Name: physiological recordings and rna sequencing of the gustatory appendages of the yellow-fever mosquito aedes aegypti
Uploaded: 2014-12-30T14:00:00.000+00:00
Duration: 9 min 9 s
Description: Watch this Scientific Journal Video about Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti at JoVE.com

The authors thank Bryan T. Vinyard of the USDA, Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Research Center, Biometrical Consulting Service, Beltsville, MD for statistical analyses. This work was supported in part by a grant to J.C.D. from the Deployed War Fighter Protection (DWFP) Research Program funded by the Department of Defense through the Armed Forces Pest Management Board (AFPMB).

1. allevamento Ae. adulti aegypti <ol><li> Le uova si schiudono in circa ¾ di pollice in acque poco profonde cassetto. Sovraffollamento ridurrà la dimensione degli adulti. </li><li> Larve posteriore a 25 ° C (12-HL: 12-HD) ed alimentare con cibo per pesci a terra. NOTA: sovralimentazione può ridurre i tassi di sopravvivenza. </li><li> Rimuovere pupe individualmente da pipetta Pasteur quotidiano e trasferimento in piatti di plastica (9 centimetri × 5,5 centimetri) all&#39;interno secchielli di contenimento con fini coperchi maglia, stabilendo così 24 gruppi di età hr. </li><li> Mangimi zanzare adulte soluzione al 10% di saccarosio dal batuffolo di cotone collocato sulle pareti schermo di un contenitore gabbia le zanzare in una camera climatica a 27 ° C e 70% di umidità relativa sotto la stessa fotoperiodo come larve. </li><li> Per elettrofisiologia, utilizzare 5-10 giorno animali vecchi. In generale, gli animali più grandi produrranno risultati migliori. Per l&#39;isolamento di RNA, usare gli animali che si trovano all&#39;interno di un unico 24 ore raggruppamento età nel vecchio campo di 5-10 giorni. </li></ol><ol><li> Determinare una concentrazione adeguata di elettroliti (ad esempio 1-10 mm) suscita l&#39;attività minima da neuroni sensoriali selezionati. 10 mM NaCl o 1mm KCl sono elettroliti ragionevoli per testare l&#39;attività di base quando si inizia un esperimento. </li><li> Sciogliere ogni chimica sperimentale nel solvente appropriato (se non l&#39;acqua, usare etanolo o DMSO) che ha poco o nessun effetto sulla attività picco rispetto all&#39;elettrolito sola. Le concentrazioni finali di 10% etanolo o 1% DMSO sono concentrazioni iniziali ragionevoli. </li><li> Selezionare appropriata (ad esempio il chinino per amaro o di saccarosio per dolci) i prodotti chimici di controllo che sono ben descritti, deterrenti alimentazione o stimolanti negli insetti. </li></ol> 3. Elettrofisiologia (registrazione punta 8; Figura 1) <ol><li> Elettrodi di vetro Form tirando meccanicamente capillari di vetro. Ottimizzare il calore e tirare le impostazioni di tirare vetro per formare recordi opportunamente sagomataElettrodi ng. Rompere con cautela fine del elettrodo di vetro con pinze sotto un microscopio da dissezione per creare elettrodo punta apertura che è sufficientemente ampia per busta bersaglio sensillum, ma abbastanza piccolo per evitare il contatto con le strutture vicine. </li><li> Al microscopio, identificare visivamente dalla morfologia e posizionare il target sensilla / sensillum per garantire la ripetibilità della registrazione punta. Animali Prep per consentire l&#39;accesso gratuito al sensilla dall&#39;angolo di entrata dell&#39;elettrodo. </li><li> Freddo insetti anestetizzare adulti in freezer a -20 ° C. Evitare danni ai neuroni periferici da sovraesposizione a temperature fredde. Tagliare sottili strisce di nastro adesivo con lama di rasoio sul vetrino. Immobilizzare intero insetto su un vetrino con strisce strette. </li><li> Inserire un filo di tungsteno affilata chimicamente nel torace dorsale o occhio per servire come indifferente (terra) dell&#39;elettrodo. </li><li> Riempire elettrodo vetro fatto a passo 3,1 con una soluzione stimolante di interesse. Utilizzando una siringa inserita, compilare capIllary dalla fine tirato a Blunt fine. Rovesciare tutte le bolle d&#39;aria, tenendo fine abbattuto. Inserire un filo d&#39;argento (chloriding opzionale) nell&#39;elettrodo di vetro fatto a passo 3.1 per servire come elettrodo di registrazione e stimolante. </li><li> Collegare gli elettrodi ad un preamplificatore che è stato progettato per registrazioni da contatto chemoreceptive sensilli negli insetti. Questo preamplificatore (300 Hz-3 kHz filtro passa-banda) ridurrà il tempo di assestamento di catturare l&#39;attività neuronale più vicino allo stimolo introduzione possibile. Raccogliere, archiviare e analizzare i segnali elettrici con un microcomputer dotato di software di registrazione picco. NOTA: Messa a terra del sistema di registrazione correttamente può migliorare drasticamente il rapporto segnale-rumore. </li><li> Stimolare sensilla con una soluzione di controllo (solo elettrolita) per garantire la funzionalità. Conte picchi a partire 200 msec seguito l&#39;artefatto stimolo per tutte le preparazioni. </li><li> Casuale