In vitro culture and low temperature incubation tolerance of staged embryos of the silkworm, Bombyx mori

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1 Journal of Insect Biotechnology and Sericology 85, (2016): Short Communication In vitro culture and low temperature incubation tolerance of staged embryos of the silkworm, Bombyx mori Hisayoshi Fukumori, Tsuguru Fujii and Yutaka Banno * Silkworm resource division, Institute of Genetic Resources, Kyushu University Graduate School of Bio Resources and Bioenvironmental Science, Hakozaki, Higashi-ku, Fukuoka , Japan (Received February 23, 2016; Accepted March 10, 2016) Key words: egg dechorionation, embryo culture, cryopreservation, Bombyx mori INTRODUCTION Development of cryopreservation for the silkworm, Bombyx mori, has been anticipated for a long time, and now two methods are available. One uses frozen sperm (Takemura and Kanda, 1999; Takemura et al., 2000) and the other uses frozen ovaries (Mochida et al., 2003, Banno et al., 2013). These methods are already in practice for most silkworm strains, however, a small number of strains are not available due to their low tolerance to low temperature. It is important to develop a greater variety of methods for safe preservation of the large collections of silkworm bio-resources. Recently, larval hatching from cryopreserved embryos kept in liquid nitrogen was reported in Drosophila melanogaster (Mazur et al., 1992) and in Pectinophora gossypiella (Rajamohan et al., 2013). These reports present a potential alternative to those previously developed for the silkworm. To perform in vitro culture of B. mori embryos, penetration of cryoprotectant into the embryo is necessary. But silkworm embryos are enveloped with chorions (egg shells), which prevents the penetration of the cryoprotectant. To improve the permeability of embryos, first, removal of chorions is essential, and these dechorionated eggs should be cultured in vitro. The possibility of using silkworm embryos for long-term preservation was studied twenty years ago by Imanishi et al. (1996). According to their report, embryos died when they were kept at 196 C. However, their experiments were limited by the selection of the stage and basic procedures, and more detailed experimentation is needed to determine the possibility of embryo use for cryopreservation. In this study, optimal developmental stages for in vitro culture of embryos were investigated. The tolerance of the embryos for low temperature incubation was tested as the next step, along with detailed observations of their morphological features and developmental stages. *To whom correspondence should be addressed. Fax: Tel: banno@agr.kyushu-u.ac.jp MATERIALS AND METHODS Insects Silkworm strains used in this study were p22 and G01, which are maintained at the Institute of Genetic Resources, Kyushu University. Silkworm strain preservation was supported by the National BioResource Project (NBRP). The larvae were reared on mulberry leaves at C. Dechorionation of silkworm eggs Dechorionation of eggs was performed each day from diapause termination to just before hatching. The eggs were dechorionated according to a previous report (Horie et al., 2000). The surfaces of the eggs were disinfected by soaking in a 2% formaldehyde solution for 10 min and then rinsing with 70% ethyl alcohol. To remove the chorion, the eggs were dissected under a stereoscopic microscope using a surgical blade (No. 11, Feather Safety Razor Co., LTD.) and a pair of forceps. Embryos were removed from the chorions while enclosed within the serosal membranes and collected in 1 X Grace s insect medium (Gibco Life Technologies). Low temperature treatment of dechorionated eggs The dechorionated eggs were transferred to a cryogenic tube filled with cryo-protectant medium (Cell Reservoir One including DMSO; Nacalai tesque, Inc.). These cryogenic tubes were gradually cooled (1 C per min) to each targeted temperature ( 10 C, 20 C, 30 C, and 50 C) and kept for 30 min. To freeze at a much lower temperature below 50 C, cryogenic tubes were gradually cooled (1 C per min) to 80 C and kept for more than 2 hr using a BICELL freezing vessel (Nihon Freezer Co., Ltd.) and then plunged into liquid nitrogen at 196 C. The BICELL is a specialized apparatus for slow cooling (1 C/min). After the above treatment the dechorionated eggs were thawed in water at 37 C, then rinsed 3 times with 1 X Grace s insect medium to remove the cryo-protectant medium.

