How to treat insect injuries

Abstract

To provide a method for treating an insect's injury that solves the conventional problems at least partially.SOLUTION: A method for treating a wound on an insect's skin includes pasting a sheet-like biocompatible material to a wound on an insect's skin.SELECTED DRAWING: Figure 4

Description

【Technical Field】 【0001】 The present disclosure relates to a method for treating insect trauma. 【Background Art】 【0002】 Insects, especially silkworms, have been developed into a wide variety of strains and breeds, and their breeding and subculture for preservation require a great deal of labor and cost. In addition, new strains of silkworms are being developed with the spread of genetic recombination technology. Therefore, it is necessary to maintain and preserve silkworm strains and breeds stably for a long time. For example, Kyushu University has more than 450 genetic trait strains (Non-Patent Document 1), and the NARO Gene Bank has more than 540 genetic species. 【0003】 As a method for maintaining or preserving conventional silkworm strains and breeds, a method of maintaining silkworms as eggs using the diapause trait has been adopted. However, when using the diapause trait, it is necessary to breed once a year for preservation. Also, in the diapause method, there is a risk that the strain may be lost due to troubles such as diseases, natural disasters, spontaneous mutations, or human errors. Therefore, instead of the diapause method, a method of producing next-generation silkworms by cryopreserving sperm and testes and their artificial insemination or transplantation has been developed. Regarding such a method, Non-Patent Document 2 describes a manual for cryopreserving silkworm germ cells. Non-Patent Document 2 describes covering the wound formed by incising the epidermis of silkworm larvae with paraffin melted by heat. In addition, Non-Patent Document 3 describes covering the incision with paraffin wax. Thus, in the conventional method, it has been a standard method to treat the wound formed by incising the silkworm larval epidermis with heat-melted paraffin for artificial insemination or transplantation. 【0004】 Non-Patent Document 4 describes a method for transplanting ovaries of ladybug larvae. In the disclosed ovary transplantation method, it is described that the incised wound of the larval epidermis is not closed (untreated). 【0005】 On the other hand, recent advances in gene transfer and genome editing technologies have led to the creation of hundreds of transgenic silkworm lines in a single research laboratory every year. Therefore, there is a need for a simple method to maintain and preserve these numerous lines. [Prior art documents] [Non-patent literature] 【0006】 [Non-Patent Document 1] Kawaguchi, et al., (2009) J Insect Biotechnol Sericology, 78, 3 113-3 126 [Non-Patent Document 2] Mochida et al. (2014) Manual for cryopreservation of silkworm germ cells (Ver. 2014), Sericulture and Insect Biotechnology 83(3), 255-272. [Non-Patent Document 3] Fukumori, H., Lee, J., Fujii, T., Kajiura, Z. and Banno, Y. (2017) Cryobiology, 77, 71-74. [Non-Patent Document 4] Kawaguchi H, Niimi T (2018) J Insect Biotechnol Sericol 87, 35-44 [Overview of the Initiative] [Problems that the invention aims to solve] 【0007】 The dormancy method used to maintain silkworms as eggs has the aforementioned problems. Furthermore, conventional artificial insemination and transplantation methods involving insect incisions involve time-consuming processes such as ovarian collection, freezing and thawing, ovarian transplantation, and paraffin coating of the incision site. In particular, coating the incision site with paraffin wax requires heating equipment such as a hot plate or gas burner to melt the paraffin. Moreover, adjusting the temperature and amount of melted paraffin is extremely difficult, and misjudging the appropriate temperature or using the wrong amount can lead to leakage of bodily fluids and a decrease in survival rate. In other words, the melting and handling of paraffin depends on the individual skill of the worker, and the results are inconsistent in terms of transplantation efficiency and survival rate, posing problems from the standpoint of reproducibility. In addition, paraffin has a strong odor, and when melted, the odor fills the laboratory or workroom, posing problems from the hygiene and environmental perspectives of the workers. 