JP2012044889A - Method of artificially incubating dormant egg of silk worm by dimethyl sulfoxide (dmso), and method of introducing chemical material into egg - Google Patents

Method of artificially incubating dormant egg of silk worm by dimethyl sulfoxide (dmso), and method of introducing chemical material into egg Download PDF

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JP2012044889A
JP2012044889A JP2010188018A JP2010188018A JP2012044889A JP 2012044889 A JP2012044889 A JP 2012044889A JP 2010188018 A JP2010188018 A JP 2010188018A JP 2010188018 A JP2010188018 A JP 2010188018A JP 2012044889 A JP2012044889 A JP 2012044889A
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egg
dmso
dormant
dimethyl sulfoxide
silkworm
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JP5678518B2 (en
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Hiroshi Sawada
博司 澤田
Keisuke Mase
啓介 間瀬
Takayuki Yamamoto
貴之 山本
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Nihon University
Kitasato Institute
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Abstract

PROBLEM TO BE SOLVED: To provide a method of efficiently artificially incubating a dormant egg of silk worm with a simple operation, and to provide a method of introducing a chemical material.SOLUTION: The method of incubating the dormant egg of silk worm is characterized by treating the dormant egg of silk worm with solution containing dimethyl sulfoxide.

Description

本発明は、カイコ休眠卵の効率的な人工孵化方法及びカイコ休眠卵への簡便な化学物質の導入方法に関する。   The present invention relates to an efficient artificial hatching method for silkworm dormant eggs and a simple chemical substance introduction method for silkworm dormant eggs.

カイコは、完全変態の昆虫であり、産卵後休眠卵の状態で越冬し、数ヶ月後に孵化し、１齢幼虫から５齢幼虫で脱皮した後、蛹（さなぎ）になり、その後羽化する。５齢幼虫から蛹化する際に繭を作る。この繭が絹糸である。   Silkworms are completely transformed insects that overwinter in the state of dormant eggs after spawning, hatch after several months, molt from 1st instar larvae to 5th instar larvae, then become pupae, and then emerge. Make cocoons when hatching from 5th instar larvae. This cocoon is silk thread.

休眠卵の場合産卵から孵化までの期間は通常数ヶ月ある。この間は、日本においては冬なので、幼虫のえさになる桑の葉がないため、問題がない。しかし、冬の間であっても桑の葉が存在する地域では、この期間は、養蚕ができない。そこで、インド、タイ、ベトナム等では休眠期のないカイコ（非休眠系統）が用いられているが、これらの系統では生産性および品質的にも劣っており、しかも一年中カイコ飼育すると、消毒期間が設けられないことによる病気の発生等の問題が発生している（特許文献１）。
従って、休眠性のある系統を用い、かつその期間を自由に制御できれば飼育計画も立て易くなり、生糸の生産性の向上や、品質低下の防止も期待できる。これらの観点から、休眠卵に何らかの処理をして人工的に孵化させる手段が必要とされている。 In the case of dormant eggs, the period from egg laying to hatching is usually several months. During this time, it is winter in Japan, so there is no problem because there are no mulberry leaves to feed on larvae. However, even in winter, sericulture is not possible during this period in areas where mulberry leaves are present. In India, Thailand, Vietnam, etc., silkworms with no dormancy (non-dormant lines) are used, but these lines are inferior in productivity and quality. Problems such as the occurrence of diseases due to the absence of a period have occurred (Patent Document 1).
Therefore, if a dormant line is used and its period can be controlled freely, it is easy to make a breeding plan, and improvement of raw silk productivity and prevention of quality degradation can be expected. From these viewpoints, there is a need for means for artificially hatching dormant eggs by performing some kind of treatment.

このような人工孵化手段としては、従来から休眠卵を塩酸水溶液に浸漬する、いわゆる浸酸処理法が知られている（特許文献１、２）。また、休眠卵に対して品質改良等の目的で化学物質を導入する手段としては、マイクロインジェクション法が用いられている（特許文献１、２）。   As such artificial hatching means, a so-called soaking treatment method in which a dormant egg is soaked in an aqueous hydrochloric acid solution has been known (Patent Documents 1 and 2). As a means for introducing a chemical substance into a dormant egg for the purpose of quality improvement or the like, a microinjection method is used (Patent Documents 1 and 2).