2 50 Fukumori et al. Fig. 1. Outline of egg dechorionation and culture. (1) Egg dechorionation in Grace s medium. The chorion was slashed with a surgical blade (arrow (a)) and the upper piece of chorion was removed (arrow (b)). (2) Suspension of dechorionated eggs into culture medium. (3) Transfer of dechorionated eggs into a cryogenic vial for low temperature treatment. (4) Suspension of each dechorionated egg into a small drop of medium. (5) Inversion of the dish to form hanging drops. Dechorionated egg culture Silkworm embryos are normally enveloped with chorions (egg shells). For in vitro culture of embryos, removal of chorions is essential, because the chorion prevents the penetration of the cryoprotectant. We call the removal step dechorionation. Cultures of the dechorionated eggs were made by a hanging-drop method (Takami et al., 1966); an outline is shown in Fig. 1. Five to seven small hanging drops containing 1 X Grace s insect medium were made on the underside of the cover of a new tissue culture dish (35 10 mm, Falcon) and dechorionated eggs were placed individually in the drops. Cultures were held at 25 C without change of culture medium for 10 days. Identification of the embryonic stages The embryonic stages before freezing were determined by dissecting 5 to 10 eggs sampled randomly from one group of dechorionated eggs. The embryonic stages after culture were determined by dissecting all eggs about 10 days after incubation at 25 C. The embryos were released from the serosal membranes using two pairs of forceps under a stereoscopic microscope. The developmental stages of the embryos were defined morphologically according to the stage criteria defined by Takami and Kitazawa (1960). RESULTS Hatchability of the dechorionated eggs Eggs were dechorionated at embryonic stages from diapause termination to hatching. The dechorionated eggs were cultured in hanging drops at 25 C. Hatchability after culture is shown in Fig. 2. Hatching of embryos was observed at all stages after culture at 25 C; however, hatchability varied with the stage of dechorionation. Fifty to seventy percent of cultured eggs hatched when the eggs were dechorionated at embryonic stage 17 (the longest embryo stage), stage 18 (the stage of appendage appearance in the gnathal, thoracic and abdominal regions), and stage 19 (the stage of embryo length decrease). However, hatchability declined to 31% at embryonic stage 20 (the

3 Low temperature incubation of embryo 51 Fig. 2. Hatchability of dechorionated eggs. Eggs were dechorionated at each embryonic stage and cultured in hanging drops at 25 C. For each stage, embryos were treated. Specific features of each stage (from Takami and Kitazawa (1960)) stage 17: beginning of organogenesis (the longest embryo stage) stage 18: appendage appearance in the gnathal, thoracic and abdominal regions stage 19: embryo length decrease stage 20: head and thorax are clearly demarcated and gnathal appendages group in the head region stage 21: during the embryonic reversal stage 22: termination of embryonic reversal stage 23: basal formation of setae stage 24: setae formation stage 25: tracheal fiber formation. stage when head and thorax are clearly demarcated and gnathal appendages group in the head region) to stage 21 (the embryonic reversal stage). Conversely, when eggs were dechorionated after the termination of embryonic reversal (stages 22, 24, and 25), almost all eggs hatched (90-100%). Most of the unhatched embryos, which dechorionated before stage 21, could not undergo embryonic reversal and arrested their development at stage 26 (head pigmentation). Influence of low temperature treatment on hatchability The dechorionated eggs were allowed to develop and then subjected to low temperature at 10 C, 20 C, and 30 C, for 30 min at each embryonic stage (Fig. 3). At stage 17, only 10% of the eggs treated at 20 C hatched; no eggs treated at 10 C or 30 C hatched. No embryos could hatch after low temperature treatment at stages 18 to 22. After stage 23, hatchability increased with embryonic stage for the 10 C treatment. Similarly, the hatchability of embryos treated at 20 C recovered after exposure to low temperature at stage 24. At stage 25, high hatchability Fig. 3. Hatchability of dechorionated eggs after freezing. The dechorionated eggs were subjected to 10 C ( ), 20 C ( ), and 30 C ( ) at each embryonic stage. After treatment, eggs were cultured in hanging drops at 25 C. For each point, 10 or 11 embryos were treated. was observed at 20 C for a 30 min treatment (Fig. 3). However, hatchability declined with one day of exposure to 20 C, and no larvae hatched after treatment for 7 days (data not shown). Features of embryos after low temperature treatment Most of the embryos did not hatch after low temperature treatment (for example, 20 to 30 C) at any embryonic stage. However, the unhatched embryos seemed to have undergone a small amount of development. To understand the stages of arrest in more detail, the embryos treated at 30 C for 30 min were observed under a microscope (Fig. 4). Embryos dechorionated at stages 17 and 18 progressed in development up to stages 20 or 21. When these embryos were cold treated at stages 19 to 23, most of them developed to stage 26 or 27. But almost all of these embryos were malformed. Additionally, these embryos could not undergo embryonic reversal when the eggs were treated before stage 21. When the freezing treatment was carried out for eggs at stage 24, the only stage at which hatching was observed, most unhatched embryos progressed up to stage 27 without malformation. When the eggs were frozen at stage 25, most embryos did not change shape after freezing. After freezing in liquid nitrogen ( 196 C), all embryos except for those frozen at stage 24 were dead. By contrast, dechorionated eggs frozen in liquid nitrogen at stage 24 progressed in embryonic development up to stage 26 during incubation at 25 C after cold treatment.

