Name: mass-rearing and molecular studies in tortricidae pest insects
Uploaded: 2022-03-25T15:00:00
Description: Watch this Scientific Journal Video about Mass-Rearing and Molecular Studies in Tortricidae Pest Insects at JoVE.com

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]。

1. 昆虫收集和大规模饲养
<ol><li>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。</li>
	<li>将收集的幼虫（图1C</stro...

<ol>
	<li>Gaston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+magnitude+of+global+insect+species+richness.">The magnitude of global insect species richness.</a> Conservation Biology. 5 (3), 283-296 (1991).</li><li>Pogue, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+world+revision+of+the+genus+Spodoptera+Guen&#233;e+(Lepidoptera:+Noctuidae).">A world revision of the genus Spodoptera Guen&#233;e (Lepidoptera: Noctuidae).</a> Memoirs of the American Entomological Society. 43, 1 (2002).</li><li>Matsuura, H., Naito, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+cold-hardiness+and+overwintering+of+Spodoptera+litura+F.+(Lepidoptera:+Noctuidae):+VI.+Possible+overwintering+areas+predicted+from+meteorological+data+in+Japan.">Studies on the cold-hardiness and overwintering of Spodoptera litura F. (Lepidoptera: Noctuidae): VI. Possible overwintering areas predicted from meteorological data in Japan.</a> Applied Entomology and Zoology. 32 (1), 167-177 (1997).</li><li>Mita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+construction+of+an+EST+database+for+Bombyx+mori+and+its+application.">The construction of an EST database for Bombyx mori and its application.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (24), 14121-14126 (2003).</li><li>Kawamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-quality+genome+assembly+of+the+silkworm,+Bombyx+mori.">High-quality genome assembly of the silkworm, Bombyx mori.</a> Insect Biochemistry and Molecular Biology. 107, 53-62 (2019).</li><li>Fukui, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+masculinizing+function+of+the+Ostrinia+furnacalis+Masculinizer+gene.">In vivo masculinizing function of the Ostrinia furnacalis Masculinizer gene.</a> Biochemical and Biophysical Research Communications. 503 (3), 1768-1772 (2018).</li><li>Arai, H., Ishitsubo, Y., Nakai, M., Inoue, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+disperse+eggs+from+lepidopteran+scale-like+egg+masses+and+to+observe+embryogenesis.">A simple method to disperse eggs from lepidopteran scale-like egg masses and to observe embryogenesis.</a> Entomological Science. 25 (1), 12497 (2022).</li><li>vander Geest, L. P., Evenhuis, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tortricid pests: their biology, natural enemies and control. , (1991).</li><li>Kiuchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+female-specific+piRNA+is+the+primary+determiner+of+sex+in+the+silkworm.">A single female-specific piRNA is the primary determiner of sex in the silkworm.</a> Nature. 509 (7502), 633-636 (2014).</li><li>Rand, M. D., Kearney, A. L., Dao, J., Clason, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+Drosophila+embryos+for+introduction+of+small+molecules.">Permeabilization of Drosophila embryos for introduction of small molecules.</a> Insect Biochemistry and Molecular Biology. 40 (11), 792-804 (2010).</li><li>Kageyama, D., Traut, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+sex-specific+effects+of+Wolbachia+and+interference+with+the+sex+determination+of+its+host+Ostrinia+scapulalis.">Opposite sex-specific effects of Wolbachia and interference with the sex determination of its host Ostrinia scapulalis.</a> Proceedings of the Royal Society B. 271 (1536), 251-258 (2004).</li><li>Arai, H., Lin, S. R., Nakai, M., Kunimi, Y., Inoue, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closely+related+male-killing+and+nonmale-killing+Wolbachia+strains+in+the+oriental+tea+tortrix+Homona+magnanima.">Closely related male-killing and nonmale-killing Wolbachia strains in the oriental tea tortrix Homona magnanima.</a> Microbial Ecology. 79 (4), 1011-1020 (2020).</li><li>Schalamun, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+MinION:+An+example+of+how+to+establish+long-read+sequencing+in+a+laboratory+using+challenging+plant+tissue+from+Eucalyptus+pauciflora.">Harnessing the MinION: An example of how to establish long-read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora.</a> Molecular Ecology Resources. 19 (1), 77-89 (2019).</li><li>Winnebeck, E. C., Millar, C. D., Warman, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+does+insect+RNA+look+degraded.">Why does insect RNA look degraded.</a> Journal of Insect Science. 10 (1), 159 (2010).</li><li>Ivey, C. T., Carr, D. E., Eubanks, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+inbreeding+in+Mimulus+guttatus+on+tolerance+to+herbivory+in+natural+environments.">Effects of inbreeding in Mimulus guttatus on tolerance to herbivory in natural environments.</a> Ecology. 85 (2), 567-574 (2004).</li><li>Saccheri, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbreeding+and+extinction+in+a+butterfly+metapopulation.">Inbreeding and extinction in a butterfly metapopulation.</a> Nature. 392 (6675), 491-494 (1998).</li><li>Crnokrak, P., Roff, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbreeding+depression+in+the+wild.">Inbreeding depression in the wild.</a> Heredity. 83 (3), 260-270 (1999).</li><li>Keller, L. F., Waller, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbreeding+effects+in+wild+populations.">Inbreeding effects in wild populations.</a> Trends in Ecology &amp; Evolution. 17 (5), 230-241 (2002).</li><li>Margaritis, L. H., Kafatos, F. C., Petri, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eggshell+of+Drosophila+melanogaster.+I.+Fine+structure+of+the+layers+and+regions+of+the+wild-type+eggshell.">The eggshell of Drosophila melanogaster. I. Fine structure of the layers and regions of the wild-type eggshell.</a> Journal of Cell Science. 43 (1), 1-35 (1980).</li><li>Sugimoto, T. N., Ishikawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+male-killing+Wolbachia+carries+a+feminizing+factor+and+is+associated+with+degradation+of+the+sex-determining+system+of+its+host.">A male-killing Wolbachia carries a feminizing factor and is associated with degradation of the sex-determining system of its host.</a> Biology Letters. 8 (3), 412-415 (2012).</li></ol>

