龟科害虫的大规模饲养与分子研究
Summary
本方案描述了实验室中Tortricid pest insects的饲养方法。区分昆虫性别和提取核酸以进行高通量测序的程序是使用两种tortricid害虫建立的。
Abstract
龟蛾科（鳞翅目），俗称枸杞蛾或卷叶蛾，包括许多农业和林业害虫，造成严重的农业损失。为了了解这种害虫飞蛾的生物学，基本技术的需求量很大。在这里，使用两种茶托特里克斯， Homona magnanima 和 Adoxoophyes honmai （鳞翅目：Tortricidae）开发了大规模饲养，观察和分子研究的方法。昆虫是用切片的人工饲料大规模饲养的，并通过考虑其生物学特征通过近亲繁殖来维持100多年。昆虫具有各种性别二态性;因此，在发育阶段很难区分性别，这阻碍了随后的测定。本研究强调，通过观察睾丸或乳酸醋酸睾丸染色来观察雌性特异性W染色体，可以确定tortricids幼虫的性别。此外，使用性别确定方法，本研究能够从性别确定的胚胎中提取核酸并应用于高通量测序。这些提示适用于其他害虫昆虫，将有助于进一步的形态学和遗传研究。
Introduction
鳞翅目昆虫占所有描述的生物物种10以上1，某些分类群物种对植物造成严重损害和严重农业损失2，3。虽然已经使用像蚕 Bombyx mori4，5这样的模型昆虫进行了分子和遗传研究，但害虫昆虫仍未得到调查，部分原因是饲养和处理6，7的困难。因此，有必要进行基础研究和技术来了解这种非模型害虫昆虫的生物学。
龟蛾科（鳞翅目），俗称桔梗蛾或卷叶蛾，包括许多农林害虫8。在昆虫分类群中，东方茶托特里克斯 Homona magnanima Diakonoff和夏季水果托特里克斯 Adoxophyes honmai Yasuda是严重的多食性害虫，已知会破坏东亚的茶树7。这两个物种产下扁平和椭圆形的鳞片状卵簇（或卵团），由由母体分泌物覆盖的薄，柔软和脆弱的卵组成。虽然胚胎发生阶段对昆虫发育和性别决定至关重要9，但卵的结构阻止了进一步分析昆虫的生物学。克服困难，进一步研究害虫排卵这种复杂的卵块是重要的。
在这里，为了了解Tortricids的生物学，已经使用 A. honmai 和 H. magnanima开发了大规模饲养，观察和分子研究的方法。首先，大规模饲养方法通过近交将两只托里克斯维持在100代以上。从连接的鳞片状卵块中分离卵有助于使用先前从苍蝇中使用的技术开发的碱性和有机溶剂对托里克斯的胚胎发生观察10。此外，本研究通过使用乳酸 – 醋酸睾丸激素11开发鳞翅目雌性性染色质染色质的染色方法，建立了小胚胎的性别歧视。通过结合这些方法，从性别决定的胚胎中提取高质量和大量的核酸，否则很难建立6。提取的RNA用于下一代测序。总的来说，这里介绍的方法适用于其他鳞翅目昆虫和其他昆虫分类群。
Protocol
1. 昆虫收集和大规模饲养 按照先前发表的参考文献8，12从田地中收集Tortricid昆虫。注意：从受损的茶叶中收集H. magnanima 和 A. honmai 幼虫（图1A）;使用4 W便携式紫外线（365nm波长，见 材料表， 图1B）吸引成年人。 将收集的幼虫（图1C</stro…
Representative Results
建立东道主线及其维护田间收集的幼虫的生存能力因田间位置，季节和饲养条件而异（例如，台湾桃园90%的生存能力，如Arai等人12所示）。大约30%-50%的货币对将像往常一样产生下一代。对于 H. magnanima 和 A. honmai，母系已经通过近交维持了100多代。 形态学观察和性别决定用氢氧化钾（3或5M KOH）或次氯酸钠（1.2%Cl<su…
Discussion
Tortricid包括几种农业和林业害虫;本研究提出了几代人饲养Tortrix的方法，观察昆虫的胚胎发生和性别，并使用成熟的胚胎进行分子分析。
害虫研究的障碍之一是建立饲养方法。特别是，近亲繁殖有时会对物种的适应性产生负面影响。近交系的适应性下降，称为近亲繁殖抑制，已在各种植物和动物中广泛观察到，包括昆虫15，16，<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望感谢日本科学促进会（JSPS）青年科学家研究奖学金的支持[拨款号19J13123和21J00895]。
Materials
| 1/2 ounce cup | FP CHUPA | CP070009 | insect rearing; https://www.askul.co.jp/p/6010417/ |
| 1/2 ounce cup lid | FP CHUPA | CP070011 | insect rearing; https://www.askul.co.jp/p/6010434/?int_id=recom_DtTogether |
| 99.7% acetic acid | FUJIFILM Wako Chemicals Co., Osaka, Japan | 36289 | fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01ALF036289.html |
| Agilent 2100 Bioanalyzer | Agilent Technologies | not shown | Nucleic acids quantification; https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument |
| Agilent RNA6000 nano kit | Agilent Technologies | 5067-1511 | Nucleic acids quantification; https://www.agilent.com/cs/library/usermanuals/Public/G2938-90034_RNA6000Nano_KG |
| benzalkonium chloride solution | Nihon Pharmaceutical Co., Ltd | No.4987123116046 | Sterilization; https://www.nihon-pharm.co.jp/consumer/products/disinfection.html |
| Cotton | AOUME | 8-1611-02 | insect rearing; https://item.rakuten.co.jp/athlete-med/10006937/?scid=af_pc_etc&sc2id=af_113_0_1 |
| DAPI solution | Dojindo, Tokyo, Japan | 340-07971 | stainings; https://www.dojindo.co.jp/products/D523/ |
