ברצוננו להודות מישל הירח (אוניברסיטת ואנדרבילט) לקבלת סיוע עם הצילום. אוניברסיטת ואנדרבילט סיפק תמיכה ברשות הפלסטינית, SSLB נתמך על ידי DGE-1445197.

<ol>
	<li>Brisson, J. A., Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pea+aphid,+Acyrthosiphon+pisum.:+an+emerging+genomic+model+system+for+ecological,+developmental+and+evolutionary+studies.">The pea aphid, Acyrthosiphon pisum.: an emerging genomic model system for ecological, developmental and evolutionary studies.</a> BioEssays. 28, 747-755 (2006).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aphid Ecology: An Optimization Approach. , (1985).</li><li>Consortium, T. I. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Sequence+of+the+Pea+Aphid+Acyrthosiphon+pisum.">Genome Sequence of the Pea Aphid Acyrthosiphon pisum.</a> PLoS Biology. , 1000313 (2010).</li><li>Srinivasan, D. G., Brisson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aphids:+A+Model+for+Polyphenism+and+Epigenetics.">Aphids: A Model for Polyphenism and Epigenetics.</a> Genetics Research International. 2012, 1-12 (2012).</li><li>Wenger, J. A., 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=Whole+genome+sequence+of+the+soybean+aphid,+Aphis+glycines.">Whole genome sequence of the soybean aphid, Aphis glycines.</a> Insect Biochememistry and Molecular Biology. , 1-10 (2017).</li><li>Li, Z. -. Q., 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=Ecological+adaption+analysis+of+the+cotton+aphid+(Aphis+gossypii)+in+different+phenotypes+by+transcriptome+comparison.">Ecological adaption analysis of the cotton aphid (Aphis gossypii) in different phenotypes by transcriptome comparison.</a> PLoS ONE. 8, 83180 (2013).</li><li>Wang, D., Liu, Q., Jones, H. D., Bruce, T., Xia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptomic+analyses+revealed+divergences+of+two+agriculturally+important+aphid+species.">Comparative transcriptomic analyses revealed divergences of two agriculturally important aphid species.</a> BMC Genomics. 15 (1), 1023-1024 (2014).</li><li>Wu, S., 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=De+novo+characterization+of+the+pine+aphid+Cinara+pinitabulaeformis+Zhang+et+Zhang+transcriptome+and+analysis+of+genes+relevant+to+pesticides.">De novo characterization of the pine aphid Cinara pinitabulaeformis Zhang et Zhang transcriptome and analysis of genes relevant to pesticides.</a> PLoS ONE. 12, 0178496-0178517 (2017).</li><li>Birnbaum, S. S. L., Rinker, D. C., Gerardo, N. M., Abbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profile+and+differential+fitness+in+a+specialist+milkweed+insect+across+host+plants+varying+in+toxicity.">Transcriptional profile and differential fitness in a specialist milkweed insect across host plants varying in toxicity.</a> Molecular Ecology. , (2017).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect Herbivore-Host Dynamics. , (2005).</li><li>Goggin, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-aphid+interactions:+molecular+and+ecological+perspectives.">Plant-aphid interactions: molecular and ecological perspectives.</a> Current Opinion in Plant Biology. 10, 399-408 (2007).</li><li>Will, T., Furch, A., Zimmermann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+phloem-feeding+insects+face+the+challenge+of+phloem-located+defenses.">How phloem-feeding insects face the challenge of phloem-located defenses.</a> Frontiers in Plant Science. 4, 1-12 (2013).</li><li>Webster, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+olfaction+in+aphid+host+location.">The role of olfaction in aphid host location.</a> Physiological Entomology. 37, 10-18 (2012).</li><li>Agrawal, A. A., Petschenka, G., Bingham, R. A., Weber, M. G., Rasmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+cardenolides:+chemical+ecology+and+coevolution+of+specialized+plant-herbivore+interactions.">Toxic cardenolides: chemical ecology and coevolution of specialized plant-herbivore interactions.</a> New Phytologist. 194, 28-45 (2012).</li><li>Dobler, S., Petschenka, G., Pankoke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coping+with+toxic+plant+compounds-+the+insect's+perspective+on+iridoid+glycosides+and+cardenolides.">Coping with toxic plant compounds- the insect's perspective on iridoid glycosides and cardenolides.</a> Phytochemistry. 72, 1593-1604 (2011).</li><li>Opitz, S. E. W., M&#252;ller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chemistry+and+insect+sequestration.">Plant chemistry and insect sequestration.</a> Chemoecology. 19, 117-154 (2009).</li><li>Birnbaum, S. S. L., Abbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+adaptations+toward+plant+toxins+in+milkweed-herbivores+systems+-+a+review.">Insect adaptations toward plant toxins in milkweed-herbivores systems - a review.</a> Entomologia Experimentalis et Applicata. 58, 579-610 (2018).