This work was supported by the National Institutes of Health grant 5R21AI081041-02 and Georgetown University.

1. Larvenaufzucht von zwei A. albopictus Gruppen zum Erwachsenwerden <ol><li> Stellen Sie zwei Photoperiode Schränke mit programmierbaren Beleuchtung bei 21 ° C für eine optimale Diapause Ausdruck 16 und ca. 80% relativer Luftfeuchtigkeit. <ol><li> Programmieren Sie einen Schrank für einen 16L: 8D-Licht: Dunkel-Zyklus (ein nicht-Diapause induziert LD Photoperiode). Stellen Sie das zweite Gehäuse für eine 8L: 16D Licht: Dunkel-Zyklus (Diapause induziert) 13. </li><li> &quot;Licht an&quot; Programm zur gleichen Zeit in beiden Schränke auf circadiane Zeit zwischen Photoperioden zu synchronisieren. </li></ol></li><li> Berechnen Sie die Menge der Eier erforderlich sind, um das Experiment durchzuführen. Ziel für 300 bis 500 Eier pro Käfig. Mindestens drei replizieren Diapause Käfige und drei replizieren nicht Diapause Käfige für die RNA-Generation benötigt. Dieser beläuft sich auf 1,800-3,000 Eier pro Experiment ca.. <ol><li> Hatch die Eier durch Eintauchen Ei Papiere in ca. 500 ml VE H 2 O </li><li> Hinzufügen &lt;em&gt; ca. wie zuvor 1 ml Essen Aufschlämmung, bestehend aus Grundfutter und Artemia beschrieben 24. Decken Sie Behälter mit Mesh, und halten Sie die Masche an Ort und Stelle mit einem Gummiband. </li><li> Platzieren Sie in der Photoperiode LD Schrank für ca. 24 Std. </li></ol></li><li> Übertragungs geschlüpften Larven zu 10 x 10 x 2 cm Petrischalen mit ca. 90 ml gefüllt entionisiertem H 2 O. <ol><li> Halten Sie ca. 30 Larven pro Schale. Übertragen Larven zu reinigen Gerichte jeden 48-72 Stunden, zum Beispiel jeden Montag, Mittwoch und Freitag (MWF) 24. </li><li> Feed ca. 1 ml Essen Aufschlämmung, bestehend aus Boden Hundefutter und Artemia in VE-Wasser jeden MWF wie zuvor beschrieben 24. </li></ol></li><li> Bis drei Minuten vor vier Erwachsenen Käfige diese Einstellung für jedes Photoperiode Behandlung, wobei jeder Käfig einen biologischen Replikation. <ol><li> Von gegenüberliegenden Seiten des 9,5 l Eimer, schneiden Sie ein 10-für-14 cm Loch, und ein weiteres Loch mit 15 cm Durchmesser. Cover das erste mit Mesh. Geschnitten etwa eine Fußlänge eines orthopädischen Strumpf und kleben einem Ende um die Innenseite der anderen Bohrung. </li><li> Schneiden Sie das Fußende der Strumpf aus und Knoten geschlossen - offen nur bei Zugriff auf das Innere des Käfigs erforderlich. Für den Käfig Deckel, schneiden Sie den gesamten Innenraum, so dass nur die Felge, und ersetzen Sie den Innenraum Kunststoff mit Mesh-24. </li><li> Achten Sie auf die Photoperiode, replizieren Zahl, Käfig Startdatum und andere Informationen, die für das Experiment mit wasserfesten Stift auf der Seite des Käfigs. </li></ol></li><li> Den Boden der Erwachsenen Käfige mit feuchtem Filterpapier. Befeuchten des Filterpapiers mit ausreichend entionisiertem H 2 O, lokale Luftfeuchtigkeit im Käfigs zu erhöhen, aber vermeiden stehendes Wasser, das Eiablage auf dem Filterpapier 24 stimulieren kann. Überprüfen Sie das Filterpapier täglich zum Trocknen, wieder nass, wenn notwendig. </li><li> Um eine ausreichende Eier für eine RNA-Bibliothek zu erstellen, mindestens 100 Frauen / 9,5 L cage, mit nicht mehr als 500 Mücken pro Käfig. </li><li> Sammeln Puppen MWF und in eine kleine Tasse sauber H 2 O bei einer Dichte von nicht mehr als 50 Puppen pro 25 ml H 2 O Übertragen Sie die Puppen cup einem Erwachsenen Käfig. Platz Käfige in der jeweiligen Photoperiode Schrank - A. albopictus Puppen sind lichtempfindlich 15. </li><li> Stellen Sie sicher, dass täglich H 2 O in Tassen ist sauber und klar, und entfernen abgestorbene Puppen, weil Aufbau von toten Puppen können Massensterben verursachen. Entfernen H 2 O Tassen, nachdem alle Puppen entstehen. </li><li> Platzieren organische Rosinen auf dem oberen Gitter des Käfigs Zucker zur taucht Erwachsenen bereitzustellen. Überwachen Sie Rosinen, und ändern Sie sie alle 3-5 Tage, um Schimmel Ansammlung zu verhindern. </li></ol> 2. Wartung der Erwachsene zu Paarung und Eierproduktion zulassen <ol><li> Pflegen Käfigen bei hoher Feuchtigkeit (ca. 80%) durch Auskleiden der Käfigunterteil mit einem feuchten Filterpapier, und den Zugriff auf Nicht-schimmelig Rosinen, wie oben beschrieben (Abschnitons 1.5 und 1.9). </li></ol> 3. Blut-Fütterung <ol><li> Bereiten Sie sich darauf Blutzufuhr Weibchen zwischen zwei bis sechs Tage nach dem Schlüpfen, um sicherzustellen, dass Frauen haben mindestens acht eindeutige kurze Tage ausgesetzt worden, bevor der Eiablage beginnt für nahezu 100% Diapause Eier 14. </li><li> Bereiten Sie die Hemotek Membran Zuführsystem. Stecken Sie die