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

Gli autori desiderano riconoscere il sostegno della Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists [Numero di sovvenzione 19J13123 e 21J00895].

1. Raccolta e allevamento di massa degli insetti
<ol><li>Raccogli gli insetti tortricidi dai campi seguendo i riferimenti 8,12 precedentemente pubblicati. NOTA: le larve di H. magnanima e A. honmai sono raccolte da foglie di tè danneggiate (Figura 1A); gli adulti sono attratti utilizzando la luce UV portatile da 4 W (lunghezza d'onda di 365 nm, vedi Tabella dei materiali, <str...

<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 comprende diversi parassiti agricoli e forestali; il presente studio ha presentato metodi per allevare tortrix nel corso delle generazioni, osservare l'embriogenesi e il sesso degli insetti e condurre analisi molecolari utilizzando embrioni maturi.
Uno degli ostacoli per lo studio degli insetti nocivi è quello di stabilire metodi di allevamento. Soprattutto, la consanguineità a volte influisce negativamente sulla forma fisica della specie. La riduzione della forma fisica da parte d...

Gli insetti lepidotteri rappresentano oltre il 10% di tutte le specie viventi descritte1 e alcune specie di taxa causano gravi danni alle piante e gravi perdite agricole 2,3. Sebbene siano stati sviluppati studi molecolari e genetici utilizzando insetti modello come il baco da seta Bombyx mori 4,5, gli insetti parassiti rimangono non indagati, in parte a causa delle difficoltà di allevamento e manipolazione 6,7. Pertanto, sono necessari studi e tecniche fondamentali per comprendere la biologia di tali insetti parassiti non modello.
I Tortricidae (Lepidoptera), comunemente noti come tortrix o falene leafroller, comprendono molti parassiti agricoli e forestali8. Dei taxa degli insetti, il tortrix orientale del tè Homona magnanima Diakonoff e il tortrix di frutta estiva Adoxophyes honmai Yasuda sono gravi parassiti polifagi noti per danneggiare gli alberi del tè in Asia orientale7. Le due specie depongono grappoli di uova piatte e ovali simili a squame (o masse d'uovo) costituiti da uova sottili, morbide e fragili coperte da secrezioni materne. Sebbene le fasi di embriogenesi siano cruciali per lo sviluppo degli insetti e le determinazioni del sesso9, le strutture delle uova impediscono a ulteriori analisi di comprendere la biologia degli insetti. È importante superare le difficoltà per ulteriori studi sui parassiti che ovopongono una massa di uova così complessa.
Qui, per comprendere la biologia dei tortricidi, sono stati sviluppati metodi per l'allevamento di massa, osservazioni e studi molecolari utilizzando A. honmai e H. magnanima. In primo luogo, i metodi di allevamento di massa mantengono entrambi i tortricidi per oltre 100 generazioni da inbred. La separazione delle uova dalla massa uovo concatenata simile a una scala ha facilitato l'osservazione dell'embriogenesi dei tortricidi utilizzando solventi alcalini e organici precedentemente sviluppati da tecniche utilizzate nelle mosche10. Inoltre, il presente studio ha stabilito la discriminazione sessuale di piccoli embrioni sviluppando metodi di colorazione della cromatina sessuale di femmine di lepidotteri utilizzando orceina lattico-acetica11. Combinando questi metodi, gli acidi nucleici di alta qualità e quantità sono stati estratti da embrioni determinati dal sesso, che era altrimenti difficile da stabilire6. L'RNA estratto è stato utilizzato per il sequenziamento di prossima generazione. Collettivamente, i metodi qui presentati si applicano ad altri insetti lepidotteri e altri taxa di insetti.