</li><li>Rothschild, M., von Euw, J., Reichstein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+glycosides+in+the+oleander+aphid,+Aphis+nerii.">Cardiac glycosides in the oleander aphid, Aphis nerii.</a> Journal of Insect Physiology. 16, 1141-1145 (1970).</li><li>Harrison, J. S., Mondor, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+invasive+aphid+'superclone':+extremely+low+genetic+diversity+in+oleander+aphid+(Aphis+nerii)+populations+in+the+southern+United+States.">Evidence for an invasive aphid 'superclone': extremely low genetic diversity in oleander aphid (Aphis nerii) populations in the southern United States.</a> PLoS ONE. 6, 17524 (2011).</li><li>Mooney, K. A., Halitschke, R., Kessler, A., Agrawal, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+trade-offs+in+plants+mediate+the+strength+of+trophic+cascades.">Evolutionary trade-offs in plants mediate the strength of trophic cascades.</a> Science. 327, 1642-1644 (2010).</li><li>Grabherr, M. G., 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=Full-length+transcriptome+assembly+from+RNA-Seq+data+without+a+reference+genome.">Full-length transcriptome assembly from RNA-Seq data without a reference genome.</a> Nature Biotechnology. 29, 644-652 (2011).</li><li>Haas, B. J., 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=De+novo+transcript+sequence+reconstruction+from+RNA-seq+using+the+Trinity+platform+for+reference+generation+and+analysis.">De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.</a> Nature Protocols. 8, 1494-1512 (2013).</li><li>Finn, R. D., 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+Pfam+protein+families+database:+towards+a+more+sustainable+future.">The Pfam protein families database: towards a more sustainable future.</a> Nucleic Acids Research. 44, 279-285 (2016).</li><li>The UniProt Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniProt:+the+universal+protein+knowledgebase.">UniProt: the universal protein knowledgebase.</a> Nucleic Acids Research. 45, 158-169 (2017).</li><li>Johnson, 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=NCBI+BLAST:+a+better+web+interface.">NCBI BLAST: a better web interface.</a> Nucleic Acids Research. 1 (36), 5-9 (2008).</li><li>Fu, L., Niu, B., Zhu, Z., Wu, S., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD-HIT:+accelerated+for+clustering+the+next+generation+sequencing+data.">CD-HIT: accelerated for clustering the next generation sequencing data.</a> Bioinformatics. 28 (23), 3150-3152 (2012).</li><li>Sim&#227;o, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., Zdobnov, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BUSCO:+assessing+genome+assembly+and+annotation+completeness+with+single-copy+orthologs.">BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs.</a> Bioinformatics. 31, 3210-3212 (2015).</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30, 2114-2120 (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9, 357-359 (2012).</li><li>Li, H., 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+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li><li>Love, M. I., Huber, W., Anders, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderated+estimation+of+fold+change+and+dispersion+for+RNA-seq+data+with+DESeq2.">Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.</a> Genome Biology. 15, 31 (2014).</li><li>Rieu, I., Powers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+RT-PCR:+design,+calculations,+and+statistics.">Real-time quantitative RT-PCR: design, calculations, and statistics.</a> Plant Cell. 21, 1031-1033 (2009).</li><li>Malcolm, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defence+in+chewing+and+sucking+insect+herbivores:+plant-derived+cardenolides+in+the+monarch+butterfly+and+oleander+aphid.">Chemical defence in chewing and sucking insect herbivores: plant-derived cardenolides in the monarch butterfly and oleander aphid.</a> Chemoecology. 1, 12-21 (1990).</li><li>Agrawal, A. A., Underwood, N., Stinchcombe, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspecific+variation+in+the+strength+of+density+dependence+in+aphid+populations.">Intraspecific variation in the strength of density dependence in aphid populations.</a> Ecological Entomology. 29, 521-526 (2004).</li><li>Zehnder, C. B., Hunter, 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=A+comparison+of+maternal+effects+and+current+environment+on+vital+rates+of+Aphis+nerii,+the+milkweed-oleander+aphid.">A comparison of maternal effects and current environment on vital rates of Aphis nerii, the milkweed-oleander aphid.</a> Ecological Entomology. 32, 172-180 (2007).</li><li>Hartbauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+defense+of+Aphis+nerii+and+Uroleucon+hypochoeridis+(Homoptera,+Aphididae)+against+natural+enemies.">Collective defense of Aphis nerii and Uroleucon hypochoeridis (Homoptera, Aphididae) against natural enemies.</a> PLoS ONE. 5, 10417 (2010).</li></ol>