【0008】 In one embodiment, the disclosure aims to provide a method for treating insect injuries that at least partially solves the problems of the prior art. In another embodiment, the disclosure aims to provide a method for maintaining insect strains that at least partially solves the problems of the prior art. [Means for solving the problem] 【0009】 As a result of diligent research to solve the aforementioned problems, the inventors have found, as an example, that wounds in silkworms can be easily treated by covering them with anti-drying paper or fibroin film instead of heat-meltable paraffin, and have completed the present invention, which includes this as one embodiment. Furthermore, the inventors have found that insect strains can be easily maintained by covering wounds in insects with anti-drying paper or fibroin film, and have completed the present invention, which includes this as one embodiment. 【0010】 This disclosure includes the following embodiments. [1] A method for treating a wound on the exoskeleton of an insect, comprising applying a sheet-shaped biocompatible material to the wound on the exoskeleton of the insect. [2] A method for transplanting insect ovaries, (i) The process of surgically incising the epidermis of the recipient insect, (ii) The process of transplanting ovaries collected from donor insects into dissected recipient insects, and (iii) A step of applying a sheet-shaped biocompatible material to the wound on the epidermis of the incised recipient insect, The method, including the method described above. [3] The method according to Embodiment 1 or 2, wherein the biocompatible material having a sheet shape includes a material selected from the group consisting of anti-drying paper, paraffin paper, woven fabric, nonwoven fabric, paper, natural resin, synthetic resin, and fibroin film. [4] The method according to any one of embodiments 1 to 3, wherein the insect is an insect of the order Lepidoptera or Coleoptera. [5] The method according to any one of embodiments 1 to 4, wherein the insect is in the larval or pupal stage. [6] An insect ovarian transplantation method according to any of Embodiments 2 to 5, wherein only one ovary is transplanted as the ovary collected from the donor insect. [7] An insect wound treatment kit for use in a method for treating wounds on the epidermis of insects according to any one of Embodiments 1 to 5, comprising a biocompatible material having a sheet shape. [8] A kit for treating insect wounds, for use in an insect ovarian transplantation method according to any of Embodiments 2 to 6, comprising a biocompatible material having a sheet shape. [9] An insect wound treatment kit comprising a sheet-shaped biocompatible material and instructions, wherein the instructions describe applying the sheet-shaped biocompatible material to the wound of the insect.

[10] An insect ovarian transplant kit comprising a sheet-shaped biocompatible material and instructions, wherein the instructions describe applying the sheet-shaped biocompatible material to the wound of the insect.

[11] The kit according to Embodiment 9 or 10, wherein the biocompatible material having a sheet shape includes a material selected from the group consisting of anti-drying paper, paraffin paper, woven fabric, nonwoven fabric, paper, natural resin, synthetic resin, and fibroin film.

[12] The kit according to any one of embodiments 9 to 11, wherein the insect is an insect of the order Lepidoptera or Coleoptera.

[13] The kit according to any one of embodiments 9 to 12, wherein the insect is in the larval or pupal stage. [Effects of the Invention] 【0011】 The effects of this disclosure include the ability to treat injured insects with simple procedures, and the avoidance of various problems associated with heat-melted paraffin. [Brief explanation of the drawing] 【0012】 [Figure 1] This figure shows the conventional method and the method disclosed herein. After ovarian transplantation, the incision site was covered with soluble paraffin in A, and with anti-drying paper in B. Figure C shows the results of comparing the survival rates of both methods. Surgically treated larvae were reared until they became adults, and their survival rates were compared. The survival rate was calculated from the number of adults that were reared and the number of treated larvae. The values ​​are shown as the mean and standard deviation (±SD) of five independent experiments. The number of individuals in each experiment was 9 to 13. ns: Not statistically significant (by Student's t-test). [Figure 2]The method of the present disclosure with reduced burden by incision is shown. In Fig. 2A, a schematic diagram of the somite to be incised is shown. In group A, both ovaries of the recipient itself were pulled out from the larva, connected to the donor ovary expressing DsRed by a string body, excised, and then the donor ovary was inserted into the body of the larva. In group B, the operation was performed in the same manner as in group A, except that only one ovary