特開２００２−６８９１６号公報JP 2002-68916 A 特開２００３−８８２７４号公報JP 2003-88274 A

しかしながら、前述の浸酸処理法においては、浸酸処理の際に台紙から休眠卵の脱落を防ぐための前処理としてホルマリン処理や塩酸の比重を調整する等の操作が必要である。これらの薬品は劇物であることから、安全環境対策が必要となり、その経費や労力の負担軽減が求められている。
また、マイクロインジェクション法に関しては、カイコ卵は卵殻が厚く丈夫なため、卵殻を傷つけずに化学物質の導入を行うことは困難であった。 However, in the above-described immersion treatment method, operations such as formalin treatment and adjustment of the specific gravity of hydrochloric acid are necessary as pretreatment for preventing falling-off of dormant eggs from the mount during the immersion treatment. Since these chemicals are deleterious substances, it is necessary to take safe environmental measures, and there is a demand for reducing the cost and labor.
Regarding the microinjection method, silkworm eggs have thick and strong eggshells, and it was difficult to introduce chemical substances without damaging eggshells.

従って、本発明の課題は、簡便な操作で効率良く、カイコ休眠卵を人工的に孵化できる方法及び化学物質導入方法を提供することにある。   Accordingly, an object of the present invention is to provide a method and a chemical substance introduction method capable of artificially hatching silkworm dormant eggs with a simple operation and efficiently.

そこで本発明者は、休眠卵に対して種々の成分を接触させて休眠解除効果について検討してきたところ、休眠卵をジメチルスルホキシド含有液で処理すれば極めて高い確率で休眠が解除され、人工孵化できることを見出した。また、ジメチルスルホキシド含有液に対象化学物質を共存させれば、当該対象化学物質が効率良く人工孵化したカイコ卵中に導入されることも見出した。   Therefore, the present inventor has examined the dormancy release effect by bringing various components into contact with the dormant egg. If the dormant egg is treated with a dimethyl sulfoxide-containing solution, the dormancy is released with a very high probability and can be artificially hatched. I found. It was also found that if a target chemical substance is allowed to coexist in a dimethyl sulfoxide-containing liquid, the target chemical substance is efficiently introduced into an artificially hatched silkworm egg.

すなわち、本発明は、カイコ休眠卵をジメチルスルホキシド含有液で処理することを特徴とするカイコ休眠卵の人工孵化方法を提供するものである。
また、本発明は、カイコ休眠卵を、対象化学物質及びジメチルスルホキシドの含有液で処理することを特徴とするカイコ卵への対象化学物質導入方法を提供するものである。 That is, the present invention provides an artificial hatching method for silkworm diapause eggs characterized by treating silkworm diapause eggs with a dimethyl sulfoxide-containing solution.
The present invention also provides a method for introducing a target chemical substance into a silkworm egg, wherein the silkworm dormant egg is treated with a liquid containing the target chemical substance and dimethyl sulfoxide.

本発明の人工孵化方法によれば、産卵されたカイコ休眠卵をジメチルスルホキシド（ＤＭＳＯ）含有液に浸漬、又は当該液を塗布するだけで簡便かつ効率良く、休眠を解除でき人工孵化が可能であり、安全性も高い。またＤＭＳＯによる人工孵化は、産下後２４時間以内の時期に可能であり、従来の塩酸処理方法とは作用機序が相違する。また、本発明の化学物質導入方法によれば、高価な装置や熟練した技術が必要なく、効率良く、遺伝子や酵素阻害剤等の化学物質をカイコ卵に導入可能である。   According to the artificial hatching method of the present invention, it is possible to release the dormancy simply and efficiently by simply immersing the laying silkworm dormant egg in a dimethyl sulfoxide (DMSO) -containing solution or applying the solution, and artificial hatching is possible. High safety. Artificial hatching with DMSO is possible within 24 hours after delivery, and the mechanism of action is different from conventional hydrochloric acid treatment methods. Moreover, according to the chemical substance introduction method of the present invention, it is possible to efficiently introduce chemical substances such as genes and enzyme inhibitors into silkworm eggs without the need for expensive equipment and skilled techniques.