המחברים אין לחשוף.

זה זמן הוכר כי aposematic nerii א יכולה לספק תובנות דפוסים ומנגנונים של התנגדות צמח ההגנות ו פחמיות כימי במיוחד18,37. לאחרונה הופיעו מספר משאבים גנומית nerii א10, המציע הזדמנויות חדש אקולוגי ופונקציונליים גנומית מחקרים המשתמשים nerii א כמודל. א?...

כנימות הן חרקים קטנים, hemimetabolous ליישב על משפחות צמחים מגוונים ברחבי העולם. הם ייחודי עבור מספר תכונות, ביותר ובמיוחד שלהם מחזורי החיים מורכבים המערבים רביית בתולים מחזורית polyphenisms דיסקרטית, ואת שלהם symbioses תזונתי פעל עם endosymbionts בקטריאלי או שמרים לספק חומרים מזינים חסרים הדיאטה שלהם של הצמח sap1. בעוד רוב הכנימות הם מומחים הצמח הפונדקאי, מינים מסוימים עובדים כלליים הם מזיקים חשוב חיתוך, גרימת נזק כלכלי ניכר על היבול גם ישירות או באמצעות גורמי מחלה, וירוסים הם וקטור2. הפרסום של הגנום כנימות עלה הראשון בשנת 2010 מימדיה אפונה Acyrthosiphon אפון3, מסומן ציון דרך חשוב בחקר הביולוגיה כנימות עלה משום שסיפקה את המשאבים גנומית לשאלות המיעון על החרק עיבודים ל אורח החיים אוכלי עשב, כולל אלה שעשויים להוביל אסטרטגיות שליטה טובה יותר4. מאז, צברו משאבים גנומית נוספים עם הפרסום של הגנום מוערת על סויה כנימות עלה Aphis glycines5ומשאבים הגנום כולו מהעדכונים הזמינים עבור שלוש-כנימות עלה עוד מינים (המידע cerasi (כנימות עלה דובדבן שחור), המידע שיפורט להלן מתייחס (כנימות עלה אפרסק-תפוחי אדמה), Rhopalosiphum padi (כנימות עלה ענביה-שיבולת שועל)6. ערך דה נובו transcriptomic משאבים זמינים גם עבור מספר מינים אחרים כנימות עלה (e.g.,Aphis gossypii (כותנה כנימות עלה)7, Sitobion avenae (כנימות עלה תבואה)8, Cinara pinitabulaeformis (כנימות עלה אורן)9, Aphis nerii (כנימות עלה לבנבן-הרדוף)10).
כנימות עשו גם תרומות מתמשך להבנה שלנו את האינטראקציות צמח-חרק ואקולוגיה של חיי הצמחים11. תחום אחד שבו כנימות לתרומות חשוב במיוחד הוא בהבנתנו. אקולוגיה כימית של אינטראקציות צמח המארח. עיבודים אקספרס מגוונות חרקים אוכלי עשב עבור להתגבר על ההגנות צמח, וחלק אפילו לצרף צמח ההגנות שלהם ליהנות12,13,14. לדוגמה, מימדיה לבנבן-הרדוף Aphis nerii, הוא בהיר צהוב, פולשני כנימות עלה, נמצאו באזורים אקלים ואזורים טרופיים ברחבי העולם זה והצנרות על צמחים ממשפחת לבנבן (הרדופיים). צמחים ממשפחת הרדופיים התפתחו ההגנות כימיים מגוונים, לרבות latex חלבי גליקוסיד הידועה בשם cardenolides, אשר מאגד המוביל הקטיון Na, K-ATPase ומהוות deterrents יעיל ומתוכנן אוכלי עשב15, 16. לבנבן מומחים לבטא מצבים שונים של התנגדות cardenolides, חלקם באופן סלקטיבי או פסיבי לצבור או לשנות cardenolides ברקמות שלהם כאמצעי להרתיע טריפה או הטבות אחרות17. Nerii א נחשב לבודד cardenolides בדרך זו, אף מנגנוני והיתרונות פונקציונלי לא ברור10,18.
בהינתן המשאבים גנומית בהישג יד, א nerii מספק מדגם ניסיוני מעולה לצורך המחקר של המנגנונים המולקולריים והגנטיים מעורבת ביחסי הגומלין כימותרפיה-אקולוגית בין צמחים פונדקאים רעילים מומחה שלהם אוכלי עשב. ראוי לציין כי, בעוד שבחלקם המחקרים המוקדמים של א nerii התמקדו פחמיות של cardenolides19, מאז, מחקרים של nerii א סיפקו תובנות לתוך מערך רחב של שאלות אבולוציונית וגם אקולוגית, כולל המבנה הגנטי של חרקים פולשנית20 והגומלין בין רגולציה מלמטה ולא מלמעלה על צפיפות הרביוור21. Nerii א ולכן מועמד טוב בתור מודל ניסיוני עבור מערך רחב במיוחד של מחקרים של אינטראקציות חרק-צמח. קריטי להצלחה של כל מחקר עם nerii א הוא התרבות זהיר של כנימות עלה אוכלוסיות, כולל התרבות של צמחים אשר תלויים הכנימות, וכן של דור יעילה של נתונים באיכות גבוהה - omic. המטרה שלנו היא להנחות את הקורא דרך שניהם. המפורטות להלן הן שיטות דור ותחזוקה של התרבויות צמח, כנימות עלה החממה, מעבדה, DNA, RNA עקירות, ניתוח microsatellite, דה נובו transcriptome הרכבה, ביאור, transcriptome ניתוח ביטוי דיפרנציאלי, ואימות qPCR באופן שונה באה לידי ביטוי גנים. בעוד שיטות אלה כתובות א neriiculturing כללי, חילוץ, שיטות ניתוח יכול להרחיב מגוון מינים כנימות עלה.