Zuführungseinheiten in die Stromversorgung. Die Temperatur einer jeden Einheit auf 37 ° C mit der Stellschraube. Mit einem elektronischen Thermometer und Sonde, um die Temperatur der Zuführungseinheit während der Kalibrierung zu messen. </li><li> Bereiten Sie die Mahlzeit Reservoir. Dehnen Sie ein Quadrat von Kollagen Fütterung Membran über der Öffnung der Mahlzeit Behälter und sichern Sie sie mit einem O-Ring. Die Ecken, um Falten zu entfernen Ziehen; schneiden Sie die überschüssige Membran mit einer Schere. HINWEIS: Collagen Membran kann nicht gut für alle Mückenarten zu arbeiten, und es kann notwendig sein, verschiedene Arten versucht, die optimale Membran zu finden, wenn die Arbeit mit einemaußer A. Arten albopictus. Parafilm funktioniert gut mit Culex pipiens. <ol><li> Wenn Blut wird bei Raumtemperatur für mindestens 1 Stunde vor der Verwendung gefroren gelagert, Tauwetter. </li><li> Halten Sie den Behälter, so dass die Membran nach unten zeigt, werden nicht unterstützt, und die Einfüllöffnungen nach oben zeigt. Verwenden einer Transferpipette oder Spritze, um das Reservoir mit etwa 3 ml Vollblut von Hühnern, Natriumcitrat als Antikoagulans einfüllen. Siegel Einfüllöffnungen mit Kunststoffstopfen. </li></ol></li><li> Befestigen Sie das vorbereitet Reservoir zur Zuführung durch Aufschrauben auf das Gestüt auf der Wärmeübertragungsplatte auf der Unterseite des Förderers. Kehren Sie die Zuführung und setzen Sie ihn auf dem Käfig, Membranseite nach unten, so dass Mücken können durch die Maschen des Käfigs zu ernähren. Halten Sie die Zuführung auf den Käfig ca. 45 min zu Fütterung zu maximieren. </li></ol> 4. Anregung Eiablage <ol><li> Vier bis fünf Tage nach der Blutmahlzeit, rüsten jeden Käfig mit einem dunkel gefärbten 50 ml cmit rohen Samenkeimung Papier (Ei Papier) oder strukturierte nicht-gebleichten Papiertuch ausgekleidet und füllen auf halbem Weg mit VE-Wasser 9. Wenn mehr als 250 Mücken sind in einem Käfig, verwenden Sie zwei Tassen. HINWEIS: Bei kleinen Käfigen oder Einzel weibliche Fläschchen kann &quot;Heu-Infusion&quot; in Eiablagebehälter Eiablage durch den Geruch der mikrobiellen Flora 25 zu erhöhen. </li></ol> 5. sammeln und speichern Eggs <ol><li> Beginnen Eiersammlung innerhalb von 4-5 Tagen von blutsaugenden, weil die Eierproduktion Spitzen typischerweise etwa fünf Tage nach Blut Fütterung und dann nachlässt in der nächsten Woche. </li><li> Vary Eiersammlung Frequenz auf Notwendigkeiten des Experiments. Für allgemeine Zwecke, sammeln Eier Papiere auf einer MWF Zeitplan. Entfernen Ei Papiere aus jedem Käfig und ersetzen mit frischem Papier. Zeigen kürzlich entfernt Papiere in Petrischalen und lagern in SD Photoperiode Schrank auf verwirrende Auswirkungen ei der Lagerung zu vermeiden. </li><li> Lassen Sie Eier Papieren neu<html> Haupt nass für 2 Tage nach der Eiablage an serösen Nagelhautbildung, die Ei Austrocknung Widerstand 26 erhöht ermöglichen. </li><li> Etwa 48 Stunden nach der Sammlung, trockene Eier im Freien. Trocknen Sie das Papier, so dass es schlaff und leicht feucht anfühlen, aber nicht so feucht, dass das Papier dunkel von H 2 O oder stimuliert Schlüpfen der Eier. HINWEIS: Ein 6.5 &quot;x 4&quot; Papier kann etwa 3,5 Stunden dauern, um zu trocknen. Seien Sie vorsichtig, nicht zu über-trocken Ei Papiere, da dies in Ei Austrocknung 27 führen. </li><li> Reserve zusätzliche Eier sowohl von LD und SD-Photoperioden zu Diapause Häufigkeit zu beurteilen und interpretieren die photoperiodische Effekt (siehe Mess Diapause, Abschnitt 6). </li><li> Für langfristige Lagerung, halten Ei Papiere bei 21 ° C und ca. 80% Luftfeuchtigkeit in Petrischalen. Halten Sie Petrischalen in einer Tupperware Vorratsbehälter mit einer Flasche Wasser auf lokale Feuchtigkeit zu halten, wie der Embryonalentwicklung dauert vier bis fünf Tage bei 21 ° C. </li></ol>ove_title &quot;&gt; 6. Messen Sie Diapause Inzidenz <ol><li> Verwenden Sie zusätzliche vorbehalten Embryonen (siehe Stimulieren Eiablage, Abschnitt 4), die 7 bis 20 Tage alt, um die Diapause Antwort zu quantifizieren sind. </li><li> Notieren Sie sich die Anzahl der auf jedem Ei papier Eier. </li><li> Stimulieren Sie Eier, die von Notlaufeigenschaften einzelnen Ei Papiere in einem 90 ml Petrischale mit ca. 80 ml ​​VE H 2 O schlüpfen In etwa 0,25 ml Lebensmittel Gülle. </li><li> Nach 24 Stunden tally die Anzahl der schraffierten ersten Larvenstadium. Legen Sie die Petrischale auf eine schwarze Oberfläche zu Larven zu visualisieren und legen Sie eine Lichtquelle auf einer Seite der Schale. Die Larven werden von der Lichtquelle weg bewegen, was eine klare Bilanz von einzelnen Larven. Entfernen Sie einzelne Larven