was transplanted from the donor, and the other ovary not expressing DsRed was left as the recipient's own ovary. Fig. 2B shows the difference in eggs obtained from group A and group B. All eggs in group A were derived from the donor ovary expressing DsRed. In group B, eggs of different colors were seen in about half, that is, eggs derived from the donor ovary and eggs derived from the recipient ovary were seen. The scale bar indicates 5 mm. [Figure 3] A photograph showing the scale of the desiccation-proof paper is shown. [Figure 4] A photograph of a silkworm with a wound covered with paraffin or desiccation-proof paper is shown. [Figure 5] Survival rates when covered with molten paraffin, when covered with a fibroin film, and when covered with desiccation-proof paper after surgical treatment are shown. The vertical axis represents the survival rate (%). [Figure 6] Survival rates when covered with molten paraffin, when covered with desiccation-proof paper, and when nothing is done after injuring the somite are shown. The vertical axis represents the survival rate (%). 【Mode for Carrying Out the Invention】 【0013】 In one embodiment, the present disclosure provides a method for treating a wound on the epidermis of an insect, which includes attaching a biocompatible material having a sheet shape to the wound on the epidermis of the insect. The wound on the epidermis of the insect can be covered with the biocompatible material having a sheet shape. Thereby, for example, the outflow of body fluid from the wound can be prevented, the invasion of pathogens can be prevented, and drying can be prevented. Also, for example, the wound healing of the insect's wound can be promoted. In the present disclosure, biocompatibility means that when the material comes into contact with a living body, it does not cause any adverse effects or side reactions to the living body, or does not cause a foreign body reaction, and performs the intended function. 【0014】 In one embodiment, this disclosure provides a method for insect ovarian transplantation. This method may include (i) surgically incising the epidermis of a recipient insect, (ii) transplanting an ovary taken from a donor insect into the incised recipient insect, and (iii) applying a sheet-shaped biocompatible material to the wound in the epidermis of the incised recipient insect. All steps except (iii) can be carried out in accordance with conventional methods. See, for example, Non-Patent Documents 2 and 3. 【0015】 To use donor ovaries for transplantation, the frozen ovaries are thawed and then transplanted into another female silkworm (also called the recipient). During this process, the recipient's own ovaries (one or both) can be removed. The recipient silkworm can then be reared to obtain adult silkworms. An example of a silkworm ovary transplantation method is described below. However, the method disclosed is not limited to this example. 【0016】 Ovarian harvesting Ovarian harvesting and transplantation can be performed in a clean bench. In the case of silkworms, larvae from the second day of the fourth instar to the second day of the fifth instar can be used for ovarian harvesting. The larvae are placed on ice and anesthetized. The epidermis around the stellate pattern on the eighth segment of the larva is disinfected by wiping it with cotton wool soaked in 75% alcohol. Under a microscope, the epidermis around the stellate pattern is incised with a scalpel, and the ovary can be extracted by placing it on tweezers. At this time, the oviduct (dumb) extending from the ovary may also be extracted in a longer length and can be cut with scissors. The ovary with the oviduct attached can be collected in a petri dish containing Grace insect medium. 【0017】 Ovarian freezing The excised ovaries can be stored for a long period under liquid nitrogen in addition to a cryoprotective agent. Dimethyl sulfoxide (DMSO) can be used as a cryoprotective agent, for example, at concentrations of 1-2 M or 1.5 M, but is not limited to this. The ovaries can be transferred to a freezing container containing a suitable culture medium (e.g., Grace's insect medium) containing a cryoprotective agent. The freezing container tube containing the ovaries can be transferred to a separate container that has been pre-cooled to 10-15°C, and then placed in a deep freezer at -80°C for a specified period of time or longer. The cryopreservation cane is placed in a polystyrene box filled with liquid nitrogen. Next, the freezing tube is removed from the separate container stored in the -80°C deep freezer and placed in the polystyrene box filled with liquid nitrogen. The freezing tube is attached to the cryopreservation cane using tweezers or similar tools in liquid nitrogen. The cryopreservation cane with the attached freezing tube can be stored in a liquid nitrogen storage container. 