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，<...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/2 ounce cup</td>
			<td>FP CHUPA</td>
			<td>CP070009</td>
			<td>insect rearing; https://www.askul.co.jp/p/6010417/</td>
		</tr>
		<tr>
			<td>1/2 ounce cup lid</td>
			<td>FP CHUPA</td>
			<td>CP070011</td>
			<td>insect rearing; https://www.askul.co.jp/p/6010434/?int_id=recom_DtTogether</td>
		</tr>
		<tr>
			<td>99.7% acetic acid</td>
			<td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td>
			<td>36289</td>
			<td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01ALF036289.html</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>not shown</td>
			<td>Nucleic acids quantification; https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument</td>
		</tr>
		<tr>
			<td>Agilent RNA6000 nano kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1511</td>
			<td>Nucleic acids quantification; https://www.agilent.com/cs/library/usermanuals/Public/G2938-90034_RNA6000Nano_KG 
			.pdf</td>
		</tr>
		<tr>
			<td>benzalkonium chloride solution</td>
			<td>Nihon Pharmaceutical Co., Ltd</td>
			<td>No.4987123116046</td>
			<td>Sterilization; https://www.nihon-pharm.co.jp/consumer/products/disinfection.html</td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td>AOUME</td>
			<td>8-1611-02</td>
			<td>insect rearing; https://item.rakuten.co.jp/athlete-med/10006937/?scid=af_pc_etc&#38;sc2id=af_113_0_1</td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Dojindo, Tokyo, Japan</td>
			<td>340-07971</td>
			<td>stainings; https://www.dojindo.co.jp/products/D523/</td>
		</tr>
		<tr>
			<td>Disodium Hydrogenphosphate</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>Na2HPO4; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0286.html</td>
		</tr>
		<tr>
			<td>dsDNA HS quantification kit</td>
			<td>Invitrogen</td>
			<td>Q33231</td>
			<td>Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q33230?SID=srch-srp-Q33230</td>
		</tr>
		<tr>
			<td>Econospin RNA II column</td>
			<td>Epoch Life Science Inc.</td>
			<td>EP-11201</td>
			<td>RNA extraction; http://www.epochlifescience.com/Product/SpinColumn/minispin.aspx</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td>
			<td>4.98748E+12</td>
			<td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0105-0045.html</td>
		</tr>
		<tr>
			<td>Ethylenediamine-N,N,N&#39;,N&#39;-tetraacetic Acid Tetrasodium Salt Tetrahydrate (4NA)</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>Cell lysis buffer (EDTA); https://labchem-wako.fujifilm.com/jp/product/detail/W01T02N003.html</td>
		</tr>
		<tr>
			<td>Glassine paper</td>
			<td>HEIKO</td>
			<td>2100010</td>
			<td>insect rearing; https://www.monotaro.com/p/8927/0964/?utm_id=g_pla&#38; 
			utm_medium=cpc&#38;utm_source= 
			Adw</td>
		</tr>
		<tr>
			<td>heat block WSC-2620 PowerBLOCK</td>
			<td>ATTO, Tokyo, Japan</td>
			<td>4002620</td>
			<td>incubation; https://www.attoeng.site/</td>
		</tr>
		<tr>
			<td>heptane</td>
			<td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td>
			<td>4.98748E+12</td>
			<td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0108-0015.html</td>
		</tr>
		<tr>
			<td>INSECTA LF</td>
			<td>Nosan Co., Ltd</td>
			<td>not shown</td>
			<td>Artificial diet; https://www.nosan.co.jp/business/fodder/ist.htm</td>
		</tr>
		<tr>
			<td>ISOGENII</td>
			<td>Nippon Gene</td>
			<td>311-07361</td>
			<td>RNA extraction; https://www.nippongene.com/siyaku/product/extraction/isogen2/isogen2.html</td>
		</tr>
		<tr>
			<td>isopropanol</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>nucleic acids extraction; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0232-0004.html</td>
		</tr>
		<tr>
			<td>Lactic acid</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>Stainings; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0112-0005.html</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td>
			<td>4.98748E+12</td>
			<td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0113-0182.html</td>
		</tr>
		<tr>
			<td>MSV-3500 vortex</td>
			<td>Biosan</td>
			<td>BS-010210-TAK</td>
			<td>Voltex mixer; https://biosan.lv/products/-msv-3500-multi-speed-vortex/</td>