mit einer Pipette beim Zählen erzählt einzelnen Larven zu verhindern. </li><li> Platz Ei Papiere in eine neue Petrischale und wieder trocken. Re-Eier brüten nach ca. 1 Woche wieder übereinstimmen Eiern geschlüpft Verwendung des vorstehenden Verfahrens. </li><li> Platz Ei Papiere mit dem restlichen u<html> n-geschlüpften Eier in neue 90 ml Petrischalen mit ca. 80 ml ​​Bleichlösung 28. Sicherzustellen, dass die Eier Papiere vollständig in der Bleichlösung eingetaucht und lassen im Abzug über Nacht, um den Geruch der Bleich vermeiden. HINWEIS: Bleichlösung kann für ~ 1 Woche bei 4 ° C gelagert werden, sollte aber ansonsten frisch hergestellt werden. </li><li> Überprüfen Sie Eier mit einem Lichtmikroskop als Bleich das Chorion zu löschen und Visualisierung von embryonierten, un-geschlüpften Eiern. Wenn das Ei embryonierten, wird das Ei eine cremefarbene, mit den Augen als zwei kleine schwarze Punkte gegenübereinander auf der Rückenseite erscheinen. Tally die Anzahl der nicht-schraffierten, embryonierten Eiern 13. </li><li> Bestimmen Diapause Einfall mit der folgenden Formel:% Diapause = keine. embryonierte un-Eier ausgebrütet / (Nr. geschlüpften Eier + Nr. embryonierte un-Eier ausgebrütet) x 100 13. </li></ol> 7. RNA-Extraktion aus Eier / Larven pharate NHALT &quot;&gt; Hinweis: Verwenden Trizol in einer Sterilbank. <ol><li> Pinsel Mücke Eier enthalten entwickelnden Embryonen oder pharate Larven an bestimmten Entwicklungszeitpunkten vom Ei Papiere Glasschleifer mit einer Kamelhaarbürste. Schleifen Sie die Eier in Trizol (1 ml pro 50 bis 100 mg Gewebe), bis sie vollständig pulverisiert. Verwenden Sie mindestens 400 Eier pro Bibliothek, um eine ausreichende RNA ergeben. <ol><li> Alternativ Snap freeze Eier in flüssigem Stickstoff und bei -80 ° C in Mikrozentrifugenröhrchen für bis zu einem Monat vor dem Mahlen in Trizol gespeichert. </li></ol></li><li> Führen RNA-Extraktion in Trizol gefolgt von Isopropanolfällung nach Herstelleranweisungen. </li><li> Behandeln Sie die Bank mit RNase Dekontaminationslösung oder andere Mittel, um alle restlichen Nukleasen zu entfernen, um RNA-Abbau zu vermeiden. <ol><li> Behandeln Sie die extrahierte RNA mit DNase. Nach den Anweisungen des Herstellers, Inkubation der RNA-Proben mit DNase für 30 min bei 37 ° C. Verwenden Sie 1 und #181; l DNase für bis zu 10 &amp; mgr; g RNA in einer 50 ul Reaktion. Erhöhung der Menge an DNase, wenn es mehr als 10 &amp; mgr; g RNA in einer Reaktion. </li><li> Inaktivierung von DNase durch Zugabe von 5 ul suspendiert DNase-Inaktivierung Reagenz. Inkubiere 5 Minuten bei Raumtemperatur, dreimal während der Inkubationsperiode (leichter Verwirbelung). </li><li> Zentrifugieren bei 10.000 × g für 1,5 min. Die überstehende, die die behandelten RNA Proben in frische Röhrchen zur anschließenden Schritten. </li></ol></li><li> Bewerten Sie die Qualität der Gesamt-RNA-Proben mittels Fluorometrie. Senden Sie die Proben auf eine Spezialanlage mit geeignete Instrument für diese Aufgabe. Die Anlage wird auf dem Chip-Gelelektrophorese durchgeführt, um die Größe der RNA-Spezies in der Probe durch Fluoreszenzfarbstoff in der Chip instilliert visualisiert bestimmen. Die Ergebnisse werden als ein Elektropherogramm zurückgegeben. <ol><li> Bestimmung der Integrität der Gesamt-RNA-Proben durch das Vorhandensein oder Fehlen von Abbauprodukten, wie durch das Vorhandensein o belegenf Peaks zwischen 18S und 5S rRNA Peaks auf der resultierenden Elektropherogramm (1B). </li></ol></li></ol> 8. RNA Sequenzierung <ol><li> Senden der Gesamt-RNA-Proben mit einer ausreichend hohen Qualität (1A) und Menge (in der Regel&gt; 3 ug pro Bibliothek) an eine kommerzielle Ordnungszentrum für den Bau von angereichertem Paired-End mRNA-Bibliotheken und mRNA-Sequenzierung nach Standardprotokollen. </li><li> Wenn mehr als eine Spur wird für die Sequenzierung eines einzigen Experiment verwendet wird, aufgeteilt einzelnen Bibliotheken in zwei Fahrspuren für die Sequenzierung für technische Unterschiede zwischen den Fahrspuren während der Sequenzierung zu berücksichtigen. </li></ol> 9. Illumina lesen Reinigung ANMERKUNG: Abbildung 2 zeigt die Bioinformatik Teil dieses Protokolls. Für eine vollständige Liste aller Programme und Ressourcen in der Bioinformatik Abschnitt dieses Protokoll verwendet, auf die Tabelle 1 verwiesen. InAußerdem STADT Datei 1 enthält Befehlszeilenbeispiele für jede der folgenden Bioinformatik Protokollschritte. <ol><li> Verwenden ssaha2 29 (Tabelle 1), um Gruppen aus 95% Identität oder höher, um der NCBI UniVec Core-Datenbank identifiziert (Tabelle 1), A. albopictus rRNA-Sequenz (GenBank # L22060.1) und Sequenzierung Adaptern (detaillierte Befehlszeilenbeispiele in STADT Datei-1 zur Verfügung gestellt). Entfernen Lesepaare mit Streichhölzern mit Perl oder eine ähnliche Scripting-Tool, beispielsweise durch Anpassung der bereitgestellten Perl-Skript (Supplemental Datei 2). </li><li> Reinigen Sie verbleibende liest mit dem SolexaQA Paket 30 (Tabelle 1; Supplemental Datei 1): schneiden Regionen mit phred Punktzahl Äquivalent von weniger als 20 mit den Standardeinstellungen des DynamicTrim.pl. <ol><li> Entfernen Sie liest die kürzer als 25 bp mit LengthSort.pl auf Vorwärts- und Rückwärts liest gleichzeitig. Bewerten Sie die Qualität der fastq gereinigt Dateien mit FastQC (Tabelle 1 </strong&gt;) - insbesondere, ob die pro-Basensequenz Qualität und die pro-Sequenz, Qualitätswerte sind über 20. </li></ol></li></ol> 10. Digitale Normalisierung <ol><li> Führen Sie eine Runde von digitalen Normalisierung auf dem gereinigten liest mit Hilfe des Khmer-Werkzeug 31 (Tabelle 1; Supplemental Datei 1), insbesondere normalize-by-median.py (mit k-mer Größe 20, eine Abdeckung Cut-off von 20, und x = 1e10). </li><li> Alternativ, wenn eine Maschine mit hoher RAM zur Verfügung steht (Hunderte von GB), verwenden Sie Trinity normalize_by_kmer_coverage.pl Skript (Tabelle 1). </li></ol> 11. De Novo Transkriptom Assembly <ol><li> Beim Zugang zu einem Computer oder einem Computer-Cluster mit bis zu 256 GB RAM und 24 CPUs, abhängig von der Größe der Anordnung. </li><li> Verwenden Trinity 32 (Tabelle 1; Supplemental Datei-1), um die digital normalisierte Lese in Contigs gesetzt zu montieren. So verkleinernSpeichernutzung, verwenden --min_kmer_cov 2. </li></ol> 12. Montage Bewertung <ol><li> Führen assemblathon_stats.pl vom Assemblathon2 Projekt 33 auf der Fläche zu Trinity-Ausgang. Dieses Skript führt Grundrechenarten relevant Auswertung Montagequalität, wie die Anzahl der Gerüste, N50, Montage Zusammensetzung und (Tabelle 1; Supplemental Datei 1). </li></ol> 13. Markieren des Assembled Transkriptom <ol><li> Führen BLASTX (Tabelle 1) der Anordnung gegenüber einem Referenzprotein-Set; für Moskitos, Drosophila melanogaster, Anopheles gambiae, Culex pipiens und Aedes aegypti eignen Referenzen. Genauer gesagt, formatieren Sie das Referenzprotein fasta Datei für Explosion, gefolgt von blastx (Supplemental Datei 1). </li></ol> 14. Karte Liest der Versammlung Mit RSEM 34 (Tabelle 1) <ol><li> Erstellen Sie eine Datei &quot;transcript-to-Gen-Karte&quot;, in dem dererste Spalte enthält die Referenz-Gen-IDs und die zweite Spalte die Fläche zu IDs. In einem Spreadsheet-Editor, tauschen die ersten und zweiten Spalte von der BLASTX Ausgang, und schreiben Sie diese Spalten in eine TXT-Datei. Verwenden Sie die Linebreak-Programm um Zeilenumbrüche in der resultierenden Textformat an den Unix-Format zu konvertieren. </li><li> Erstellen Sie eine Referenzdatensatz aus der Transkriptom fasta Datei mit dem RSEM-Vorbereitung Verweis Skript, in der RSEM Paket (Supplemental Datei 1) zur Verfügung gestellt. </li><li> Berechnen Sie die Expressionswerte für jede Bibliothek mit dem Befehl RSEM-calculate-Ausdruck, in der RSEM Paket (Supplemental Datei 1) zur Verfügung gestellt. Wie liest, verwenden Sie die gepaart fastq Dateien aus dem Lesereinigungsschritt (Schritt 9.2) ergibt. <ol><li> Wenn RNA aus einer biologischen Replikation wurde für die Sequenzierung in zwei Bahnen aufgeteilt, umfassen sowohl fastq Dateien im Ausdruck Berechnung, um eine einzelne Datei zu generieren. </li></ol></li><li> Konvertieren Sie die Ausdruck ergibt sich aus jeder Bibliothek zu einer Matrix leicht von anderen p verarbeitetROGRAMME mit dem mitgelieferten Skript RSEM-generate-Data-Matrix, in der RSEM Paket (Supplemental Datei 1) zur Verfügung gestellt. </li></ol> 15. Differential Expressionsanalyse <ol><li> Installieren R und Kantenschneider (Tabelle 1). </li><li> Verwenden read.delim die RSEM Ergebnisse aus Schritt 14.3 (Supplemental Datei-1) zu laden. Falls erforderlich, ergänzen die Zähler auf die nächste ganze Zahl. HINWEIS: Der Kantenschneider Leitfaden empfiehlt die Begrenzung der Datenmenge, um Gene mit hoch genug zum Ausdruck Bedeutung zu erkennen. </li><li> Um die Daten für Edger zu formatieren, erzeugen eine DGEList Objekt aus der geladenen Datei (Supplemental Datei 1). Dann normalisieren die Daten mit TMM Normalisierung (Supplemental Datei 1). Schätzen Sie die gemeinsame und tagwise Dispersionen der Daten (Supplemental Datei 1). </li><li> Identifizieren differentiell exprimierte Gene mit einem Benjamini-Hochberg korrigierten p-Wert &lt;0,05 (Supplemental Datei 1). Zeichnen Sie die Verteilung der log-fach Wechsel vs. Fülle (Supplemental Datei 1). </li></ol>