【0018】 Thawing and transplantation of frozen ovaries The cryopreservation cane, with the freezing tube still attached, can be removed from the liquid nitrogen and directly immersed in warm water at approximately 37°C. The thawed ovaries can be transferred from the freezing tube to a petri dish along with an insect culture medium containing a cryoprotectant. The liquid in the petri dish can then be changed to dilute the cryoprotectant (freeze-resistant agent). A staining solution (e.g., Nile Blue) diluted in the insect culture medium can be added to the petri dish containing the thawed ovaries to stain the ovaries and meristems. The recipient larva can be anesthetized by placing it on ice for one hour. The epidermis of the stellate-patterned area of ​​the larva can be disinfected by wiping it with cotton soaked in 75% alcohol, and then it can be incised, the ovaries removed without cutting the meristems, and placed on the dorsal side. In one embodiment, when incising the recipient, the incision can be made without crossing any segments. The donor ovaries can then be placed over the wound in the recipient. The meristems can then be intertwined, the excess portions can be trimmed, and the donor ovaries can be inserted into the recipient's body through the wound. 【0019】 Next, the recipient's wound is covered. In conventional methods, the wound is covered with soluble paraffin. In the method of this disclosure, the wound is covered with a sheet-shaped biocompatible material instead of soluble paraffin. In other words, a sheet-shaped biocompatible material is applied to the wound on the insect's epidermis. The sheet-shaped biocompatible material can be cut to an appropriate size in advance (see, for example, Figure 3). In one embodiment, the sheet-shaped biocompatible material can have a length of 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, for example, 1 cm or more, or a length of 1 cm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, for example, 1 mm or less, but the dimensions are not limited to these. In one embodiment, the biocompatible material having a sheet shape can have a width of 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, for example, 1 cm or more, or a width of 1 cm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, for example, 1 mm or less, but the dimensions are not limited to these. Biocompatible materials having sheet shapes of various dimensions can be prepared, and a biocompatible material having a sheet shape appropriate to the size and shape of the wound can be used. The covering operation can be performed using appropriate instruments, such as tweezers. The covering operation may also be performed under a microscope. After treatment, the insect can be left undisturbed. The insect can also be fed after the wound has dried. 【0020】 In one embodiment, the biocompatible material having a sheet shape may include a material selected from the group consisting of anti-drying paper, paraffin paper, woven fabric, nonwoven fabric, paper, natural resin, synthetic resin, and fibroin film. These may be commercially available. In one embodiment, the biocompatible material having a sheet shape may be a biodegradable material. 【0021】 In one embodiment, the insect is an insect of the order Lepidoptera. Lepidoptera refers to insects belonging to the taxonomic order Lepidoptera, which includes butterflies and moths. Butterflies include insects belonging to the families Nymphalidae, Papilionidae, Pieridae, Lycaenidae, and Hesperiidae. Moths include insects belonging to families such as Saturniidae, Bombycidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Geometridae, Archtiidae, Noctuidae, Pyralidae, and Sphingidae. For example, among moths, this includes species belonging to the genera Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically silkworms, Bombycidae, mulberry silkworms (Bombyx mandarina), the Japanese silkworm (Samia cynthia; including Samia cynthia ricini and hybrids of Samia cynthia and Samia cynthia), the Japanese oak silkworm (Antheraea yamamai), the Japanese silkworm (Antheraea pernyi), the small Japanese silkworm (Saturnia japonica), and the Japanese luna moth (Actias gnoma), but is not limited to these.The order Coleoptera includes the families Scarabaeidae (e.g., rhinoceros beetle (Trypoxylus dichotomus), scarab beetle (Mimela splendens)); Lucanidae (stag beetle family); Cerambycidae (longhorn beetle family); Dytiscidae (diving beetle family); Carabidae (ground beetle family); Lampyridae (firefly family) (e.g., firefly, Genji firefly (Luciola cruciata), Heike firefly (Luciola lateralis), Hime firefly (Luciola parvula), North American firefly (Photuris)); Coccinellidae (ladybug family) (e.g., seven-spotted ladybug (Coccinella septempunctata), two-spotted ladybug (Harmonia axyridis), white-spotted ladybug (Vibidia) Examples include, but are not limited to, the superfamily Curculionoidea and the family Chrysomelidae. In one embodiment, the insect is a silkworm. In this specification, insect refers to the donor and / or recipient. 