4 52 Fukumori et al. Fig. 4. Embryonic features. (A) Features of embryos at stages 17 to 25 without low temperature treatment. (B) Typical features of embryos with low temperature treatment ( 30 C, 30 min). Low temperature treatment was done at stages 17 to 25 shown in (A). At all stages, the treated embryos showed a little development; however, none hatched. Specific features of each stage were written in Fig. 2. CONCLUSION 1. We reconfirmed an in vitro hanging drop system for dechorionated silkworm eggs and determined that in vitro culture of embryos from all developmental stages after diapause termination was possible. The ratio of hatching after culture was highest (~90%) with establishment of in vitro culture at stages 22 to 25. The earlier stages of embryos might be damaged by a surgical blade, result in the low hatchability.

5 Low temperature incubation of embryo The embryos showed tolerance to a short treatment at low temperature (optimum of 30 min, 20 C). On the other hand, no embryo could hatch after long treatment, such as 7 days. Although this will restrict some applications, many of the cold treated embryos progressed 2-6 stages in development, suggesting further refinement of conditions for cold treatment may be successful. 3. Based on the results reported here, dechorionated embryos were recognized to have tolerance for low temperature treatment; however it was not high. Our study indicates that stages are the only ones possible for further attempts to use silkworm embryos for long term preservation. ACKNOWLEDGEMENTS We are sincerely grateful to Dr. Y. Mochida and Dr. Y. Takemura of Institute of Sericulture for their kind help and advice regarding the methods of egg dechorionation and in vitro culture. We would like to thank Dr. Marian R Goldsmith of University of Rhode Island for critical reading of manuscript. This work was supported by JSPS KAKENHI Grant Number Silkworm strains used in this study were supported by the National Bio- Resource Project (NBRP) of the MEXT, Japan. REFERENCES Banno, Y., Nagasaki, K., Tsukada, M., Minohara, Y., Banno, J., Nishikawa, K., Yamamoto, K., Tamura, K. and Fujii, T. (2013) Development of a method for long-term preservation of Bombyx mori silkworm strains using frozen ovaries. Cryobiology, 66, Horie, Y., Kanda, T. and Mochida, Y. (2000) Sorbitol as an arrester of embryonic development in diapausing eggs of the silkworm, Bombyx mori. J. Insect Physiol., 46, Imanishi, S., Sinbo, H. and Kiguchi, K. (1996) Cryopreservation of silkworm eggs. J. Seric. Sci. Jpn., 65, (in Japanese). Mazur, P., Cole, K.W., Hall, J.W., Schreuders, P.D. and Mahowald, A.P. (1992) Cryobiological preservation of Drosophila embryos. Science, 258, Mochida, Y., Takemura, Y., Kanda, T. and Horie, Y. (2003) Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm, Bombyx mori. Cryobiology, 46, Rajamohan, A., Rinehart, J.P., Foster, S.P. and Leopold, R.A. (2013) Permeability barriers to embryo cryopreservation of Pectinophora gossypiella (Lepidoptera: Gelechiidae). J. Econ. Entomol., 106, Takami, T. and Kitazawa, T. (1960). The embryos features during embryogenesis in the silkworm, Bombyx mori. Sanshi Shikenjo Hokoku, 75, 1-31 (in Japanese). Takami, T., Sugiyama, H., Kitazawa, T. and Kanda, T. (1966) Infection of cultured Silkworm eggs with the nuclear-polyhedrosis virus. Jap. J. Appl. Ent. Zool., 10, (in Japanese). Takemura, Y. and Kanda, T. (1999) Artificial insemination and oviposition using cryopreserved sperm in several strains of the silkworm, Bombyx mori. J. Seric. Sci. Jpn., 68, (in Japanese). Takemura, Y., Kanda, T. and Horie, Y. (2000) Artificial insemination using cryopreserved sperm in the silkworm, Bombyx mori. J. Insect Physiol., 46,