1 SUB-52 others 2 SUB-11 ness. Rearing cases were kept clean by removing residues from A. yamamai larvae, disinfecting by Fig. 2. Ranking of cocoon weight in female group in 2013, red bar: SUB-52 group records the third spaying with 70% EtOH and exchanging old Q. place in cocoon weight, green bar: SUB-11 group acutissima branches for new ones every morning. records the heaviest cocoon weight, blue bar: Groups of A. yamamai were released to the groups of other strains that not referred to the paper. oak field at the different Q. acutissima trees in According to the analysis of variance (ANOVA; each group when the 4th instar larva was first ap- Tukey-Kramer method), cocoon weight of SUB-11 was significantly heavier than that of SUB-52 and peared in the group to reduce the labor for rearing.