種々の物質処理による孵化率を示す。The hatching rate by various substance treatments is shown. 産下後ＤＭＳＯ処理までの時間と孵化率との関係を示す。The relationship between postpartum DMSO processing time and hatching rate is shown. 孵化率とＤＭＳＯ濃度との関係を示す。The relationship between hatching rate and DMSO concentration is shown. ＤＭＳＯ浸漬時間と孵化率との関係を示す。The relationship between DMSO immersion time and hatching rate is shown.

本発明の人工孵化方法は、カイコ休眠卵をＤＭＳＯ含有液で処理することを特徴とする。
用いられるカイコ休眠卵は、休眠系統のカイコの休眠卵であればよい。カイコの種類としては、Bombyx属に属するもの、例えばカイコガ以外に、クワコ等が挙げられる。 The artificial hatching method of the present invention is characterized in that silkworm dormant eggs are treated with a DMSO-containing solution.
The silkworm diapause egg used may be a diapause egg of a dormant strain silkworm. Examples of silkworm types include those belonging to the genus Bombyx, such as silkworms in addition to silkworms.

ＤＭＳＯ含有液による処理は、産下後２４時間以内のカイコ休眠卵に対して行うのが、孵化率の点から好ましい。産下後２４時間を超える休眠卵に対してＤＭＳＯ含有液処理しても十分な孵化率は得られない。一方、従来法の塩酸処理は産下後６０時間程度まで孵化可能である。このことから、本発明方法と塩酸処理による人工孵化の作用機序は相違するものと考えられる。   The treatment with the DMSO-containing solution is preferably performed on silkworm dormant eggs within 24 hours after delivery from the viewpoint of hatching rate. Even if a DMSO-containing liquid treatment is performed on a dormant egg exceeding 24 hours after delivery, a sufficient hatching rate cannot be obtained. On the other hand, the conventional hydrochloric acid treatment can be hatched for up to about 60 hours after delivery. From this, it is thought that the mechanism of action of artificial hatching by the method of the present invention and hydrochloric acid treatment is different.

用いられるＤＭＳＯ含有液としては、ＤＭＳＯ水溶液又はＤＭＳＯ液が挙げられる。ＤＭＳＯ含有液中のＤＭＳＯ濃度は、孵化率の点から３０〜１００質量％が良く、特に７５〜１００質量％が好ましい。   Examples of the DMSO-containing liquid used include a DMSO aqueous solution or a DMSO liquid. The DMSO concentration in the DMSO-containing liquid is preferably from 30 to 100% by mass, particularly preferably from 75 to 100% by mass, from the viewpoint of hatchability.

ＤＭＳＯ含有液による処理の手段としては、休眠卵にＤＭＳＯ含有液が５分以上接触していればよく、例えば休眠卵をＤＭＳＯ含有液に浸漬する、休眠卵にＤＭＳＯ含有液を塗布する等の手段が挙げられる。ＤＭＳＯ含有液の使用量は、休眠卵１重量部あたり２重量部以上であればよく、特に５〜１０重量部であるのが好ましい。   The treatment with the DMSO-containing liquid may be any treatment as long as the DMSO-containing liquid is in contact with the dormant egg for 5 minutes or longer. For example, the dormant egg is immersed in the DMSO-containing liquid, or the DMSO-containing liquid is applied to the dormant egg. Is mentioned. The amount of DMSO-containing liquid used may be 2 parts by weight or more per 1 part by weight of the dormant egg, and is preferably 5 to 10 parts by weight.

ＤＭＳＯ含有液による休眠卵の処理時間（接触時間）は、５分以上であればよく、孵化率の点から１０〜１２０分が好ましく、特に３０〜４５分が好ましい。   The treatment time (contact time) of the dormant egg with the DMSO-containing solution may be 5 minutes or more, preferably 10 to 120 minutes, particularly preferably 30 to 45 minutes from the viewpoint of hatching rate.

ＤＭＳＯ含有液による処理は、室温、例えば１５〜３０℃の温度条件で行うことができ、特に２０〜２５℃が好ましい。   The treatment with the DMSO-containing liquid can be performed at room temperature, for example, at a temperature of 15 to 30 ° C., and 20 to 25 ° C. is particularly preferable.