<table><tbody><tr><td>Sun Gro Fafard Germination Mix</td><td>Hummert International</td><td>10-0952-2</td><td></td></tr><tr><td>Sun Gro Fafard 3B/ Metro Mix</td><td>Hummert International</td><td>10-0951-2</td><td></td></tr><tr><td>2&quot; x 4&quot; Round Standard Pot</td><td>Anderson Pots</td><td>1503</td><td></td></tr><tr><td>DreamTaq DNA Polymerase</td><td>ThermoFisher Scientific</td><td>EP0701</td><td></td></tr><tr><td>Trizol</td><td>ThermoFisher Scientific</td><td>15596026</td><td></td></tr><tr><td>SuperScript&amp;reg; III First-Strand Synthesis kit</td><td>ThermoFisher Scientific</td><td>18080051</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>ThermoFisher Scientific</td><td>4367659</td><td></td></tr></tbody></table>

maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions

כנימות הן ניסיוניות מעולה למגוון רחב של שאלות ביולוגיות החל את האבולוציה של symbioses ופיתוח של polyphenisms לשאלות המקיפים של החרק אינטראקציות עם צמחים פונדקאים שלהם. גנומית משאבים זמינים עבור מספר מינים כנימות עלה, עם ההתקדמות רצף הדור הבא, מחקרים transcriptomic להיות מורחבות כדי אורגניזמים שאינם-דגם חסרי הגנום. יתר על כן, תרבויות כנימות עלה יכול להיות שנאספו מהשטח וגדלתי במעבדה לשימוש בניסויים organismal מולקולרית, כדי לגשר על הפער בין מחקרים אקולוגיים גנטי. בפעם האחרונה, כנימות רבים יכול להישמר במעבדה על צמחים פונדקאים המועדפת שלהם במחזורי החיים הנצחי, parthenogenic המאפשר השוואות של asexually מתרבה אחרים. Aphis nerii, מימדיה לבנבן-הרדוף מספק מודל אחד כזה ללמוד אינטראקציות חרקים עם צמחים רעילים באמצעות ניסויים הן organismal והן מולקולרית. שיטות דור ותחזוקה של תרבויות החממה, מעבדה, עקירות DNA ו- RNA, ניתוח microsatellite, דה נובו transcriptome הרכבה, ביאור, transcriptome דיפרנציאלית הביטוי צמח, כנימות עלה ניתוח ואימות qPCR של גנים ביטוי באופן שונה המתוארים הינם שנדונו כאן.

צמח תרבויות: זרעים ייקח בערך שבועיים עד ארבעה שבועות, תלוי בעונה, לגדול גדול מספיק כדי להיות מחדש בעציץ (איור 1א'). שתילים מחדש בעציץ ייקח עוד שבועיים עד ארבעה שבועות לגדול לגודל האופטימלי עבור תרבויות כנימות עלה (איור 1<stron...

Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

מימדיה Aphis nerii והצנרות על הגן מאוד צמחים ממשפחת dogbane (Apocyanaceae), ומספק הזדמנויות רבות ללמוד אינטראקציות צמח-חרקים. כאן, אנו מציגים סדרת פרוטוקולים עבור שמירה על צמח, כנימות עלה תרבויות, ואת הדור ניתוח של מולקולרית ונתונים - omic עבור nerii א.

שמירה על תרבויות ביולוגי מדידה ביטוי גנים ב Aphis nerii: מערכת Non-מודל עבור אינטראקציות צמח-חרק

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

