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

1. larvale allevamento di due A. albopictus gruppi di età adulta <ol><li> Impostare due armadi fotoperiodo con illuminazione programmabile a 21 ° C per l&#39;espressione ottimale diapausa 16 e circa l&#39;80% di umidità relativa. <ol><li> Programma un armadio per un 16L: luce 8D: ciclo scuro (un non-diapausa indurre LD fotoperiodo). Impostare il secondo armadio per 8L: 16D luce: ciclo scuro (diapausa indurre) 13. </li><li> &#39;Luci&#39; del programma contemporaneamente in entrambe le armadi per sincronizzare l&#39;ora circadiano tra fotoperiodi. </li></ol></li><li> Calcolare la quantità di uova necessarie per eseguire l&#39;esperimento. Obiettivo per 300-500 uova per gabbia. Almeno tre replicare gabbie diapausa e tre replicano gabbie non diapausa sono necessari per la produzione di RNA. Questo ammonta a circa il 1,800-3,000 uova per esperimento. <ol><li> Hatch le uova immergendo carte uovo in circa 500 ml di H 2 O. deionizzata </li><li> Aggiungi &lt;em&gt; ca. 1 ml sospensione degli alimenti costituiti da cibo per cani a terra e il gambero di salamoia come descritto in precedenza 24. Coprire il contenitore con rete, e mantenere la maglia in posizione con un elastico. </li><li> Mettere nell&#39;armadio LD fotoperiodo per circa 24 ore. </li></ol></li><li> Trasferimento larve schiuse a 10 x 10 x 2 cm Petri piatti pieni di ca. 90 ml di acqua deionizzata H 2 O. <ol><li> Mantenere ca. 30 larve per piatto. Trasferimento larve per pulire piatti ogni 48-72 ore, ad esempio ogni Lunedi, Mercoledì e Venerdì (MWF) 24. </li><li> Alimentazione circa 1 ml sospensione degli alimenti costituiti da cibo per cani a terra e artemie in acqua deionizzata ogni MWF come descritto in precedenza 24. </li></ol></li><li> Impostare 3-4 gabbie per adulti per ogni trattamento fotoperiodo, dove ogni gabbia comprende una replica biologica. <ol><li> Da bande opposte 9.5 L secchi, tagliare una 10-by-14 centimetri foro, e un altro foro di diametro 15 cm. Conel corso dei primi con rete. Tagliare circa un lunghezza di piede di una calza ortopedica, e incollate un&#39;estremità intorno l&#39;interno del foro. </li><li> Tagliare l&#39;estremità di piede della calza off e nodo arresto - aperto solo quando è necessario accedere all&#39;interno della gabbia. Per il coperchio della gabbia, tagliare fuori tutto l&#39;interno, lasciando solo il bordo, e sostituire la plastica interno con rete 24. </li><li> Notare il fotoperiodo, replicare numero, data di inizio della gabbia, e altre informazioni pertinenti per l&#39;esperimento con pennarello indelebile sul lato della gabbia. </li></ol></li><li> Foderare il fondo delle gabbie adulti con carta da filtro umida. Inumidire la carta da filtro con abbastanza deionizzata H 2 O per aumentare l&#39;umidità locale nella gabbia, ma evitare acqua stagnante, che può stimolare ovideposizione sulla carta da filtro 24. Controllare la carta da filtro giornaliera per essiccamento, ri-bagnato quando necessario. </li><li> Per produrre uova sufficienti per una libreria di RNA, comprende almeno 100 donne / 9.5 L cage, con non più di 500 zanzare per gabbia. </li><li> Raccogliere pupe MWF e posto in una tazzina di H 2 O pulita con una densità di non oltre il 50 pupe per 25 ml di H 2 O. Trasferire la coppa pupe di una gabbia adulto. Posizionare gabbie nel rispettivo cabinet fotoperiodo - A. albopictus pupe sono fotosensibile 15. </li><li> Assicurarsi quotidiana che H 2 O in tazze è pulito e chiaro, e rimuovere pupe morti, perché l&#39;accumulo di pupe morta può causare la mortalità di massa. Rimuovere H 2 O tazze dopo tutti pupe emergono. </li><li> Mettere uvetta organici sulla maglia superiore della gabbia di fornire zucchero per adulti emerse. Monitorare uvetta, e cambiarle ogni 3-5 giorni per evitare l&#39;accumulo di muffe. </li></ol> 2. Manutenzione di adulti per consentire l&#39;accoppiamento e Egg Production <ol><li> Mantenere gabbie a umidità elevata (circa. 