上記の休眠解除により人工的に孵化したカイコは、通常に１齢幼虫から５齢幼虫へと脱皮し、繭を形成し、変態する。従って、人工孵化後は、常法に従って飼育すればよい。   Silkworms artificially hatched by the release of dormancy usually molt from 1st instar larvae to 5th instar larvae, form cocoons and transform. Therefore, after artificial hatching, it may be raised according to a conventional method.

前述の人工孵化方法において、ＤＭＳＯ含有液中に対象化学物質を含有させておき、前記と同様の処理をすれば、当該対象化学物質をカイコ卵に効率良く導入することができる。用いられる対象化学物質としては、ＤＭＳＯ含有液に可溶性な化学物質全てであり、例えば外来遺伝子、変異誘発性物質、各種酵素阻害剤等が挙げられる。当該化学物質導入方法における処理時期、処理時間、処理量、処理温度等は前記の人工孵化方法と同様である。   In the aforementioned artificial hatching method, if the target chemical substance is contained in the DMSO-containing liquid and the same treatment as described above is performed, the target chemical substance can be efficiently introduced into the silkworm egg. The target chemical substances used are all chemical substances that are soluble in the DMSO-containing liquid, and examples thereof include foreign genes, mutagenic substances, various enzyme inhibitors, and the like. The treatment time, treatment time, treatment amount, treatment temperature, etc. in the chemical substance introduction method are the same as in the artificial hatching method.

対象化学物質のＤＭＳＯ含有液中の濃度は、特に限定されず、カイコ卵の孵化率に影響を及ぼす最低濃度から飽和溶液までであり、例えば０．００１〜６０質量％とすることができる。   The concentration of the target chemical substance in the DMSO-containing liquid is not particularly limited, and is from the lowest concentration that affects the hatching rate of silkworm eggs to a saturated solution, and can be, for example, 0.001 to 60% by mass.

次に実施例を挙げて、本発明をさらに詳細に説明するが、本発明はこれら実施例に限定されるものではない。   EXAMPLES Next, although an Example is given and this invention is demonstrated still in detail, this invention is not limited to these Examples.

実施例１
産下後２０時間のカイコ二化性系統（大造種）の休眠卵１ｇに対し、２２％塩酸、７５％ＤＭＳＯ水溶液、７５％ジメチルホルムアミド（ＤＭＦ）水溶液、及び７５％ジメチルスルフィド（ＤＭＳ）水溶液を、それぞれ１０ｍＬずつ添加し、室温下４５分間浸漬した。その後、孵化した幼虫を数えることにより孵化率を測定した。
結果を図１に示す。図１から明らかなように、ＤＭＳＯ処理により塩酸処理とほぼ同じ効率で休眠が解除され、人工孵化することが判明した。ＤＭＳＯの類似化合物であるＤＭＦやＤＭＳではほとんど孵化しなかったことから、本発明の効果はＤＭＳＯ特異的であると考えられる。 Example 1
22 g hydrochloric acid, 75% DMSO aqueous solution, 75% dimethylformamide (DMF) aqueous solution, and 75% dimethyl sulfide (DMS) aqueous solution were added to 1 g of the diapause eggs of the silkworm binarizing strain (large artificial species) 20 hours after delivery. 10 mL each was added and immersed for 45 minutes at room temperature. Thereafter, the hatching rate was measured by counting the hatched larvae.
The results are shown in FIG. As is clear from FIG. 1, it was found that dormancy was released by DMSO treatment with almost the same efficiency as hydrochloric acid treatment, and artificial hatching occurred. Since DMF and DMS, which are similar compounds of DMSO, hardly hatched, the effect of the present invention is considered to be DMSO specific.