<ol>
	<li>Bilyk, K. T., Cheng, C. H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+of+gene+expression+in+extreme+cold+-+reference+transcriptome+for+the+high-Antarctic+cryopelagic+notothenioid+fish+Pagothenia+borchgrevinki.">Model of gene expression in extreme cold - reference transcriptome for the high-Antarctic cryopelagic notothenioid fish Pagothenia borchgrevinki.</a> BMC Genomics. 14, 634 (2013).</li><li>Chapman, M. A., Hiscock, S. J., Filatov, 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=Genomic+divergence+during+speciation+driven+by+adaptation+to+altitude.">Genomic divergence during speciation driven by adaptation to altitude.</a> Mol. Biol. Evol. 30, 2553-2567 (2013).</li><li>Schwarz, 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=Sympatric+ecological+speciation+meets+pyrosequencing:+sampling+the+transcriptome+of+the+apple+maggot+Rhagoletis+pomonella.">Sympatric ecological speciation meets pyrosequencing: sampling the transcriptome of the apple maggot Rhagoletis pomonella.</a> BMC Genomics. 10, 633 (2009).</li><li>Barshis, D. 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=Genomic+basis+for+coral+resilience+to+climate+change.">Genomic basis for coral resilience to climate change.</a> P. Natl Acad. Sci. USA. 110, 1387-1392 (2013).</li><li>Urbanski, J. M., Aruda, A., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptional+element+of+the+diapause+program+in+the+Asian+tiger+mosquito,+Aedes+albopictus,+identified+by+suppressive+subtractive+hybridization.">A transcriptional element of the diapause program in the Asian tiger mosquito, Aedes albopictus, identified by suppressive subtractive hybridization.</a> J. Insect Physiol. 56, 1147-1154 (2010).</li><li>Huang, Y. 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+duck+genome+and+transcriptome+provide+insight+into+an+avian+influenza+virus+reservoir+species.">The duck genome and transcriptome provide insight into an avian influenza virus reservoir species.</a> Nat. Genet. 45, 776-783 (2013).</li><li>Sessions, O. 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=Host+cell+transcriptome+profile+during+wild-type+and+attenuated+dengue+virus+infection.">Host cell transcriptome profile during wild-type and attenuated dengue virus infection.</a> PLoS Negl. Trop. Dis. 7 (3), 2107 (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Elsik, C. G., Denlinger, D. L., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+sequencing+reveals+complex+mechanisms+of+diapause+preparation+in+the+invasive+mosquito,+Aedes+albopictus.">Deep sequencing reveals complex mechanisms of diapause preparation in the invasive mosquito, Aedes albopictus.</a> P. R. Soc B. 280, (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Elsik, C. G., Denlinger, D. L., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+sequencing+as+a+platform+to+elucidate+molecular+components+of+the+diapause+response+in+Aedes+albopictus.">Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in Aedes albopictus.</a> Physiol. Entomol. 38, 173-181 (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Denlinger, D. L., Elsik, C. G., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+de+novo+transcriptome+of+the+Asian+tiger+mosquito,+Aedes+albopictus,+to+identify+candidate+transcripts+for+diapause+preparation.">A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation.</a> BMC Genomics. 12, 619 (2011).</li><li>Benedict, M. Q., Levine, R. S., Hawley, W. A., Lounibos, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spread+of+the+tiger:+Global+risk+of+invasion+by+the+mosquito+Aedes+albopictus.">Spread of the tiger: Global risk of invasion by the mosquito Aedes albopictus.</a> Vector-Borne Zoonot. 7, 76-85 (2007).</li><li>Lounibos, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasions+by+insect+vectors+of+human+disease.">Invasions by insect vectors of human disease.</a> Annu. Rev. Entomol. 47, 233-266 (2002).</li><li>Urbanski, J. 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=Rapid+adaptive+evolution+of+photoperiodic+response+during+invasion+and+range+expansion+across+a+climatic+gradient.">Rapid adaptive evolution of photoperiodic response during invasion and range expansion across a climatic gradient.</a> Am. Nat. 179, 490-500 (2012).</li><li>Lounibos, L. P., Escher, R. L., Lourenco-de-Oliveria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+evolution+of+photoperiodic+diapause+in+temperate+and+tropical+invasive+populations+of+Aedes+albopictus+(Diptera+Culicidae).">Asymmetric evolution of photoperiodic diapause in temperate and tropical invasive populations of Aedes albopictus (Diptera Culicidae).</a> Ann. Entomol. Soc. Am. 96, 512-518 (2003).</li><li>Mori, A., Oda, T., Wada, 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> Studies on the egg diapause and overwintering of Aedes albopictus in Nagasaki. Trop. Med. 23, 79-90 (1981).</li><li>Pumpuni, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Factors influencing photoperiodic control of egg diapause in Aedes albopictus [dissertation]. , (1989).</li><li>Wang, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+influence+of+photoperiod+on+egg+diapause+in+Aedes+albopictus+Skuse.">Observations on the influence of photoperiod on egg diapause in Aedes albopictus Skuse.</a> Acta Entomol. Sinica. 15, 75-77 (1966).</li><li>Sota, T., Mogi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival-time+and+resistance+to+desiccation+of+diapause+and+non-diapause+eggs+of+temperate+Aedes+(Stegomyia)+mosquitoes.">Survival-time and resistance to desiccation of diapause and non-diapause eggs of temperate Aedes (Stegomyia) mosquitoes.</a> Entomol. Exp. Appl. 63, 155-161 (1992).</li><li>Reynolds, J. A., Poelchau, M. F., Rahman, Z., Armbruster, P. A., Denlinger, 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=Transcript+profiling+reveals+mechanisms+for+lipid+conservation+during+diapause+in+the+mosquito,+Aedes+albopictus.">Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus.</a> J. Insect Physiol. 58, 966-973 (2012).</li><li>Andrewartha, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diapause+in+relation+to+the+ecology+of+insects.">Diapause in relation to the ecology of insects.</a> Biol. Rev. 27, 50-107 (1952).</li><li>Danks, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+Dormancy:+An+Ecological+Perspective.">Insect Dormancy: An Ecological Perspective.</a> Biological Survey of Canada (Terrestrial Arthropods). , (1987).</li><li>Denlinger, 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=Regulation+of+diapause.">Regulation of diapause.</a> Ann. Rev. Entomol. 47, 93-122 (2002).</li><li>Rev Entomol, A. n. 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> 59, 93-122 (2014).</li><li>Armbruster, P. A., Conn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+variation+of+larval+growth+in+North+American+Aedes+albopictus+(Diptera).">Geographic variation of larval growth in North American Aedes albopictus (Diptera).</a> Culicidae). Ann. Entomol. Soc. Am. 99, 1234-1243 (2006).</li><li>Reiter, P., Amador, M. A., Colon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+CDC+ovitrap+with+hay+infusions+for+daily+monitoring+of+Aedes+aegypti+populations.">Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations.</a> J. Am. Mosquito Contr. Association. 7 (1), 52 (1991).</li><li>Rezende, G. L., 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=Embryonic+desiccation+resistance+in+Aedes+aegypti:+presumptive+role+of+the+chitinized+serosal+cuticle.">Embryonic desiccation resistance in Aedes aegypti: presumptive role of the chitinized serosal cuticle.</a> BMC Dev. Biol. 8, 182 (2008).</li><li>Munstermann, L., Crampton, J., Beard, C., C, L. o. u. i. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Care+and+maintenance+of+Aedes+mosquito+colonies.">Care and maintenance of Aedes mosquito colonies.</a> The Molecular Biology of Insect Disease Vectors. , 13-20 (1997).</li><li>Trpis, 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+new+bleaching+and+decalcifying+method+for+general+use+in+zoology.">A new bleaching and decalcifying method for general use in zoology.</a> Can. J. Zoolog. 48, 892-893 (1970).</li><li>Ning, Z. M., Cox, A. J., Mullikin, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SSAHA:+A+fast+search+method+for+large+DNA+databases.">SSAHA: A fast search method for large DNA databases.</a> Genome Res. 11 (10), 1725-1729 (2001).</li><li>Cox, M. P., Peterson, D. A., Biggs, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SolexaQA:+At-a-glance+quality+assessment+of+Illumina+second-generation+sequencing+data.">SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data.</a> BMC Bioinformatics. 11, 485 (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> Nat. Biotechnol. 29 (7), 644-652 (2011).</li><li>Bradnam, K. R., 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=Assemblathon+2:+evaluating+de+novo+methods+of+genome+assembly+in+three+vertebrate+species.">Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species.</a> GigaScience. 2, 10 (2013).</li><li>Li, B., Dewey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSEM:+accurate+transcript+quantification+from+RNA-Seq+data+with+or+without+a+reference+genome.">RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.</a> BMC Bioinformatics. 12, 323 (2011).</li><li>White, B. N., De Lucca, F. L., Turner, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+analysis+of+RNA.">Preparation and analysis of RNA.</a> Analytical Biochemistry of Insects. , (1977).</li><li>Hawley, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+Aedes+albopictus.">The biology of Aedes albopictus.</a> J. Am. Mosq. Contr. Assoc. 4, 1-39 (1988).</li><li>Dowling, Z., Ladeau, S. L., Armbruster, P., Biehler, D., Leisnham, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Socioeconomic+status+affect+mosquito+(Diptera:Culicidae)+larval+habitat+type+availability+and+infestation+level.">Socioeconomic status affect mosquito (Diptera:Culicidae) larval habitat type availability and infestation level.</a> J. Med Entomol. 50, 764-772 (2013).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2,4,10.+Bloodfeeding:+Membrane+apparatuses+and+animals.">Chapter 2,4,10. Bloodfeeding: Membrane apparatuses and animals.</a> Methods in Anopheles Research. , (2010).</li><li>Das, S., Garver, S., Ramirez, J. R., Xi, Z., Dimopolous, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dengue+infections+in+mosquitoes+(A.+aegypti)+and+infection+phenotype+determination.">Protocol for dengue infections in mosquitoes (A. aegypti) and infection phenotype determination.</a> J. Vis. Exp. 5 (220), (2007).</li><li>Goff, S. 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=The+iPlant+collaborative:+cyberinfrastructure+for+plant+biology.">The iPlant collaborative: cyberinfrastructure for plant biology.</a> Plant Sci. 2 (34), (2011).</li><li>Robinson, M. D., McCarthy, D. J., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edgeR:+a+Bioconductor+package+for+differential+expression+analysis+of+digital+gene+expression+data.">edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.</a> Bioinformatics. 26, 139-140 (2010).</li><li>Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCHIME+improves+sensitivity+and+speed+of+chimera+detection.">UCHIME improves sensitivity and speed of chimera detection.</a> Bioinformatics. 27 (16), 2194-2220 (2011).</li><li>Goecks, 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=Galaxy:+a+comprehensive+approach+for+supporting+accessible,+reproducible,+and+transparent+computational+research+in+the+life+sciences.">Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences.</a> Genome Biol. 11 (8), 86 (2010).</li></ol>