80%) allineando il fondo della gabbia con una carta da filtro umida e fornire accesso a uva non-muffa, come descritto sopra (Sezioons 1.5 e 1.9). </li></ol> 3. si nutrono di sangue <ol><li> Preparatevi a femmine di sangue-alimentazione tra 2-6 giorni dopo eclosion per garantire che le femmine sono stati esposti ad almeno otto giorni prima della deposizione delle uova brevi inequivocabili inizia per quasi 100 uova% diapausa 14. </li><li> Preparare il sistema di alimentazione Hemotek membrana. Collegare le unità di alimentazione alla presa di corrente. Regolare la temperatura di ciascuna unità di 37 ° C con la vite di regolazione. Utilizzare un termometro elettronico e sonda per misurare la temperatura del gruppo di alimentazione durante la calibrazione. </li><li> Preparare il serbatoio pasto. Allungare un quadrato di membrana di collagene alimentazione sopra l&#39;apertura del serbatoio pasto e fissarlo con un O-ring. Tirare con attenzione gli angoli per rimuovere le rughe; tagliare la membrana in eccesso con le forbici. NOTA: membrana collagene non può funzionare bene per tutte le specie di zanzara, e può essere necessario provare diversi tipi di trovare membrana ottimale se si lavora con unspecie diverse da A. albopictus. Parafilm funziona bene con Culex pipiens. <ol><li> Se il sangue è conservato congelato, scongelare a temperatura ambiente per almeno 1 ora prima di utilizzare. </li><li> Tenere il serbatoio in modo che la membrana è rivolta verso il basso, e le porte di riempimento non supportati sono rivolti verso l&#39;alto. Utilizzare una pipetta di trasferimento o una siringa per riempire il serbatoio con circa 3 ml di sangue intero da polli che ha citrato di sodio come anticoagulante. Sigillare porte di riempimento con tappi di plastica. </li></ol></li><li> Fissare il serbatoio preparata all&#39;alimentatore avvitandolo sul perno sulla piastra di trasferimento di calore sul fondo dell&#39;alimentatore. Invertire l&#39;alimentatore e posizionarlo sulla parte superiore della gabbia, lato membrana verso il basso, in modo che le zanzare possono alimentare attraverso le maglie della gabbia. Tenere l&#39;alimentatore sulla gabbia per circa 45 min per massimizzare alimentazione. </li></ol> 4. Stimolare Deposizione delle uova <ol><li> Quattro o cinque giorni dopo pasto di sangue, dotare ogni gabbia con un colore scuro 50 ml cup foderata con carta greggi germinazione dei semi (carta d&#39;uovo) o il tovagliolo strutturato non sbiancata carta e riempire a metà con acqua deionizzata 9. Se più di 250 zanzare sono in una gabbia, utilizzare due tazze. NOTA: Per le piccole gabbie o fiale singolo femminile, &quot;infuso fieno&quot; in contenitori di deposizione delle uova può aumentare deposizione causa l&#39;odore della flora microbica 25. </li></ol> 5. Raccogliere e deposito Uova <ol><li> Iniziare la raccolta delle uova entro 4-5 giorni di alimentazione sangue, perché la produzione di uova picchi tipicamente di circa cinque giorni dopo l&#39;alimentazione di sangue, e poi scompare nel corso della settimana prossima. </li><li> Variare frequenza di raccolta delle uova sulla necessità dell&#39;esperimento. Per scopi generali, raccogliere documenti di uova su una pianificazione MWF. Rimuovere carte uovo da ogni gabbia e sostituirla con carta nuova. Posizionare i documenti rimossi di recente in piastre di Petri e conservare in cabinet fotoperiodo SD per evitare effetti confondenti di stoccaggio delle uova. </li><li> Lasciare carte uovo di ri<html> principale bagnato per 2 giorni dopo la deposizione delle uova per permettere la formazione sierosa cuticola, che aumenta la resistenza uovo essiccazione 26. </li><li> Raccolta postale circa 48 ore, uova asciugare all&#39;aria aperta. Essiccare la carta in modo che sia molle e leggermente umido al tatto, ma non così bagnato che la carta è scuro da H 2 O o stimola la schiusa delle uova. NOTA: A &quot;x 4&quot; carta 6.5 potrebbe richiedere circa 3,5 ore ad asciugare. Fare attenzione a non troppo secca carte d&#39;uovo, in quanto questo si tradurrà in uovo essiccazione 27. </li><li> Riserva uova aggiuntivi sia da LD e SD fotoperiodi per valutare l&#39;incidenza diapausa e interpretano l&#39;effetto photoperiodic (vedi Misurazione diapausa, sezione 6). </li><li> Per la conservazione a lungo termine, tenere le carte d&#39;uovo a 21 ° C e circa l&#39;80% di umidità in piastre di Petri. Tenere piastre di Petri in un contenitore Tupperware con un pallone di acqua per mantenere l&#39;umidità locale come lo sviluppo embrionale richiede 4-5 giorni a 21 ° C. </li></ol>ove_title &quot;&gt; 6. Misura diapausa Incidenza <ol><li> Utilizzare ulteriori embrioni riservati (cfr Stimolare Deposizione delle uova, sezione 4) che sono 7-20 giorni per quantificare la risposta diapausa. </li><li> Registrare il numero di uova presenti su ogni carta uovo. </li><li> Stimolare le uova per schiudersi immergendo completamente carte uovo individuali in un piatto di 90 ml di Petri con circa 80 ml ​​di H 2 O. deionizzata Aggiungere circa 0,25 ml cibo liquami. </li><li> Dopo 24 ore coincidere il numero di tratteggiata prima larve instar. Porre la capsula Petri su una superficie nera per visualizzare larve e posizionare una sorgente di luce su un lato del piatto. Le larve si allontanerà dalla fonte di luce, consentendo una chiara conteggio delle larve individuale. Rimuovere larve