実施例２
産下後０〜７２時間の大造種の休眠卵１ｇに対し、１００％ＤＭＳＯ水溶液を１０ｍＬ添加し、室温下３０分間浸漬した。その後、実施例１と同様にして孵化率を測定した。
結果を図２に示す。図２に示すように、産下後２４時間以内の休眠卵に対するＤＭＳＯ処理では高率で孵化したが、産下後２４時間経過後にＤＭＳＯ処理すると孵化率は大きく低下した。本発明方法による人工孵化は、休眠の初期過程にのみ作用していることが判明した。 Example 2
10 g of a 100% DMSO aqueous solution was added to 1 g of a large-sized diapause egg of 0 to 72 hours after delivery and immersed for 30 minutes at room temperature. Thereafter, the hatching rate was measured in the same manner as in Example 1.
The results are shown in FIG. As shown in FIG. 2, the DMSO treatment on dormant eggs within 24 hours after delivery hatched at a high rate, but the hatching rate significantly decreased when DMSO treatment was performed 24 hours after delivery. It has been found that artificial hatching by the method of the present invention acts only on the initial process of dormancy.

実施例３
産下後１２時間の大造種の休眠卵１ｇに対し、０〜１００％ＤＭＳＯ水溶液を、それぞれ１０ｍＬずつ添加し、室温下３０分間浸漬した。その後、実施例１と同様にして孵化率を測定した。なお、同様の操作を非休眠卵に対しても行った。
結果を図３に示す。図３に示すように、休眠卵（Ｄｉａｐａｕｓｅ）では、ＤＭＳＯ濃度が５０質量％以上、さらに７５質量％以上、特に１００質量％で孵化率が向上した。一方、非休眠卵（Ｎｏｎ−ｄｉａｐａｕｓｅ）ではＤＭＳＯ濃度が高くなると逆に孵化率は低下した。 Example 3
10 mL of a 0-100% DMSO aqueous solution was added to each 1 g of large-sized diapause eggs 12 hours after delivery and immersed for 30 minutes at room temperature. Thereafter, the hatching rate was measured in the same manner as in Example 1. The same operation was performed for non-dormant eggs.
The results are shown in FIG. As shown in FIG. 3, in the dormant egg (Diapause), the hatching rate was improved when the DMSO concentration was 50 mass% or more, further 75 mass% or more, particularly 100 mass%. On the other hand, in the case of non-diapause eggs, the hatching rate decreased with increasing DMSO concentration.

実施例４
産下後１２時間の大造種の休眠卵１ｇに対し、１００％ＤＭＳＯ水溶液を１０ｍＬ添加し、室温下０〜１２０分間浸漬した。その後、実施例１同様にして孵化率を測定した。 結果を図４に示す。図４に示すように、ＤＭＳＯ浸漬時間は５分以上、さらに１５〜１２０分、特に３０〜６０分で高い孵化率が得られた。 Example 4
10 g of a 100% DMSO aqueous solution was added to 1 g of a large-sized dormant egg 12 hours after delivery, and immersed for 0 to 120 minutes at room temperature. Thereafter, the hatching rate was measured in the same manner as in Example 1. The results are shown in FIG. As shown in FIG. 4, a high hatching rate was obtained when the DMSO immersion time was 5 minutes or longer, further 15 to 120 minutes, particularly 30 to 60 minutes.

Claims (7)

