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

يود المؤلفون الاعتراف بالدعم المقدم من زمالات أبحاث الجمعية اليابانية لتعزيز العلوم (JSPS) للعلماء الشباب [رقم المنحة 19J13123 و 21J00895].

1. جمع الحشرات والتربية الجماعية
<ol><li>جمع الحشرات tortricid من الحقول التالية نشرت سابقا المراجع 8,12. ملاحظة: تجمع يرقات H. magnanima و A. honmai من أوراق الشاي التالفة (الشكل 1A) ؛ ينجذب البالغون باستخدام ضوء الأشعة فوق البنفسجية المحمو...

<ol>
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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>

يضم Tortricid العديد من الآفات الزراعية والحرجية. قدمت هذه الدراسة طرقا لتربية التورتريكس على مر الأجيال، ومراقبة التكوين الجنيني وجنس الحشرات، وإجراء التحليل الجزيئي باستخدام الأجنة الناضجة.
واحدة من العقبات التي تحول دون دراسة الحشرات الآفات هي إنشاء طرق التربية. على وجه ال...

تمثل حشرات Lepidopteran أكثر من 10٪ من جميع الأنواع الحية الموصوفة1 ، وتسبب بعض أنواع الأنواع أضرارا جسيمة للنباتات وخسائر زراعية خطيرة 2,3. على الرغم من أن الدراسات الجزيئية والجينية قد تم تطويرها باستخدام الحشرات النموذجية مثل دودة القز Bombyx mori4,5 ، إلا أن حشرات الآفات لا تزال غير محققة ، ويرجع ذلك جزئيا إلى صعوبات تربية ومناولة 6,7. لذلك ، فإن الدراسات والتقنيات الأساسية ضرورية لفهم بيولوجيا حشرات الآفات غير النموذجية هذه.
يضم Tortricidae (Lepidoptera) ، المعروف باسم tortrix أو عث أسطوانة الأوراق ، العديد من الآفات الزراعية والحرجية8. من بين أنواع الحشرات ، فإن تورتريكس الشاي الشرقي Homona magnanima Diakonoff و tortrix الفاكهة الصيفية Adoxophyes honmai Yasuda هي آفات polyphagous خطيرة معروفة بإتلاف أشجار الشاي في شرق آسيا7. يضع النوعان مجموعات بيض مسطحة وبيضاوية تشبه المقياس (أو كتل البيض) تتكون من بيض رقيق وناعم وهش تغطيه إفرازات الأمهات. على الرغم من أن مراحل التكوين الجنيني حاسمة لتطور الحشرات وتحديد الجنس9 ، إلا أن هياكل البيض تمنع إجراء مزيد من التحليل من فهم بيولوجيا الحشرات. من المهم التغلب على الصعوبات لإجراء مزيد من الدراسة حول الآفات التي تبيض مثل هذه الكتلة البيضاء المعقدة.
هنا ، لفهم بيولوجيا tortricids ، تم تطوير طرق للتربية الجماعية ، والملاحظات ، والدراسات الجزيئية باستخدام A. honmai و H. magnanima. أولا ، تحافظ طرق التربية الجماعية على كل من tortricids على مدى 100 جيل عن طريق التنشئة. وقد سهل فصل البيض عن كتلة البيض المتسلسلة الشبيهة بالمقياس مراقبة التكوين الجنيني للتورتريسيدات باستخدام المذيبات القلوية والعضوية التي تم تطويرها سابقا من التقنيات المستخدمة في الذباب10. بالإضافة إلى ذلك ، أثبتت هذه الدراسة التمييز الجنسي للأجنة الصغيرة من خلال تطوير طرق تلطيخ الكروماتين الجنسي للإناث lepidopteran باستخدام orcein اللاكتيك الخليك11. من خلال الجمع بين هذه الطرق ، تم استخراج الأحماض النووية عالية الجودة والكمية من الأجنة المحددة للجنس ، والتي كان من الصعب تأسيسها6. تم استخدام الحمض النووي الريبي المستخرج لتسلسل الجيل التالي. بشكل جماعي ، تنطبق الطرق المعروضة هنا على حشرات lepidopteran الأخرى وأنواع الحشرات الأخرى.

<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) ، المعروف باسم tortrix أو عث الأسطوانة الورقية ، يضم العديد من الآفات الزراعية والحرجية ، والتي تسبب خسائر زراعية خطيرة. لفهم بيولوجيا عث الآفات هذه ، كان هناك طلب كبير على التقنيات الأساسية. هنا ، يتم تطوير طرق للتربية الجماعية والملاحظات والدراسات الجزيئية باستخدام اثنين من تورتريكس الشاي ، Homona magnanima و Adoxophyes honmai (Lepidoptera: Tortricidae). تم تربية الحشرات على نطاق واسع مع نظام غذائي اصطناعي شرائح والحفاظ عليها عن طريق زواج الأقارب لأكثر من 100 جيل من خلال النظر في خصائصها البيولوجية. الحشرات لديها إزدواج الشكل الجنسي المختلفة. وبالتالي من الصعب التمييز بين الجنس خلال مراحل النمو ، والتي حالت دون إجراء فحوصات لاحقة. وأبرز هذا العمل أنه يمكن تحديد جنس يرقات التورتريسيدات من خلال مراقبة الخصيتين أو تلطيخ أورسين اللاكتيك - الخليك لتصور كروموسوم W الخاص بالإناث. علاوة على ذلك ، باستخدام طرق تحديد الجنس ، مكنت هذه الدراسة من استخراج الأحماض النووية من الأجنة المحددة جنسيا وتطبيقها نحو تسلسل الإنتاجية العالية. هذه النصائح قابلة للتطبيق على حشرات الآفات الأخرى وستسهل المزيد من الدراسات المورفولوجية والجينية.