		</tr>
		<tr>
			<td>Nano Photometer NP 80</td>
			<td>Implen</td>
			<td>not shown</td>
			<td>Nucleic acids quantification; https://www.implen.de/product-page/implen-nanophotometer-np80-microvolume-cuvette-spectrophotometer/tech-specs/</td>
		</tr>
		<tr>
			<td>Natural pack wide</td>
			<td>Inomata chemical</td>
			<td>1859</td>
			<td>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</td>
		</tr>
		<tr>
			<td>NEBNext Ultra II RNA Library Prep Kit for Illumina</td>
			<td>New England BioLabs</td>
			<td>E7770S</td>
			<td>Library preparation; https://www.nebj.jp/products/detail/2039</td>
		</tr>
		<tr>
			<td>orcein</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>Stainings; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0115-0094.html</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>160-16061</td>
			<td>fixation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-1606.html</td>
		</tr>
		<tr>
			<td>Polyoxyethylene(20) Sorbitan Monolaurate</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>Tween-20; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-2121.html</td>
		</tr>
		<tr>
			<td>Portable UV Black Light (4W, 365nm wavelength)</td>
			<td>Southwalker Co., Ltd., Kanagawa, Japan</td>
			<td>not shown</td>
			<td>Insect collection; http://www.southwalker.com/shopping/?pid=1364614057-467328</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>KCl; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-0354.html</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>KH2PO4; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0116-0424.html</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>Invitrogen, MA, USA</td>
			<td>P36965</td>
			<td>antifade; https://www.thermofisher.com/order/catalog/product/P36965</td>
		</tr>
		<tr>
			<td>Proteinase K Solution</td>
			<td>Merck</td>
			<td>71049-4CN</td>
			<td>DNA extraction; https://www.merckmillipore.com/JP/ja/product/Proteinase-K-Solution-600-mAU-ml,EMD_BIO-71049</td>
		</tr>
		<tr>
			<td>protein precipitation solution</td>
			<td>Qiagen</td>
			<td>158912</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Qubit V4</td>
			<td>Invitrogen</td>
			<td>Q33238</td>
			<td>Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q33238</td>
		</tr>
		<tr>
			<td>rifampicin</td>
			<td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td>
			<td>4.98748E+12</td>
			<td>Sterilization; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0118-0100.html</td>
		</tr>
		<tr>
			<td>RNA HS quantification kit</td>
			<td>Invitrogen</td>
			<td>Q32855</td>
			<td>Nucleic acids quantification; https://www.thermofisher.com/order/catalog/product/Q32852</td>
		</tr>
		<tr>
			<td>RNase solution</td>
			<td>Nippon Gene</td>
			<td>313-01461</td>
			<td>RNA extraction; https://www.nippongene.com/siyaku/product/modifying-enzymes/rnase-a/rnase-s.html</td>
		</tr>
		<tr>
			<td>Silk Mate 2S</td>
			<td>Nosan Co., Ltd</td>
			<td>not shown</td>
			<td>Artificial diet; https://www.nosan.co.jp/business/fodder/ist.htm</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>NaCl; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0166.html</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>Cell lysis buffer (SDS); https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-1398.html</td>
		</tr>
		<tr>
			<td>sodium hypochlorite aqueous solution</td>
			<td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td>
			<td>4.98748E+12</td>
			<td>egg separation; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0119-0220.html</td>
		</tr>
		<tr>
			<td>Tetracycline Hydrochloride</td>
			<td>FUJIFILM Wako Chemicals Co., Osaka, Japan</td>
			<td>4.98748E+12</td>
			<td>Sterilization; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0120-1656.html</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>FUJIFILM Wako Chemicals Co.</td>
			<td>4.98748E+12</td>
			<td>Cell lysis buffer; https://labchem-wako.fujifilm.com/jp/product/detail/W01W0120-1536.html</td>
		</tr>
		<tr>
			<td>ultra-pure distilled water</td>
			<td>Invitrogen</td>
			<td>10977023</td>
			<td>RNA extraction; https://www.thermofisher.com/order/catalog/product/10977015</td>
		</tr>
	</tbody>
</table>