【0022】 In one embodiment, the insect may be in the larval or pupal stage. In one embodiment, the step of applying a sheet-shaped biocompatible material to a wound on the insect's epidermis can be performed at room temperature. 【0023】 In one embodiment, the insect ovarian transplantation method of the present disclosure may involve transplanting only one or both ovaries, taken from a donor insect, into a recipient insect. 【0024】 In one embodiment, an insect wound treatment kit is provided for use in a method of treating wounds on the epidermis of insects according to the Disclosure, comprising a biocompatible material having a sheet shape. In another embodiment, an insect wound treatment kit is provided for use in an insect ovary transplantation method according to the Disclosure, comprising a biocompatible material having a sheet shape. 【0025】 In one embodiment, the Disclosure provides an insect wound treatment kit comprising a sheet-shaped biocompatible material and instructions. The instructions may describe applying the sheet-shaped biocompatible material to the insect wound. In another embodiment, the Disclosure provides an insect ovary transplantation kit comprising a sheet-shaped biocompatible material and instructions. The instructions may describe applying the sheet-shaped biocompatible material to the insect wound. 【0026】 In the kit of this disclosure, the biocompatible material in sheet form may include materials selected from the group consisting of anti-drying paper, paraffin paper, woven fabric, nonwoven fabric, paper, natural resin, synthetic resin, and fibroin film. In the kit of this disclosure, the insect may be a Lepidoptera or Coleoptera insect. In the kit of this disclosure, the insect may be an insect in the larval or pupal stage. The paper used does not necessarily have to be anti-drying paper; any biocompatible, sheet-like paper of sufficient size to cover a wound may be used. Furthermore, this technique can be used not only to repair wounds caused by gonad transplantation but also to seal cuts of about the size of a single segment in insect larvae. 【0027】 The method disclosed herein eliminates the need to heat-melt paraffin with a gas burner or heater each time to treat insect injuries, and also eliminates the need to adjust the amount of melted paraffin used. This allows for the treatment of insects with postoperative injuries, such as those from gonad transplantation, through simple operation. Therefore, insect surgery can be performed more easily than with conventional techniques. For example, in one embodiment, the survival rate of insects treated by the method disclosed herein may be improved (compared to, for example, untreated insects). In another exemplary embodiment, it is possible to treat a large number of insects in a short time (compared to using melted paraffin). The novel technology disclosed herein does not show a decrease in the survival rate of treated insects (compared to untreated insects or insects using melted paraffin), demonstrating the high usefulness of the method disclosed herein. Furthermore, an advantage of this disclosure is that the operation can be performed at room temperature and does not require special temperature control. Another advantage of this disclosure is that it avoids the odor of heat-melted paraffin that fills laboratories or workrooms when handling paraffin, thus improving the working environment for workers. 【0028】 The present disclosure will be further explained below with reference to examples and comparative examples. However, the present disclosure shall not be construed as being limited to the following examples. [Examples] 【0029】 [Example 1] material and method Silkworms were reared on artificial feed called "Silkmate" (Nippon Nosan Kogyo) at 28°C (up to the 4th instar) or 25°C (after the 5th instar) under a photoperiod of 12 hours of light and 12 hours of darkness. The w1-cs strain (w-cs) was used as the recipient, and the AyFib-431a strain (Tsubota et al., 2014, G3 (Bethesda), 4, 1347-1357) was used as the donor. The AyFib-431a strain is a strain that ubiquitously expresses the red fluorescent protein DsRed. The w-cs strain was bred by crossing a male of the wc strain (Uchino et al., 2008, Insect biochem mol biol, 38, 1165-1173), which has white eggs and diapause characteristics, with a female of the C146 strain, which has a sex-limiting larval marker, and then backcrossing the resulting female with a male of the wc strain more than six times. These lineages are maintained by NARO. 