إنشاء الخطوط المضيفة وصيانتها وتعزى صلاحية اليرقات المجمعة ميدانيا بشكل مختلف إلى الموقع الميداني والمواسم وظروف التربية (على سبيل المثال، 90٪ من قابلية البقاء في تايوان، تاويوان، كما هو موضح في Arai et al.12). ما يقرب من 30٪ -50٪ من الأزواج ستولد الجيل التالي كالمعتاد. بال?...

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

يصف هذا البروتوكول طريقة تربية حشرات الآفات التورتسيد في المختبرات. يتم تحديد إجراءات التمييز بين جنس الحشرات واستخراج الأحماض النووية لتسلسل الإنتاجية العالية باستخدام آفتين ممزقتين.

التربية الجماعية والدراسات الجزيئية في حشرات آفات Tortricidae

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

نحن نقدم كتلا أساسية ، ولكن عالية الطلب على التربية الجماعية والملاحظات والدراسات الجزيئية باستخدام كتل Tortrix الخطيرة للآفات ، لدراسة بيولوجيتها. حتى الآن ، منع هيكل بيض الحشرات إجراء مزيد من التحليل. والجدير بالذكر أننا مكننا من دراسة البويضات الرقيقة والناعمة والهشة التي يغطيها إفراز الأمهات.هناك تقنيات بسيطة من المتوقع أن تكون قابلة للتطبيق لمزيد من الأبحاث حول tortrix ، وتجربة الحشرات الأخرى وغيرها من الأصناف. ابدأ بوضع أنثى واثنين من الذكور في كوب بلاستيكي سعة 120 ملليلتر مع قطعة من ورق البارافين لإنشاء خط ماترا. قم بتقطيع ما يقرب من 60 جراما من الوجبات الغذائية الاصطناعية باستخدام مبشرة للتربية الجماعية.ضع كتلة البيض المملوءة بالأجنة الناضجة على النظام الغذائي الاصطناعي المقطع إلى شرائح في وعاء بلاستيكي. ضع أوراق البارافين على كتلة البيض مع الوجبات الغذائية المقطعة. ضع 15 ذكرا و 10 إناث في صندوق بلاستيكي للتزاوج عند 25 درجة مئوية.جمع البيض كل خمسة إلى سبعة أيام وتكرار خطوات التربية الجماعية لكل جيل. نقع كتلة البيض في 1000 ميكرولتر من محلول مائي هيبوكلوريت الصوديوم بنسبة 1.2٪ لمدة 10 دقائق أو في 1000 ميكرولتر من خمسة محلول مائي هيدروكسيد البوتاسيوم المولي لمدة 30 دقيقة لفصل البيض. اغسل البيض المنفصل في 1000 ميكرولتر من PBSt.نقع البيض في خليط الوزن حسب الحجم من 500 ميكرولتر من 100 ٪ من الهيبتان و 500 ميكرولتر من محلول 4 ٪ Paraformaldehyde PBSt. اخلط لمدة 10 دقائق بمعدل 1500 دورة في الدقيقة باستخدام خلاط دوامة. نقع البيض في خليط من 500 ميكرولتر من 100٪ هيبتان و 500 ميكرولتر من الميثانول 100٪.اخلط لمدة 10 دقائق بمعدل 1500 دورة في الدقيقة. اغسل البيض مرتين ب 1000 ميكرولتر من الميثانول بنسبة 100٪ وخزنه عند أربع درجات مئوية في 100٪ ميثانول. نقع البيض بالتتابع في 99٪ 70٪ و 50٪ من الإيثانول و PBSt لمدة خمس دقائق لكل منهما للمهب بالماء.اغسل البيض باستخدام PBSt. اغمر البيض في ميكروغرام واحد لكل محلول DAPI ملليلتر لمدة خمس دقائق ، ثم اغسل البيض باستخدام PBS مرتين. نقع البيض في 20 ميكرولتر من محلول 1.25٪ من اللاكتيك أو الخليك أو CN ، لتصور heterochromatin حتى تظهر النوى لونا أحمر لامعا.انقل البيض الملون باستخدام ماصة إلى شريحة زجاجية ، ثم أرفقها بكاشف مضاد للتلاشي وزجاج غطاء. استخراج يرقات Ferrate و Amonamagnetama و Adoxophyes honmai من كتل البيض باستخدام الملقط. تشريح اليرقات على الشريحة الزجاجية.إصلاح الأنسجة بمزيج من واحد إلى ثلاثة 99.7٪ من حمض الزهد في 100٪ ميثانول لمدة خمس دقائق. قم بتلطيخ أولئك الذين لديهم وزن 1.25٪ بالوزن محلول اللاكتيك أو الزاهد أو CN حتى يتم تلطيخ النوى. تحديد جنس كل عينة من خلال مراقبة وجود heterochromatin للإناث أو غياب للذكور تحت المجهر.لاستخراج الحمض النووي والحمض النووي الريبي من يرقات Ferrate المحددة بالجنس ، قم بتجميع 12 يرقات ذكور أو أنثى محددة من قبل الجنس ، وأضف إما المخزن المؤقت لتحلل الخلايا أو كواشف استخراج الحمض النووي الريبي في أنبوب واحد 1.5 ملليلتر. تتيح خطوات التثبيت والتخلل والتلطيخ تصور النوى باستخدام محلول DAPI. كشف تلطيخ اللاكتيك أو الزهد أو CN باستخدام يرقات Ferrate المشوهة أن الإناث تظهر heterochromatin كنقطة ، لكن الذكور يفتقرون إلى heterochromatin.حسنت البروتوكولات المعدلة باستخدام عمود الدوران جودة الحمض النووي الريبي الذي ينتج 900 إلى 1500 نانوغرام من المنتج بنسبة 260 إلى 200 من 1.9 إلى 2.1 ، ونسبة 260 إلى 280 من 1.9 إلى 2.3. وكانت نسبة تركيز الحمض النووي الريبي للبروتوكول المعدل أقل من 1.2، مما يفي بمتطلبات الجودة لتسلسل الجيل التالي. تم تأكيد سلامة الحمض النووي الريبي المستخرج من يرقات Ferrate المحددة جنسيا باستخدام البروتوكول المعدل باستخدام الرحلان الكهربائي للرقاقة الدقيقة.تم تأكيد المكتبات المعدة للحصول على قراءات عالية من درجة Q 30. تساهم رسالتنا في الدراسات الأساسية للحشرات وأدوات الآفات. علاوة على ذلك ، فإن بروتوكولاتنا لديها القدرة على استخدامها لتقييم المبيدات الكيميائية الفعالة أو الميكروبات بين الخلايا أثناء التكوين الجنيني.