| Disodium Hydrogenphosphate | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | Na2HPO4; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0286.html |
| dsDNA HS quantification kit | Invitrogen | Q33231 | Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q33230?SID=srch-srp-Q33230 |
| Econospin RNA II column | Epoch Life Science Inc. | EP-11201 | RNA extraction; http://www.epochlifescience.com/Product/SpinColumn/minispin.aspx |
| Ethanol | FUJIFILM Wako Chemicals Co., Osaka, Japan | 4.98748E+12 | fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0105-0045.html |
| Ethylenediamine-N,N,N',N'-tetraacetic Acid Tetrasodium Salt Tetrahydrate (4NA) | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | Cell lysis buffer (EDTA); https://labchem-wako.fujifilm.com/jp/product/detail/W01T02N003.html |
| Glassine paper | HEIKO | 2100010 | insect rearing; https://www.monotaro.com/p/8927/0964/?utm_id=g_pla& utm_medium=cpc&utm_source= Adw |
| heat block WSC-2620 PowerBLOCK | ATTO, Tokyo, Japan | 4002620 | incubation; https://www.attoeng.site/ |
| heptane | FUJIFILM Wako Chemicals Co., Osaka, Japan | 4.98748E+12 | fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0108-0015.html |
| INSECTA LF | Nosan Co., Ltd | not shown | Artificial diet; https://www.nosan.co.jp/business/fodder/ist.htm |
| ISOGENII | Nippon Gene | 311-07361 | RNA extraction; https://www.nippongene.com/siyaku/product/extraction/isogen2/isogen2.html |
| isopropanol | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | nucleic acids extraction; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0232-0004.html |
| Lactic acid | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | Stainings; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0112-0005.html |
| methanol | FUJIFILM Wako Chemicals Co., Osaka, Japan | 4.98748E+12 | fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0113-0182.html |
| MSV-3500 vortex | Biosan | BS-010210-TAK | Voltex mixer; https://biosan.lv/products/-msv-3500-multi-speed-vortex/ |
| Nano Photometer NP 80 | Implen | not shown | Nucleic acids quantification; https://www.implen.de/product-page/implen-nanophotometer-np80-microvolume-cuvette-spectrophotometer/tech-specs/ |
| Natural pack wide | Inomata chemical | 1859 | insect rearing; https://www.monotaro.com/g/03035766/?t.q=%E3%83%8A%E3%83%81%E3%83%A5%E3% 83%A9%E3%83%AB%E3%83%91% E3%83%83%E3%82%AF%E3%83% AF%E3%82%A4%E3%83%89 |
| NEBNext Ultra II RNA Library Prep Kit for Illumina | New England BioLabs | E7770S | Library preparation; https://www.nebj.jp/products/detail/2039 |
| orcein | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | Stainings; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0115-0094.html |
| Paraformaldehyde | FUJIFILM Wako Chemicals Co. | 160-16061 | fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-1606.html |
| Polyoxyethylene(20) Sorbitan Monolaurate | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | Tween-20; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-2121.html |