【0030】 Ovarian cryopreservation and transplantation Ovaries with string bodies were collected from the second day of the third instar larvae or the first day of the fourth instar larvae. After being submerged in the cryoprotective agent "Cell Reservoir One (with DMSO)" (Nacalai Tesque) for 30 minutes, the ovaries were frozen at -80°C using the freezing container "BICELL" (Nippon Freezer) and finally stored in a liquid nitrogen tank. Basically, except for the method of sealing the wound, transplantation was performed using the method of Bannno et al. (2013, Cryobiology, 66, 283-287). That is, instead of using the method of using heat-soluble paraffin reported by Bannno et al., we tried a method of using anti-drying paper (Toa, Gifu) or fibroin film made from silkworm silk on the incision site and placing it on top of the silkworm wound. 【0031】 Screening of transgenic silkworms To detect offspring derived from donor ovaries expressing the fluorescent protein DsRed, an Olympus SZX16 was used. Images were taken using an Olympus DP71 equipped with a DsRed filter (excitation: 530 / 50 nm, emission: 575 nm). 【0032】 Results and Discussion Dryproof paper Conventional methods involved covering surgical incisions with molten paraffin (Mochida et al., 2003, Cryobiology, 46, 153-160). However, using too much molten paraffin resulted in a low survival rate, making it difficult to effectively seal the incision. Therefore, the inventors investigated a new method for sealing the incision. As shown in Figure 1, instead of the conventional method (Figure 1A), a simple covering method using moisture-proof paper (also called anti-drying paper) was attempted (Figure 1B). In the case of paraffin, since its melting point is approximately 56-58°C, solid paraffin was first heated to approximately 110°C to melt it. Then, a small amount was taken with a spatula and placed on the adult insect's wound while allowing it to cool appropriately before it hardened. In the case of moisture-proof paper, a piece of appropriately sized moisture-proof paper was placed directly on the adult insect's wound. 【0033】 Comparing the survival rates of adult insects using both methods, the average survival rate with molten paraffin was 66.9% (SE 24.8), while the survival rate with anti-drying paper was 73.1% (SE 14.7) (Figure 1C). In other words, the survival rate of adult insects was almost the same with both methods. Furthermore, the number of eggs laid was also examined, but there was no significant difference between these methods (data not shown). Sealing with anti-drying paper is far superior to sealing with molten paraffin in that it is easy to operate and does not require equipment to dissolve paraffin. 【0034】 Furthermore, since good results were obtained when using anti-drying paper, the inventors considered that similar effects could be obtained with other sheet-shaped biocompatible materials, and therefore tried fibroin film as a sheet-shaped biocompatible material. As a result, even when using fibroin film, the survival rate (emergence rate) of surgically treated individuals was equivalent to that of dissolved paraffin (see description below and Figure 5). Dissolved paraffin is difficult to control the amount applied to the wound and the work is complicated, but like anti-drying paper, fibroin film is very easy to use as insect wounds can be treated simply by placing a pre-prepared piece on the wound. In addition, both anti-drying paper and fibroin film are sheet-shaped biocompatible materials, and therefore, it is thought that similar effects can be obtained when using other sheet-shaped biocompatible materials, such as paraffin paper, woven fabric, nonwoven fabric, paper, biocompatible natural resin, and biocompatible synthetic resin. However, the materials disclosed herein are not limited to these. 【0035】 Ovarian transplant To expand the opportunities for reproduction from frozen ovaries, the AyFib-431a strain was used as a donor and the w-cs strain as a recipient. One donor ovary was transplanted into a recipient larva (whose own ovary had been removed) (Figure 2A). The resulting adult worms were mated with w-cs adults, and the eggs of their offspring were collected. Donor-derived eggs could be detected by checking for DsRed expression (Figure 2B). 【0036】 Figure 3 shows a photograph illustrating the scale of the anti-drying paper. Figure 4 shows a photograph of a silkworm with a wound covered with paraffin or anti-drying paper. 