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

Research

JoVE Journal

Environment

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

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7.
T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 &#176;C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the&#160;tomato Solanum lycopersicum)11. Together, these&#160;model organisms could provide insights into molecular bases of plant-pest interactions.
Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.

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

Protocols for efficient preparation of homogenous samples of spider mites, infestation of experimental plants, and assessment of plant damage, as required for studies of plant-pest interaction were developed.

تطبيق رصدت اثنين من العنكبوت سوس<em&gt; الخاشية urticae</em&gt; للدراسات التفاعل النبات من الآفات

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

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

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

الفيزيائية والكيميائية والبيولوجية توصيف ستة Biochars إنتاج لمعالجة المواقع الملوثة

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

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may be treated with simple systems designed to promote sorption of contaminants to vegetation and soils and promote infiltration. Two example systems are described: a bioswale treatment system for urban stormwater treatment, and a vegetated drainage ditch for treating agriculture irrigation runoff. Both have similar attributes that reduce contaminant loading in runoff: vegetation that results in sorption of the contaminants to the soil and plant surfaces, and water infiltration. These systems may also include the integration of granulated activated carbon as a polishing step to remove residual contaminants. Implementation of these systems in agriculture and urban watersheds requires system monitoring to verify treatment efficacy. This includes chemical monitoring for specific contaminants responsible for toxicity. The current paper emphasizes monitoring of current use pesticides since these are responsible for surface water toxicity to aquatic invertebrates.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

نظم معالجة الخضروات لإزالة الملوثات المرتبطة بالسمية السطحية للمياه في الزراعة والجريان السطحي في المناطق الحضرية

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

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 rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

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

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

منهجية "وضع جداول الحياة" "الحشرات لاطئة" في "الحقل باستخدام" الذبابة،، حيث دراسةفي "القطن كنموذج النظام"

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

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

الإجراءات المختبرية التبخير للسيطرة على الآفات مع غاز أكسيد النيتريك

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

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

طرق التسليم الشفوي الحمض النووي الريبي مزدوج-الذين تقطعت بهم السبل للحث على تدخل الجيش الملكي النيبالي في لحاء وتغذية النبات-ساب الحشرات هيميبتيران

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

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

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

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

اختبار الإصابة بالنبات: الرش والجرح بوساطة التلقيح بمسببات الأمراض النباتية رمادية ماجنابورثي

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

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

حصاد السم السموم من قاتل الحشرات وغيرها من الحشرات هيتيروبتيران

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

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

بروتوكول تجريبي لفحص ملفات تعريف الاستجابة السلوكية في الأسماك اليرقات: تطبيق على الكافيين منشط للأعصاب

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

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 an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

توليد هومو-وهيتيروجرافتس بين البطيخ والقرع زجاجة لدراسة MicroRNAs المراعية للبرد

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 ...