The authors wish to acknowledge support from the Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists [Grant Number 19J13123 and 21J00895].

1. Insect collection and mass rearing
<ol>
	<li>Collect tortricid insects from fields following previously published References8,12. 
	NOTE:&#160;H. magnanima and A. honmai larvae are collected from damaged tea leaves (Figure 1A); adults are attracted using 4 W portable UV light (365 nm wavelength, see Table of Materials, Figure 1B).</li>
	<li>Re...

<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 comprises several agricultural and forestry pests; the present study presented methods to rear tortrix over generations, observe embryogenesis and sex of the insects, and conduct molecular analysis using matured embryos.
One of the obstacles for pest insect study is to establish rearing methods. Especially, inbreeding sometimes affect the fitness of the species negatively. The fitness reduction by the inbred, called inbreeding depression, has widely been observed in various plants an...

Lepidopteran insects represent more than 10% of all described living species1, and certain taxa species cause severe damage to plants and serious agricultural losses2,3. Although molecular and genetic studies have been developed using model insects such as the silkworm Bombyx mori4,5, pest insects remain uninvestigated, partly because of the difficulties for rearing and handling6,7. Therefore, fundamental studies and techniques are necessary to understand the biology of such non-model pest insects.
The Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests8. Of the insect taxa, the oriental tea tortrix Homona magnanima Diakonoff and the summer fruit tortrix Adoxophyes honmai Yasuda are serious polyphagous pests known to damage tea trees in East Asia7. The two species lay flat and oval scale-like egg clusters (or egg masses) consisting of thin, soft, and fragile eggs covered by maternal secretions. Although embryogenesis stages are crucial for insect development and sex determinations9, structures of the eggs prevent further analysis from understanding the biology of the insects. It is important to overcome the difficulties for further study on pests ovipositing such complex egg mass.
Here, to understand the biology of tortricids, methods for mass rearing, observations, and molecular studies have been developed using A. honmai and H. magnanima. First, mass rearing methods maintain both tortricids over 100 generations by inbred. The separation of eggs from the concatenated scale-like egg mass facilitated embryogenesis observation of the tortricids using alkaline and organic solvents previously developed from techniques used in flies10. In addition, the present study established sex discrimination of small embryos by developing staining methods of the sex chromatin of lepidopteran females using lactic-acetic orcein11. By combining these methods, high quality and quantity nucleic acids were extracted from sex determined embryos, which was otherwise difficult to establish6. The extracted RNA was utilized for next-generation sequencing. Collectively, the methods presented here apply to other lepidopteran insects and other insect taxa.

<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

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

Establishment of host lines and their maintenance 
The viability of field-collected larvae is differently attributed to field location, seasons, and rearing conditions (e.g., 90% of viability in Taiwan, Taoyuan, as shown in Arai et al.12). Approximately 30%-50% of pairs will generate the next generation as usual. For H. magnanima and A. honmai, matrilines have been maintained by inbreeding for over 100 generations.
Morphol...

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Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

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

We present fundamental, but highly in demand masses for mass rearing, observations and molecular studies using serious pest Tortrix masses, to study their biology. So far, the structure of the insect eggs have prevented further analysis. Notably, we enabled the study of thin, soft and fragile eggs covered by maternal secretion.There is simple techniques expected to be applicable for further research on tortrix, trying other insects and other taxa. Begin by placing a female and two males in a 120 milliliter plastic cup with a piece of paraffin paper to establish a Matra line. Slice approximately 60 grams of artificial diets using a Grater for mass rearing.Place the egg mass filled with matured embryos on the sliced artificial diet in a plastic container. Place paraffin papers on the egg mass with the sliced diets. Place 15 males and 10 females in a plastic box for mating at 25 degrees Celsius.Collect eggs every five to seven days and repeat mass rearing steps for each generation. Soak the egg mass in 1000 microliters of 1.2%sodium hypochlorite aqueous solution for 10 minutes or into 1000 microliters of five molar potassium hydroxide aqueous solution for 30 minutes to separate the eggs. Wash the separated eggs in 1000 microliters of PBSt.Soak eggs in a weight by volume mixture of 500 microliters of 100%heptane and 500 microliters of 4%Paraformaldehyde PBSt solution. Mix for 10 minutes at 1500 rotations per minute using a vortex mixer. Soak the eggs into a mixture of 500 microliters of 100%heptane and 500 microliters of 100%methanol.Mix for 10 minutes at 1500 rotations per minute. Wash the eggs twice with 1000 microliters of 100%methanol and store them at four degrees Celsius in 100%methanol. Soak the eggs sequentially in 99%70%and 50%ethanol and PBSt for five minutes each for hydrophilization.Wash the eggs with PBSt. Immerse the eggs in one microgram per milliliter DAPI solution for five minutes, and then wash the eggs with PBS twice. Soak the eggs in 20 microliters of 1.25%lactic, acetic or CN solution, to visualize heterochromatin until the nuclei exhibit a brilliant red color.Transfer the stained eggs using a pipette to a glass slide, then enclosed them with an anti-fade reagent and a cover glass. Extract Ferrate, Amonamagnetama and Adoxophyes honmai larvae from the egg masses using forceps. Dissect the larvae on the glass slide.Fix the tissues with a mixture of one to three 99.7%ascetic acid in 100%methanol for five minutes. Stain those with 1.25%weight by weight lactic, ascetic, or CN solution until the nuclei are stained. Determine the sex of each specimen by observing the presence of heterochromatin for females or absence for males under a microscope.To extract DNA and RNA from sex determined Ferrate larvae, pool 12 sex determined male or female ferrate larvae, and add either cell lysis buffer or RNA extraction reagents into one 1.5 milliliter tube. The fixation, Permeabilization and staining steps enable the visualization of nuclei with DAPI solution. Lactic, ascetic or CN staining using the dissected Ferrate larvae revealed that females exhibit heterochromatin as a dot, but males lack the heterochromatin.The modified protocols using a spin column improved the quality of the RNA producing 900 to 1500 nanograms of product with a 260 to 200 ratio of 1.9 to 2.1, and a 260 to 280 ratio of 1.9 to 2.3. The RNA concentration ratio for the modified protocol was below 1.2, meeting the quality requirements for next generation sequencing. The intactness of the RNA extracted from sex determined Ferrate larvae using the modified protocol was confirmed using microchip electrophoresis.The prepared libraries were confirmed to yield high Q 30 score reads. Our message contributes to fundamental insect studies and pest tools. Moreover, our protocols have the potential to be used to assess the effective chemical pesticides or inter cell microbes during embryogenesis.

mass-rearing-and-molecular-studies-in-tortricidae-pest-insects

Research

JoVE Journal

Environment

2.6K visualizações.  Tokyo University of Agriculture and Technology. Apresentamos massas fundamentais, mas altamente demandadas para criação em massa, observações e estudos moleculares usando massas tortrix de pragas graves, para estudar sua biologia. Até agora, a estrutura dos ovos de insetos impediu uma análise mais aprofundada. Notavelmente, possibilitamos o estudo de ovos finos, macios e frágeis cobertos pela secreção materna.Espera-se que sejam aplicáveis técnicas simples para novas pesquisas sobre tortrix, experimentando outros insetos ...