individuo con una pipetta, mentre il conteggio per evitare raccontare larve individuale. </li><li> Carte Luogo d&#39;uovo in una nuova piastra di Petri e ri-asciutto. Re-hatch uova dopo ~ 1 settimana e ancora coincidono uova schiuse utilizzando il metodo di cui sopra. </li><li> Documenti Posizionare uovo con lo u rimanenti<html> uova n-covato in nuovi 90 ml capsule di Petri con una soluzione sbiancante circa 80 ml ​​28. Assicurarsi che le carte di uova sono completamente immersi in soluzione sbiancante e lasciare sotto una cappa durante la notte per evitare l&#39;odore di candeggina. NOTA: Bleaching soluzione può essere conservata per ~ 1 settimana a 4 ° C, ma dovrebbe tuttavia essere fatta fresca. </li><li> Controllare le uova con un microscopio ottico come sbiancamento cancellerà il corion e permettere la visualizzazione di embrionate, uova non-covato. Se l&#39;uovo è embrionate, l&#39;uovo avrà un colore bianco sporco, con gli occhi che appaiono come due piccoli punti neri di fronte all&#39;altro sul lato dorsale. Coincidere il numero di non-nati, uova embrionate 13. </li><li> Determinare l&#39;incidenza diapausa con la seguente formula:% diapausa = no. uova embrionate un-nati / (n. di uova schiuse + n. embrionate di uova non-covato) x 100 13. </li></ol> 7. RNA Estrazione da uova / pharate Larve ONTENUTO &quot;&gt; NOTA: Uso Trizol in una cappa a flusso laminare. <ol><li> Uova di zanzara Brush contenenti embrioni in via di sviluppo o larve pharate in distinti momenti di sviluppo dalle carte a base di uova a macine di vetro con un pennello di peli di cammello. Macinare le uova in Trizol (1 ml per 50-100 mg di tessuto) fino a completo polverizzato. Utilizzare almeno 400 uova per biblioteca per produrre RNA sufficiente. <ol><li> In alternativa, far scattare le uova congelare in azoto liquido e conservati a -80 ° C in provette da microcentrifuga fino a un mese prima della macinazione, in Trizol. </li></ol></li><li> Eseguire l&#39;estrazione di RNA in Trizol seguita da isopropanolo precipitazione secondo le istruzioni del produttore. </li><li> Trattare panchina con soluzione RNase decontaminazione o altri agenti per rimuovere eventuali residui nucleasi per evitare la degradazione dell&#39;RNA. <ol><li> Trattare l&#39;RNA estratto con DNasi. Secondo le indicazioni del produttore, incubare i campioni di RNA con DNasi per 30 minuti a 37 ° C. Utilizzare 1 &amp; #181; l DNasi fino a 10 mg di RNA in una reazione di 50 microlitri. Aumentare la quantità di DNasi se vi sono più di 10 mg di RNA in una reazione. </li><li> Inattivare DNase aggiungendo 5 ml DNasi sospeso inattivazione reagente. Incubare 5 min a temperatura ambiente, mescolando tre volte durante il periodo di incubazione (vortex). </li><li> Centrifugare a 10.000 g per 1.5 min. Trasferire il surnatante contenente i campioni di RNA trattati ai tubi freschi per fasi successive. </li></ol></li><li> Valutare la qualità dei campioni di RNA totale da fluorometria. Inviare i campioni ad un impianto di specialità con strumento adeguato per questo compito. L&#39;impianto eseguirà elettroforesi su gel-chip per determinare le dimensioni delle specie di RNA nel campione, visualizzati da colorante fluorescente instillato nel chip. I risultati saranno restituiti come elettroferogramma. <ol><li> Determinare l&#39;integrità dei campioni di RNA totale dalla presenza o l&#39;assenza di prodotti di degradazione, come dimostra la presenza opicchi F tra le 18S e 5S ribosomiale picchi RNA sul dell&#39;elettroferogramma risultante (Figura 1B). </li></ol></li></ol> 8. RNA Sequencing <ol><li> Invia campioni di RNA totale con qualità sufficientemente elevata (Figura 1A) e quantità (generalmente&gt; 3 mg per libreria) a un centro commerciale sequenziamento per la costruzione di librerie arricchiti accoppiato fascia mRNA mRNA e sequenziamento, seguendo protocolli standard. </li><li> Se più di una corsia viene utilizzato per il sequenziamento di un singolo esperimento, dividere singole biblioteche in due corsie per il sequenziamento in funzione delle variazioni tecniche tra vicoli durante la sequenza. </li></ol> 9. lllumina Leggi Cleaning NOTA: La Figura 2 riassume la parte bioinformatica di questo protocollo. Per un elenco completo di tutti i programmi e le risorse utilizzate nella sezione bioinformatica di questo protocollo, fare riferimento alla Tabella 1. InInoltre, File supplementare 1 contiene esempi riga di comando per ciascuna delle seguenti fasi di protocollo di bioinformatica. <ol><li> Utilizzare ssaha2 29 (Tabella 1) per identificare le partite di 95% di identità o superiore al database NCBI UniVec core (Tabella 1), A. albopictus sequenza rRNA (GenBank # L22060.1), e gli adattatori di sequenziamento (esempi dettagliata della riga di comando fornite in file supplementare 1). Rimuovere le coppie a capire con le partite utilizzando Perl o uno strumento di scripting simile, ad esempio adattando lo script Perl in dotazione (File supplementare 2). </li><li> Pulire rimanendo legge con il pacchetto SolexaQA 30 (Tabella 1; Supplemental File 1): tagliare le regioni con un punteggio Phred equivalente di meno di 20 utilizzando le impostazioni predefinite di DynamicTrim.pl. <ol><li> Rimuovere legge più breve di 25 bp con LengthSort.pl sia in avanti e retromarcia legge simultaneamente. Valutare la qualità dei file puliti FASTQ con FastQC (Tabella 1 </strong&gt;) - in particolare, verificare che la qualità per-base di sequenza e il punteggio di qualità per-sequenza sono al di sopra di 20. </li></ol></li></ol> 10. Digital Normalizzazione <ol><li> Effettuare un giro di normalizzazione digitale sul pulito legge utilizzando lo strumento khmer 31 (Tabella 1; Supplemental File 1), in particolare normalize-by-median.py (con dimensioni della k-mer 20, una copertura cut-off di 20, e x = 1e10). </li><li> In alternativa, se una macchina ad alta RAM è disponibile (centinaia di GB), utilizzare lo script normalize_by_kmer_coverage.pl di Trinity (Tabella 1). </li></ol> 11. De Novo trascrittoma Assembly <ol><li> Ottenere accesso a un computer o un computer cluster con fino a 256 Gb di RAM e 24 CPU, a seconda delle dimensioni del gruppo. </li><li> Utilizzare Trinity 32 (Tabella 1; File supplementare 1) per assemblare la lettura digitale normalizzato impostare in contigs. Per ridurrel&#39;utilizzo della memoria, uso --min_kmer_cov 2. </li></ol> 12. Assemblea valutazione <ol><li> Eseguire assemblathon_stats.pl dal progetto Assemblathon2 33 sull&#39;uscita contig Trinità. Questo script esegue i calcoli di base per affrontare la valutazione della qualità di assemblaggio, come il numero di ponteggi, N50, la composizione di assemblaggio, e più (Tabella 1; File supplementare 1). </li></ol> 13. Annotazione del trascrittoma Assemblato <ol><li> Eseguire BLASTX (Tabella 1) l&#39;insieme contro una serie proteina di riferimento; per le zanzare, Drosophila melanogaster, Anopheles gambiae, Culex pipiens, e Aedes aegypti sono riferimenti adeguati. In particolare, formattare il file FASTA proteina di riferimento per esplosione, seguita da BLASTX (File supplementare 1). </li></ol> 14. Map Legge all&#39;Assemblea Utilizzo RSEM 34 (Tabella 1) <ol><li> Creare un file &#39;trascrizione-to-gene-map&#39;, in cui laprima colonna contiene gli ID del gene di riferimento, e la seconda colonna della IDS contig. In un editor di fogli di calcolo, scambiare la prima e la seconda colonna della uscita BLASTX, e scrivere queste colonne in un file .txt. Utilizzare il programma per convertire linebreak interruzioni di riga nel file .txt risultante in formato Unix. </li><li> Creare un set di dati di riferimento dal file FASTA trascrittoma utilizzando lo script rsem-preparare-riferimento, fornito nella confezione RSEM (File supplementare 1). </li><li> Calcolare i valori di espressione separatamente per ciascuna libreria utilizzando il comando rsem-calcolo-espressione, fornito nella confezione RSEM (File supplementare 1). Come si legge, utilizzare i file abbinati FASTQ risultanti dal passo-lettura pulizia (punto 9.2). <ol><li> Se RNA da una replica biologico è stato diviso in due corsie per il sequenziamento, includere entrambi i file FASTQ nel calcolo dell&#39;espressione per generare un unico file. </li></ol></li><li> Convertire i risultati di espressione di ogni libreria a una matrice facilmente trattati da altri programs utilizzando lo script rsem-generate-data-matrix forniti, forniti nella confezione RSEM (File supplementare 1). </li></ol> 15. Espressione Differential Analysis <ol><li> Installare R e levigabordi (Tabella 1). </li><li> Utilizzare read.delim per caricare i risultati RSEM dal punto 14.3 (File supplementare 1). Se necessario, completano i conteggi al numero intero più vicino. NOTA: La guida Edger raccomanda di limitare il dataset di geni con abbastanza espressione elevata per rilevare significato. </li><li> Per formattare i dati per edger, generare un oggetto DGEList dal file di dati caricati (File supplementare 1). Poi, normalizzare i dati utilizzando TMM normalizzazione (File supplementare 1). Stimare le dispersioni comuni e tagwise dei dati (File supplementare 1). </li><li> Identificare geni differenzialmente espressi con un Benjamini-Hochberg corretto p-value &lt;0.05 (File supplementare 1). Tracciare la distribuzione di log-fold-change vs. abbondanza (File supplementare 1). </li></ol>