カイコ休眠卵をジメチルスルホキシド含有液で処理することを特徴とするカイコ休眠卵の人工孵化方法。   A method for artificially hatching silkworm dormant eggs, comprising treating silkworm dormant eggs with a solution containing dimethyl sulfoxide. ジメチルスルホキシド含有液処理が、産下後２４時間以内である請求項１記載の人工孵化方法。   The artificial hatching method according to claim 1, wherein the dimethyl sulfoxide-containing liquid treatment is within 24 hours after delivery. ジメチルスルホキシド含有液中のジメチルスルホキシド濃度が５０〜１００質量％である請求項１又は２記載の人工孵化方法。   The artificial hatching method according to claim 1 or 2, wherein the concentration of dimethyl sulfoxide in the dimethyl sulfoxide-containing liquid is 50 to 100% by mass. ジメチルスルホキシド含有液による処理時間が１０〜１２０分である請求項１〜３のいずれか１項記載の人工孵化方法。   The artificial hatching method according to any one of claims 1 to 3, wherein the treatment time with the dimethyl sulfoxide-containing liquid is 10 to 120 minutes. カイコ休眠卵を、対象化学物質及びジメチルスルホキシドの含有液で処理することを特徴とするカイコ卵への対象化学物質導入方法。   A method for introducing a target chemical substance into a silkworm egg, characterized by treating a silkworm dormant egg with a liquid containing the target chemical substance and dimethyl sulfoxide. 対象化学物質及びジメチルスルホキシドの含有液処理が、産下後２４時間以内である請求項５記載の対象化学物質導入方法。   The method for introducing a target chemical substance according to claim 5, wherein the treatment with the liquid containing the target chemical substance and dimethyl sulfoxide is within 24 hours after delivery. 対象化学物質及びジメチルスルホキシドの含有液による処理時間が１０〜１２０分である請求項５又は６記載の対象化学物質導入方法。   The method for introducing a target chemical substance according to claim 5 or 6, wherein the treatment time with the liquid containing the target chemical substance and dimethyl sulfoxide is 10 to 120 minutes.
JP2010188018A 2010-08-25 2010-08-25 Method for artificial hatching of silkworm dormant eggs with dimethyl sulfoxide (DMSO) and method for introducing chemical substances into eggs Expired - Fee Related JP5678518B2 (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103704181A (en) * 2013-12-09 2014-04-09 华南农业大学 Comprehensive utilization method of fresh cocoons of silkworms
CN104839100A (en) * 2015-05-24 2015-08-19 梁永宁 Silkworm cultivation method
CN105941350A (en) * 2016-05-19 2016-09-21 苏州大学 Rearing method with mulberry shoots for silkworms of all ages
CN107279075A (en) * 2017-06-28 2017-10-24 合肥市聚丰制丝有限责任公司 A kind of silkworm in spring egg hatching method
CN108477082A (en) * 2018-03-09 2018-09-04 北京中农富通园艺有限公司 The instant termination of diapause of silkworm heat shock and the method for refrigeration delay hatching
JP7475689B2 (en) 2021-02-08 2024-04-30 国立研究開発法人農業・食品産業技術総合研究機構 Method for producing eggs capable of long-term nucleic acid reception

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JPS6468301A (en) * 1987-09-09 1989-03-14 Rikagaku Kenkyusho Freeze-storage of insect ovum
JPH03266927A (en) * 1990-03-15 1991-11-27 Katakura Kogyo Kk Method for artificially hatching egg of silkworm
JP2002068916A (en) * 2000-08-30 2002-03-08 National Institute Of Agrobiological Sciences Inducer to dormant egg of insect, and method for allowing dormant egg to be born
JP2003088274A (en) * 2001-09-19 2003-03-25 National Institute Of Agrobiological Sciences Method for introducing polynucleotide to silkworm producing dormant egg

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Publication number Priority date Publication date Assignee Title
JPS6468301A (en) * 1987-09-09 1989-03-14 Rikagaku Kenkyusho Freeze-storage of insect ovum
JPH03266927A (en) * 1990-03-15 1991-11-27 Katakura Kogyo Kk Method for artificially hatching egg of silkworm
JP2002068916A (en) * 2000-08-30 2002-03-08 National Institute Of Agrobiological Sciences Inducer to dormant egg of insect, and method for allowing dormant egg to be born
JP2003088274A (en) * 2001-09-19 2003-03-25 National Institute Of Agrobiological Sciences Method for introducing polynucleotide to silkworm producing dormant egg

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103704181A (en) * 2013-12-09 2014-04-09 华南农业大学 Comprehensive utilization method of fresh cocoons of silkworms
CN104839100A (en) * 2015-05-24 2015-08-19 梁永宁 Silkworm cultivation method
CN105941350A (en) * 2016-05-19 2016-09-21 苏州大学 Rearing method with mulberry shoots for silkworms of all ages
CN107279075A (en) * 2017-06-28 2017-10-24 合肥市聚丰制丝有限责任公司 A kind of silkworm in spring egg hatching method
CN108477082A (en) * 2018-03-09 2018-09-04 北京中农富通园艺有限公司 The instant termination of diapause of silkworm heat shock and the method for refrigeration delay hatching
CN108477082B (en) * 2018-03-09 2019-12-17 北京中农富通园艺有限公司 Method for instantly relieving diapause by heat shock of silkworms and delaying incubation by refrigeration
JP7475689B2 (en) 2021-02-08 2024-04-30 国立研究開発法人農業・食品産業技術総合研究機構 Method for producing eggs capable of long-term nucleic acid reception

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