The authors have nothing to disclose.

Dieses Protokoll stellt Methoden, um differentiell exprimierte Gene aufgrund entdecken induzierte Diapause in A. photoperiodisch albopictus. Das Protokoll ist, dass es auf einzigartige Weise Moskito Aufzucht und Bioinformatik-Techniken, um alle experimentelle Aspekte eines molekularen Physiologie Programm zugänglich Anfänger machen signifikante - insbesondere für diejenigen, die sich auf die photoperiodische Diapause Antwort. Bestehende Verfahren, nach unserer Kenntnis nicht bieten so viele Details ...

Rapid advances in next-generation sequencing (NGS) technologies are providing exciting opportunities to probe the molecular underpinnings of a wide range of genetically complex ecological adaptations in a broad diversity of non-model organisms1&#8211;3. This approach is extremely powerful because it establishes a basis for population and functional genomics studies of organisms with an especially interesting and/or well-described ecology or evolutionary history, as well as organisms of practical concern, such as agricultural pests and disease vectors. Thus, NGS technologies are leading to rapid advances in the fields of ecology and have the potential to address problems such as understanding the mechanistic bases of biological responses to rapid contemporary climate change4, the spread of invasive species5, and host-pathogen interactions6,7.
The extraordinary potential of NGS technologies for addressing basic and applied questions in ecology and evolutionary biology is in part due to the fact that these approaches can be applied to any organism at a moderate cost that is feasible for most research laboratories. Furthermore, these approaches provide genome-wide information without the requirement of a priori genetic resources such as a microarray chip or complete genome sequence. Nevertheless, to maximize the productivity of NGS experiments requires careful consideration of experimental design including issues such as the developmental timing and tissue-specificity of RNA sampling. Furthermore, the technical skills required to analyze the massive amounts of data produced by these experiments, often up to several hundred million DNA sequence reads, has been a particular challenge and has limited the widespread implementation of NGS approaches.
Recent RNA-Seq studies on the transcriptional bases of diapause in the invasive and medically important mosquito Aedes albopictus provide a useful example of some of the experimental protocols that can be employed to successfully apply NGS technology to studying the molecular basis of a complex ecological adaptation in a non-model organism8&#8211;10. A. albopictus is a highly invasive species that is native to Asia but has recently invaded North America, South America, Europe, and Africa11,12. Like many temperate insects, temperate populations of A. albopictus survive through winter by entering a type of dormancy referred to as photoperiodic diapause. In A. albopictus, exposure of pupal and adult females to short (autumnal) day lengths leads to the production of diapause eggs in which embryological development is completed, but the pharate larva inside the chorion of the egg enters a developmental arrest that renders the egg refractory to hatching stimulus15&#8211;17. Diapause eggs are more desiccation resistant5,18 and contain more total lipids19 than non-diapause eggs. Photoperiodic diapause in A. albopictus is thus a maternally controlled, adaptive phenotypic plasticity that is essential for surviving the harsh conditions of winter in temperate environments. Despite the well-understood ecological significance of photoperiodic diapause in a wide range of insects20,21, the molecular basis of this crucial adaptation is not well characterized in any insect22. In organisms such as A. albopictus that undergo an embryonic diapause at the pharate larval stage, it remains a particularly compelling challenge to understand how the photoperiodic signal received by the mother is passed to the offspring and persists through the course of embryonic development to cause arrest at the pharate larval stage.
This protocol describes mosquito rearing, experimental design and bioinformatics analyses for NGS experiments (transcriptome sequencing) performed to elucidate transcriptional components of photoperiodic diapause in A. albopictus. This protocol can be used for additional studies of diapause in A. albopictus, can be adapted to investigate diapause in other closely related species such as other aedine mosquitoes that undergo egg diapause23, and is also more generally relevant to employing NGS approaches to study the transcriptional bases of any complex adaptation in any insect.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Incubator - Model 818</td>
			<td style="width:116px;">Thermo-Scientific</td>
			<td align="right" style="width:109px;">3751</td>
			<td style="width:351px;">120V</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:209px;">Controlled environment room</td>
			<td>Thermax Scientific</td>
			<td style="width:109px;">N/A</td>
			<td style="width:351px;">Walk-in controlled environment room built to custom specifications by Thermax Scientific Products. A larger alternative to an incubator. http://thermmax.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Cool Fluorescent bulb</td>
			<td style="width:116px;">Philips</td>
			<td align="right">392183</td>
			<td style="width:351px;">4 Watt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Petri Dish 100mm x 20mm</td>
			<td style="width:116px;">Fisher</td>
			<td>08-772-E</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Filter Paper 20.5cm</td>
			<td style="width:116px;">Fisher</td>
			<td>09-803-6J</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">9.5L Bucket</td>
			<td style="width:116px;">Plastican</td>
			<td style="width:109px;">Bway Products</td>
			<td style="width:351px;">http://www.bwayproducts.com/sites/portal/plastic-products/plastic-open-head-pails/117</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Utility Fabric-Mosquito Netting White</td>
			<td style="width:116px;">Joann</td>
			<td>10173292</td>
			<td style="width:351px;">http://www.joann.com/utility-fabric-mosquito-netting-white/10173292.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Orthopedic stockings</td>
			<td>Albahealth</td>
			<td style="width:109px;">23650-040</td>
			<td style="width:351px;">product no. 081420</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Organic Raisins</td>
			<td style="width:116px;">Newman&#39;s Own</td>
			<td style="width:109px;">UPC: 884284040255</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Oviposition cups (brown)</td>
			<td>Fisher Scientific</td>
			<td>03-007-52</td>
			<td style="width:351px;">The product is actually an amber 125 mL bottle that we saw the top off of.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Recycled Paper Towels</td>
			<td style="width:116px;">Seventh Generation</td>
			<td style="width:109px;">30BPT120</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Modular Mates Square Tupperware Set</td>
			<td style="width:116px;">Tupperware</td>
			<td style="width:109px;"></td>
			<td style="width:351px;">http://order.tupperware.com/pls/htprod_www/coe$www.add_items</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Glass Grinder</td>
			<td style="width:116px;">Corning Incorporated</td>
			<td style="width:109px;">7727-2</td>
			<td style="width:351px;">These Tenbroeck tissue grinders break the eggs and release RNA into the TRI Reagent.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">TRI Reagent</td>
			<td style="width:116px;">Sigma Aldrich</td>
			<td style="width:109px;">T9424</td>
			<td style="width:351px;">Apply 1ml TRI Reagent per 50-100mg of tissue. Caution - this reagent is toxic.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:209px;">TURBO DNA-free</td>
			<td style="width:116px;">Ambion/Life Technologies</td>
			<td style="width:109px;">AM1907</td>
			<td style="width:351px;">This kit generates greater yield than traditional DNase treatment followed by phenol/chloroform cleanup, and it is simpler to use.</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:209px;">RNaseZap</td>
			<td style="width:116px;">Ambion/Life Technologies</td>
			<td style="width:109px;">AM9782</td>
			<td style="width:351px;">Apply liberally on the bench surfaces and any equipment that might be in contact with the RNA samples. The solution is slightly alkaline/corrosive, can cause irritation and is harmful when swallowed.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">2100 Bioanalyzer</td>
			<td style="width:116px;">Agilent Technologies</td>
			<td style="width:109px;">G2939AA</td>
			<td style="width:351px;">Place up to 12 RNA samples on one chip.</td>
		</tr>
		<tr height="187">
			<td height="187" style="height:187px;width:209px;">Hemotek Membrane Feeder</td>
			<td style="width:116px;">Hemotek&#160;</td>
			<td style="width:109px;">5W1</td>
			<td style="width:351px;">This system&#160; provides 5 feeding stations that can be used simultaneously. Includes PS5 Power Unit and Power cord; 5 FUI Feeders + Meal Reservoirs and O-rings; Plastic Plugs, Hemotek collagen feeding membrane; Temperature setting tool; and Plug extracting tool. The company&#39;s mailing address is: Hemotek Ltd; Unit 5 Union Court; Alan Ramsbottom Way; Great Harwood; Lancashire, UK; BB6 7FD; tel: +44 1254 889 307.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Digital Thermometer and Probe</td>
			<td style="width:116px;">Hemotek&#160;</td>
			<td style="width:109px;">MT3KFU</td>
			<td style="width:351px;">MicroT3 thermometer and KFU probe. This is used to set the temperature of each FUI feeding unit.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:209px;">Chicken Whole Blood, non-sterile with Sodium Citrate</td>
			<td style="width:116px;">Pel-Freez Biologicals</td>
			<td>33130-1</td>
			<td style="width:351px;">The 500 ml of blood were frozen and stored in 20 ml aliquots at -80 degrees C for up to 1 year.&#160; Thaw blood at room temperature for at least 1 h before using.</td>
		</tr>
	</tbody>
</table>