<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), comunemente noti come tortrix o falene leafroller, comprende molti parassiti agricoli e forestali, che causano gravi perdite agricole. Per comprendere la biologia di tali falene nocive, le tecniche fondamentali sono state molto richieste. Qui, i metodi per l'allevamento di massa, le osservazioni e gli studi molecolari sono sviluppati utilizzando due tortrix del tè, Homona magnanima e Adoxophyes honmai (Lepidoptera: Tortricidae). Gli insetti sono stati allevati in massa con una dieta artificiale affettata e mantenuti dalla consanguineità per oltre 100 generazioni considerando le loro caratteristiche biologiche. Gli insetti hanno vari dimorfismi sessuali; quindi è difficile distinguere il sesso durante le fasi di sviluppo, che hanno impedito i test successivi. Il presente lavoro ha evidenziato che il sesso delle larve di tortricidi potrebbe essere determinato osservando testicoli o colorazione di orceina lattico-acetica per visualizzare il cromosoma W specifico della femmina. Inoltre, utilizzando i metodi di determinazione del sesso, il presente studio ha permesso l'estrazione di acido nucleico da embrioni determinati dal sesso e l'applicazione verso il sequenziamento ad alto rendimento. Questi suggerimenti sono applicabili ad altri insetti parassiti e faciliteranno ulteriori studi morfologici e genetici.

Creazione di linee host e loro manutenzione La vitalità delle larve raccolte sul campo è attribuita in modo diverso alla posizione del campo, alle stagioni e alle condizioni di allevamento (ad esempio, il 90% della vitalità a Taiwan, Taoyuan, come mostrato in Arai et al.12). Circa il 30%-50% delle coppie genererà la prossima generazione come al solito. Per H. magnanima e A. honmai, le matriline sono state mantenute dalla consanguineità per oltre 100 gener...

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

Il presente protocollo descrive il metodo di allevamento degli insetti nocivi tortricidi nei laboratori. Le procedure per distinguere il sesso degli insetti ed estrarre gli acidi nucleici per il sequenziamento ad alto rendimento sono stabilite utilizzando due parassiti tortricidi.