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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. 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The authors have nothing to disclose.

Questo protocollo presenta i metodi per scoprire i geni differenzialmente espressi a causa photoperiodically diapausa indotta in A. albopictus. Il protocollo è significativo in quanto combina in modo unico allevamento di zanzare e di bioinformatica tecniche per rendere tutti gli aspetti sperimentali di un programma di fisiologia molecolare accessibile agli utenti alle prime armi - in particolare quelli incentrati sulla risposta diapausa photoperiodic. Metodi esistenti, a nostra conoscenza, non forniscono più ...

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.

Fluorometria di due campioni di RNA rappresentativi mostrato due bande a circa 2.000 nt (Figura 1A, B). Il 28S insetti RNA ribosomiale è costituito da due catene polinucleotidiche tenuti insieme da legami di idrogeno, che sono facilmente interrotti da brevi riscaldamento o agenti che sconvolgono legami idrogeno 35. Le risultanti due componenti sono approssimativamente le stesse dimensioni del 18S RNA ribosomiale. Il secondo campione RNA mostrato elevati livelli di degrado (Figura 1B)...

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.

Un Sperimentale e Bioinformatica Protocollo per analisi di RNA-Seq di photoperiodic diapausa nella zanzara tigre,<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, ...

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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 Views.  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. 

Video: 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.

Protocollo per la cura dei Mosquito (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 ...

Ricerca

Biologia

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 Bioinformatica Analisi delle embrionali umane crescita Colony cellule staminali

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.

Un protocollo per la Computer-Based Protein Structure Prediction e Funzione

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 Analisi trascrittomi in trombina-trattati e di controllo dell&#39;uomo polmonari cellule endoteliali microvascolari

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.

L&#39;esaurimento delle RNA ribosomiale per metagenomiche Gut Mosquito 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.

Fisiologici Registrazioni e RNA sequenziamento delle appendici gustative del Mosquito febbre gialla<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.

Un protocollo per laboratorio Housing di turchese Killifish (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.

Protocollo sperimentale per l'utilizzo di Drosophila come sistema modello invertebrati marini per test di tossicità in laboratorio

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.

Una pipeline bioinformatica per lo studio dell'evoluzione molecolare e dell'espressione genica usando 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.

Una guida passo-passo all'elettroantennography delle zanzare

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 ...