【0037】 Table 1 shows the results of comparing survival rates and reproductive capacity using both methods. 【0038】 [Table 1] 【0039】 There was no significant difference in survival rates between live and frozen ovaries. In the case of frozen ovaries, using only one ovary from the frozen stock, rather than two, yielded results comparable to those of live ovaries, meaning that using only one ovary provides two opportunities for reproduction. Therefore, our novel method can be said to be more effective than conventional methods. 【0040】 Furthermore, the fertility of eggs from donor ovaries was examined using both transplantation methods. As shown in Table 1, the average number of fertilized eggs from one ovarian transplant (B) was approximately the same as the average number of fertilized eggs from two ovarian transplants using live eggs (A) (Experiments 1 and 2 in Table 1). Previous reports (Banno et al., 2013, Cryobiology, 66, 283-287) showed that transplanting third-instar larvae as recipients was more effective than transplanting fourth-instar larvae. Therefore, similar experiments were conducted in this study using both third-instar and fourth-instar larvae. As a result, no significant difference was observed between third-instar and fourth-instar larvae in our experiment (Experiments 3 and 4 in Table 1). 【0041】 Furthermore, Figure 5 shows the survival rates when coated with dissolved paraffin, fibroin film, and anti-drying paper. While the survival rates were relatively high in all methods, the method disclosed herein showed superior survival rates compared to conventional methods despite its simple operation. 【0042】 [Example 2] Survival rate of individuals with a single segmental injury Non-patent document 3 describes not closing the incision wound in the larval epidermis (untreated) when transplanting ladybug larval ovaries. Therefore, we also investigated the case of not closing the incision wound (untreated) in silkworms. 【0043】 The results are shown in Figure 6. In the group where no treatment was performed (the wound was left untreated), the survival rate was 20%, while in the group where anti-drying paper was used, the survival rate was 60%. This survival rate was almost the same as that of the group where dissolved paraffin was used. For a single-segment cut, simply placing anti-drying paper on the wound showed a wound healing effect or wound sealing effect comparable to that of dissolved paraffin. 【0044】 [Comparative Example] As a means of treating injuries to insect body segments, we also tried (biocompatible) adhesives and bandages, but they were difficult to handle, such as the bandages sticking to tweezers, and we were unable to effectively treat the injuries. In addition, the success rate of the experiments was low, and reproducibility was poor. 【0045】 To effectively propagate silkworms from a limited number of cryopreserved samples, a simple method and frequent opportunities for propagation (regeneration) are essential. The method using anti-drying paper or film of the present invention was effective for ovarian transplantation of transgenic silkworms. Furthermore, the ovarian transplantation method using a single ovary of the present invention was also effective for ovarian transplantation of transgenic silkworms. [Industrial applicability] 【0046】 The disclosures of documents cited herein are not considered to be related to the patentability of the present invention, but are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated herein by reference in the same way that each individual document is specifically and individually indicated to be incorporated by reference. 【0047】 Based on the foregoing, various modifications are possible to the embodiments of the Disclosure described herein without departing from the technical idea or spirit of the Disclosure, and such modifications are also incorporated into the Disclosure.

Claims

[Claim 1] A method for transplanting silkworm ovaries in the larval or pupal stage, (i) The process of surgically incising the epidermis of the recipient silkworm, (ii) The process of transplanting the ovaries taken from the donor silkworm into the incised recipient silkworm, (iii) A step of applying a sheet-shaped biocompatible material selected from the group consisting of anti-drying paper, paraffin paper, woven fabric, nonwoven fabric, paper, natural resin, synthetic resin, and fibroin film to the wound on the epidermis of the incised recipient silkworm. The method, including the method described above. [Claim 2] A method for transplanting silkworm ovaries according to claim 1, wherein only one ovary is transplanted as the ovary taken from a donor silkworm.

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