maintaining-biological-cultures-measuring-gene-expression-aphis-nerii

Research

JoVE Journal

Genetics

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

7.5K Views.  Vanderbilt University. מימדיה Aphis nerii והצנרות על הגן מאוד צמחים ממשפחת dogbane (Apocyanaceae), ומספק הזדמנויות רבות ללמוד אינטראקציות צמח-חרקים. כאן, אנו מציגים סדרת פרוטוקולים עבור שמירה על צמח, כנימות עלה תרבויות, ואת הדור ניתוח של מולקולרית ונתונים - omic עבור nerii א.

Video: Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

משלוח חומצות גרעין דרך Microinjection העובר חרקי ההדברה החקלאיים ברחבי העולם,<em&gt; זבוב הפירות הים תיכוני</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

התרומם על רגליו אחורי פעמים תקועות מציאת גן בתיווך רנ&quot;א סתר החיפושית,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

זיהוי של תנאים קריטיים לImmunostaining באפונה בכנימות עוברים: הגדלת רקמות חדירות והפחתת מכתים רקע

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Developmental Biology

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

גידול והזרקה של<em&gt; Manduca סקסטה</em&gt; זחלים להערכה ארסית קטריאלי

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Immunology and Infection

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

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

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

גידול המוני ומחקרים מולקולריים בחרקים מזיקים בטורטריים

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

Environment

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

מציאה RNAi בתיווך זוגית ג&#39;ין ומדידה בחוש טעם בדבורי דבש (<em&gt; אפיס mellifera</em&gt;)

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Neuroscience

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

תרבות ומניפולציה גנטית של נמטודות אנטומופתוגניות

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

ביטוי חולף ואת סלולר לוקליזציה של חלבונים רקומביננטי תאים בתרבית חרקים

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Biology

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

שמירה על Aedes aegypti יתושים נגועים Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

זריקות פואפאל ומבוגרים לעריכת גנים RNAi ו- CRISPR בנסוניה ויטריפניס

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

שמירה על תרבויות ביולוגי מדידה ביטוי גנים ב Aphis nerii: מערכת Non-מודל עבור אינטראקציות צמח-חרק

Summary

Explore More Videos

Chapters in this video

משלוח חומצות גרעין דרך Microinjection העובר חרקי ההדברה החקלאיים ברחבי העולם,

התרומם על רגליו אחורי פעמים תקועות מציאת גן בתיווך רנ"א סתר החיפושית,