| Portable UV Black Light (4W, 365nm wavelength) | Southwalker Co., Ltd., Kanagawa, Japan | not shown | Insect collection; http://www.southwalker.com/shopping/?pid=1364614057-467328 |
| Potassium Chloride | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | KCl; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-0354.html |
| Potassium Dihydrogen Phosphate | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | KH2PO4; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-0424.html |
| ProLong Diamond Antifade Mountant | Invitrogen, MA, USA | P36965 | antifade; https://www.thermofisher.com/order/catalog/product/P36965 |
| Proteinase K Solution | Merck | 71049-4CN | DNA extraction; https://www.merckmillipore.com/JP/ja/product/Proteinase-K-Solution-600-mAU-ml,EMD_BIO-71049 |
| protein precipitation solution | Qiagen | 158912 | DNA extraction; https://www.qiagen.com/us/products/discovery-and-translational-research/lab-essentials/buffers-reagents/puregene-accessories/?cmpid=PC_DA_NON_ BIOCOMPARE_ProductListing_ 0121_RD_MarketPlace_ProductC |
| Qubit V4 | Invitrogen | Q33238 | Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q33238 |
| rifampicin | FUJIFILM Wako Chemicals Co., Osaka, Japan | 4.98748E+12 | Sterilization; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0118-0100.html |
| RNA HS quantification kit | Invitrogen | Q32855 | Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q32852 |
| RNase solution | Nippon Gene | 313-01461 | RNA extraction; https://www.nippongene.com/siyaku/product/modifying-enzymes/rnase-a/rnase-s.html |
| Silk Mate 2S | Nosan Co., Ltd | not shown | Artificial diet; https://www.nosan.co.jp/business/fodder/ist.htm |
| Sodium Chloride | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | NaCl; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0166.html |
| Sodium Dodecyl Sulfate | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | Cell lysis buffer (SDS); https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-1398.html |
| sodium hypochlorite aqueous solution | FUJIFILM Wako Chemicals Co., Osaka, Japan | 4.98748E+12 | egg separation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0220.html |
| Tetracycline Hydrochloride | FUJIFILM Wako Chemicals Co., Osaka, Japan | 4.98748E+12 | Sterilization; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0120-1656.html |
| Tris-HCl | FUJIFILM Wako Chemicals Co. | 4.98748E+12 | Cell lysis buffer; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0120-1536.html |
| ultra-pure distilled water | Invitrogen | 10977023 | RNA extraction; https://www.thermofisher.com/order/catalog/product/10977015 |
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Cite This Article
Arai, H., Ishitsubo, Y., Nakai, M., Inoue, M. N. Mass-Rearing and Molecular Studies in Tortricidae Pest Insects. J. Vis. Exp. (181), e63737, doi:10.3791/63737 (2022).