Allevamento di massa e studi molecolari negli insetti parassiti Tortricidae

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

Presentiamo masse fondamentali, ma molto richieste per l'allevamento di massa, osservazioni e studi molecolari utilizzando masse Tortrix gravi, per studiare la loro biologia. Finora, la struttura delle uova di insetto ha impedito ulteriori analisi. In particolare, abbiamo permesso lo studio di uova sottili, morbide e fragili coperte da secrezione materna.Ci sono tecniche semplici che dovrebbero essere applicabili per ulteriori ricerche su tortrix, provando altri insetti e altri taxa. Inizia mettendo una femmina e due maschi in un bicchiere di plastica da 120 millilitri con un pezzo di carta di paraffina per stabilire una linea Matra. Affettare circa 60 grammi di diete artificiali usando una grattugia per l'allevamento di massa.Metti la massa d'uovo piena di embrioni maturi sulla dieta artificiale affettata in un contenitore di plastica. Metti le carte di paraffina sulla massa d'uovo con le diete a fette. Metti 15 maschi e 10 femmine in una scatola di plastica per l'accoppiamento a 25 gradi Celsius.Raccogli le uova ogni cinque-sette giorni e ripeti le fasi di allevamento di massa per ogni generazione. Immergere la massa d'uovo in 1000 microlitri di soluzione acquosa di ipoclorito di sodio all'1,2% per 10 minuti o in 1000 microlitri di cinque soluzione acquosa di idrossido di potassio molare per 30 minuti per separare le uova. Lavare le uova separate in 1000 microlitri di PBSt.Immergere le uova in una miscela peso per volume di 500 microlitri di 100% eptano e 500 microlitri di soluzione PBSt di paraformaldeide al 4%. Mescolare per 10 minuti a 1500 rotazioni al minuto utilizzando un miscelatore a vortice. Immergere le uova in una miscela di 500 microlitri di 100% eptano e 500 microlitri di metanolo al 100%.Mescolare per 10 minuti a 1500 rotazioni al minuto. Lavare le uova due volte con 1000 microlitri di metanolo al 100% e conservarle a quattro gradi Celsius in metanolo al 100%. Immergere le uova in sequenza in etanolo al 99% 70% e al 50% e PBSt per cinque minuti ciascuno per l'idrofilizzazione.Lavare le uova con PBSt. Immergere le uova in una soluzione DAPI di microgrammi per millilitro per cinque minuti, quindi lavare le uova con PBS due volte. Immergere le uova in 20 microlitri di soluzione lattica, acetica o CN all'1,25%, per visualizzare l'eterocromatina fino a quando i nuclei non mostrano un colore rosso brillante.Trasferire le uova colorate usando una pipetta su un vetrino, quindi racchiuderle con un reagente anti-dissolvenza e un vetro di copertura. Estrarre le larve di Ferrate, Amonamagnetama e Adoxophyes honmai dalle masse uovo usando una pinza. Seziona le larve sul vetrino.Fissare i tessuti con una miscela di uno o tre acido ascetico al 99,7% in metanolo al 100% per cinque minuti. Macchiare quelli con 1,25% di peso in soluzione lattica, ascetica o CN fino a quando i nuclei non sono macchiati. Determinare il sesso di ciascun campione osservando la presenza di eterocromatina per le femmine o l'assenza per i maschi al microscopio.Per estrarre DNA e RNA da larve di Ferrate determinate per sesso, raggruppare 12 larve di ferrate maschili o femminili determinate per sesso e aggiungere tampone di lisi cellulare o reagenti di estrazione dell'RNA in un tubo da 1,5 millilitri. Le fasi di fissazione, permeabilizzazione e colorazione consentono la visualizzazione dei nuclei con la soluzione DAPI. La colorazione lattica, ascetica o CN usando le larve di Ferrate sezionate ha rivelato che le femmine mostrano eterocromatina come un punto, ma i maschi mancano dell'eterocromatina.I protocolli modificati che utilizzano una colonna di spin hanno migliorato la qualità dell'RNA producendo da 900 a 1500 nanogrammi di prodotto con un rapporto da 260 a 200 da 1,9 a 2,1 e un rapporto da 260 a 280 da 1,9 a 2,3. Il rapporto di concentrazione dell'RNA per il protocollo modificato era inferiore a 1,2, soddisfacendo i requisiti di qualità per il sequenziamento di prossima generazione. L'integrità dell'RNA estratto dalle larve di Ferrate determinate dal sesso utilizzando il protocollo modificato è stata confermata utilizzando l'elettroforesi a microchip.Le librerie preparate sono state confermate per produrre letture con punteggio Q 30 elevato. Il nostro messaggio contribuisce agli studi fondamentali sugli insetti e agli strumenti per i parassiti. Inoltre, i nostri protocolli hanno il potenziale per essere utilizzati per valutare l'efficacia dei pesticidi chimici o dei microbi intercellulari durante l'embriogenesi.

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.

Applicazione di due-macchiato Spider Mite<em&gt; Tetranychus urticae</em&gt; Per gli Studi Plant-parassita di interazione

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

Ricerca

Ambiente

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.

Fisica, chimica e caratterizzazione biologica di Six Biochars Prodotti per il risanamento dei siti contaminati

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.

Sistemi di trattamento vegetale per la rimozione dei contaminanti associati alla tossicità dell&#39;acqua superficiale nell&#39;agricoltura e nella rottura urbana

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.

Metodologia per lo sviluppo di tavole di sopravvivenza per insetti Sessile nel campo utilizzando la Whitefly, Bemisia tabaci, in cotone come un modello di sistema

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.

Procedure di fumigazione di laboratorio per il controllo dei parassiti con Gas di ossido di azoto

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.

Metodi di consegna orale RNA double-stranded per indurre RNAi nel floema e insetti Pentatomomorfi pianta-sap-alimentazione

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.

La prova di infezione di pianta: Spray e ferita-mediata di inoculazione con la pianta patogeno Magnaporthe Grisea

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.

Raccolta le tossine veleno da Assassin Bugs e altri insetti Heteropteran

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.

Protocollo sperimentale per l'esame di profili di risposta comportamentale nel pesce larvale: applicazione alla caffeina Neuro-stimolante

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.

Generazione di omo - e Heterografts tra anguria e zucca bottiglia per lo studio dei microRNA freddo-sensible a reagire

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 ...