an experimental and bioinformatics protocol for rna-seq analyses of photoperiodic diapause in the asian tiger mosquito, aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

Fluorometrie zweier repräsentativer RNA Proben zeigten zwei Banden bei ca. 2000 nt (1A, B). Das Insekt 28S ribosomale RNA aus zwei Polynucleotidketten durch Wasserstoffbrücken, die sich leicht durch kurzes Erhitzen oder Agenten, die Wasserstoffbrückenbindungen 35 stören gestört zusammengehalten zusammen. Die resultierenden beiden Komponenten ungefähr die gleiche Größe wie der ribosomalen RNA 18S. Die zweite RNA-Probe zeigte eine hohe Abbauwerte (1B). <p class="jov...

Watch this Scientific Journal Video about An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus at JoVE.com

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Eine experimentelle und Bioinformatik-Protokoll für die RNA-seq Analysen der Photoperiodische Diapause in den asiatischen Tigermücke,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

an-experimental-bioinformatics-protocol-for-rna-seq-analyses

Research

JoVE Journal

Biology

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

13.2K Aufrufe.  Georgetown University. RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data. 

Serie: An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Protokoll für Mosquito Aufzucht (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Forschung

Biologie

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video bioinformatischen Analyse von humanen embryonalen Stammzellen Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

Ein Protokoll für die Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq Analyse Transkriptomen Thrombin-behandelt und Control Human Pulmonary mikrovaskulären Endothelzellen

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

Depletion der ribosomalen RNA für Mosquito Gut metagenomische RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Physiologische Aufnahmen und RNA-Sequenzierung der gustatorischen Anhangsgebilde der Gelbfieber-Moskito-<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Ein Protokoll für Labor Housing von Türkis Killifische (Nothobranchius Furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Experimentelles Protokoll für die Verwendung von Drosophila als ein Wirbellosen Modellsystem für Toxizitätstests im Labor

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

Eine Bioinformatik-Pipeline zur Untersuchung der molekularen Evolution und Genexpression mit RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Eine Schritt-für-Schritt-Anleitung zur Mücken-Elektroantennographie

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...