mass-rearing and molecular studies in tortricidae pest insects

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。

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...

Watch this Scientific Journal Video about Mass-Rearing and Molecular Studies in Tortricidae Pest Insects at JoVE.com

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

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。

龟科害虫的大规模饲养与分子研究

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

我们提出了基本但需求量很大的群体，用于大规模饲养，观察和分子研究，使用严重的害虫Tortrix质量，以研究它们的生物学。到目前为止，昆虫卵的结构已经阻止了进一步的分析。值得注意的是，我们研究了母体分泌物覆盖的薄，软和脆弱的卵子。有一些简单的技术有望适用于对Tortrix的进一步研究，尝试其他昆虫和其他分类群。首先将一名女性和两名男性放入一个120毫升的塑料杯中，并用一块石蜡纸建立Matra线。使用刨丝机切成约60克人造饲料进行大规模饲养。将充满成熟胚胎的卵块放在塑料容器中切片的人造日粮上。将石蜡纸放在卵块上，切片日粮。将15只雄性和10只雌性放入塑料盒中，在25摄氏度下交配。每五到七天收集一次卵子，并为每一代重复大规模饲养步骤。将蛋块浸泡在1000微升的1.2%次氯酸钠水溶液中10分钟或浸入1000微升的五摩尔氢氧化钾水溶液中30分钟以分离卵。将分离的卵在1000微升PBSt中洗涤。将鸡蛋浸泡在500微升100%庚烷和500微升4%多聚甲醛PBSt溶液的体积重量混合物中。使用涡流混合器以每分钟1500转的速度混合10分钟。将鸡蛋浸泡在500微升100%庚烷和500微升100%甲醇的混合物中。以每分钟 1500 次旋转的方式混合 10 分钟。用1000微升100%甲醇洗涤鸡蛋两次，并将其储存在100%甲醇中，温度为4摄氏度。将鸡蛋依次浸泡在99%70%和50%乙醇和PBSt中5分钟，分别进行水亲。用PBSt洗涤鸡蛋，将鸡蛋浸入每毫升DAPI溶液中一微克五分钟，然后用PBS洗涤鸡蛋两次。将卵浸泡在20微升的1.25%乳酸，乙酸或CN溶液中，以可视化异染色质，直到细胞核呈现出鲜红色。使用移液器将染色的卵转移到载玻片上，然后用防褪色试剂和盖玻片将其封闭。使用镊子从卵块中提取铁酸盐，单磁体和Adoxophyes honmai幼虫。在载玻片上解剖幼虫。用一到三种99.7%苦行子酸的混合物在100%甲醇中固定组织五分钟。用重量为1.25%重量的乳酸，苦行或CN溶液染色，直到细胞核染色。通过在显微镜下观察雌性异染色质的存在或雄性的不存在来确定每个标本的性别。为了从性别确定的铁酸盐幼虫中提取DNA和RNA，将12个性别确定的雄性或雌性铁酸盐幼虫合并，并将细胞裂解缓冲液或RNA提取试剂加入一个1.5毫升的管中。固定、透化和染色步骤使DAPI溶液的细胞核可视化成为可能。使用解剖的Ferrate幼虫进行乳酸，苦行或CN染色显示雌性表现出异染色质作为点，但雄性缺乏异染色质。使用自旋柱的修饰方案提高了RNA的质量，产生900至1500纳克的产物，260至200的比例为1.9至2.1，260至280的比例为1.9至2.3。修饰方案的RNA浓度比低于1.2，满足下一代测序的质量要求。使用改良方案从性别测定的Ferrate幼虫中提取的RNA的完整性，使用微芯片电泳确认。制备的文库被证实产生高Q 30分数读数。我们的信息有助于基本的昆虫研究和害虫工具。此外，我们的方案有可能用于评估胚胎发生过程中有效的化学农药或细胞间微生物。

Research

JoVE Journal

Environment

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

二斑叶螨的应用<em&gt;二斑叶螨</em&gt;植物病虫害的相互作用研究

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with ...

科研

环境科学

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

物理，化学和六Biochars生产的生物表征污染场地修复的

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

用于去除与农业和城市径流中地表水毒性有关的污染物的植被处理系统

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

用虱、烟粉虱在棉花中开发无梗昆虫生命表的方法作为模型系统

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

一氧化氮气体防治害虫实验室熏蒸程序

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

西瓜与瓶装葫芦之间的同源和异种移植在冷反应微 rna 研究中的应用

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...