l&#39;ordine di sostanze chimiche di prova per tutti gli esperimenti, tranne studi dose-risposta,per eliminare pregiudizi. Per gli studi dose-risposta, esporre sensilla ad aumentare concentrazioni della sostanza chimica sperimentale. Lasciare un minimo di 3 minuti tra stimoli al fine di garantire un adeguato recupero. NOTA: Questo requisito può variare a seconda degli insetti e sensilli. Soglia è definita come quella concentrazione per cui errore standard non si sovrappone con l&#39;errore standard per la concentrazione più bassa testata. </li></ol> 4. RNA Isolamento e Sequencing (Figura 2) <ol><li> Preparare i pestelli RNasi-free ermetici da loro raffreddamento in ghiaccio secco prima di tessuto disruption.Prepare accompagnano pestelli RNasi-free che si adattano provette con tappo a scatto saldamente. Tenere tubi di raccolta chiusi a meno dissezione attivamente per evitare eccessiva formazione di condensa. </li><li> Utilizzando aspiratore filtrato, mettere da 30 a 40 le zanzare-anestetizzato freddo (15 sec in -20 ° C freezer) sul palco freddo e mostra per il sesso. Luogo animali dorsale lato negativo, afferrare le ali per non danneggiare appendici gusto.</li><li> Utilizzando due coppie di pinza sottile, sezionare Paired Labella o tarsi da maschi o femmine sotto un microscopio da dissezione e limitare l&#39;inserimento di tessuti adiacenti. NOTA: 500 Labella accoppiato produce circa 800-1000 nanogrammi di mRNA totale. 400 tarsi individuale (5 segmenti ciascuno) produrranno 1,200-1,800 nanogrammi di mRNA totale. </li><li> Con un paio di Ansimare-pinze, leggermente cogliere proboscide zanzara solo prossimali di Labella esporre mandrini interni. Utilizzando altra coppia di raccolta-pinze, rimuovere Labella e aggiungere tessuto lato del tubo di raccolta a freddo. </li><li> Con un paio di pinze, leggermente cogliere gamba zanzare allo svincolo di tibia e primo segmento tarsale. Utilizzando altra coppia di pinze, rimuovere tarsi e aggiungere tessuto lato del tubo di raccolta a freddo. NOTA: Prendere in considerazione i tipi di cellule a livello di interfaccia del tessuto desiderato e indesiderati. Mentre è importante limitare l&#39;inclusione di tessuto limitrofo, è altrettanto importante non omettere porzioni di tessuto desiderato. Il ficampione finale deve rappresentare accuratamente l&#39;intero organo di interesse. Creare un confine preciso con le pinze di presa in modo tale che una pinza di raccolta possono liberare proprio raschiando o afferrare solo il tessuto desiderato. </li><li> Periodicamente, spin down tubo di raccolta brevemente 0 ° C centrifugare a 9.000 xg per mantenere il tessuto sul fondo della provetta. Se l&#39;umidità si accumula nei tubi di raccolta durante la dissezione, aggiungere 50ul di freddo reagente Trizol per coprire il tessuto raccolti. </li><li> Utilizzando anti-macchia marcatore laboratorio, etichetta chiaramente uno senza RNase 1,5 ml schiocco provetta con tappo per ogni tipo di tessuto per la raccolta. Usando 1,5 ml provetta rack e azoto liquido, congelare poi macinare tessuto in polvere fine (pezzi pregiati se in Trizol). Ripetere una volta. Portare il volume totale di Trizol a 1 ml e sospendere nuovamente il tessuto terra nel vortex. </li><li> Aggiungere 200 ul di cloroformio e mescolare accuratamente. Dopo 5 min a temperatura ambiente, centrifugare a centrifugare a 4 ° C e 11.000 xg per 15 min. Attentamenteaggiungere strato acquoso direttamente ad una colonna filtro DNA genomico. Spin down a 11.000 x g per 5 min. Aggiungere uguale volume di RNase-free etanolo al 70% a flusso continuo e mescolare pipetta. Trasferire il liquido a una colonna collezione RNA e continuare l&#39;estrazione di RNA secondo le istruzioni fornite. </li><li> Quantificare e valutare la purezza di RNA totale su uno spettrofotometro di precisione e procedere con qualsiasi metodo di sequenziamento RNA standard. </li></ol> 5. quantitativa RT-PCR convalida di RNA Sequencing (Figura 3) <ol><li> Selezionare come molti geni come possibile confrontare i livelli di espressione genica relativi su una gamma dinamica, sia in previsto abbondanza sequenza e la funzione del gene chemosensory presunta. </li><li> Coppie di progettazione di primer per ogni gene target per amplificare uno specifico prodotto della PCR 100-180 paio di basi. Escludi non specifico di amplificazione gDNA garantendo almeno un fondo per set estende un confine introne. In alternativa, utilizzare il trattamento DNasi per ridurre la contaminazione genomico. </li><li> Raccoglieretessuto insetto come descritto nel paragrafo precedente. Calcolare la quantità di tessuto è necessario per fornire abbastanza RNA per completare tutte le reazioni qRT-PCR. NOTA: triplice copia biologica e reazioni triplicate tecnici forniranno la massima fiducia nei risultati. </li><li> Calcola relativa quantificazione gene come X bersaglio - (Ct [destinazione] -Ct [riferimento]) per ogni gene target e media per ogni replica, sia biologico e tecnico. Utilizzare gene housekeeping (s) per normalizzare valori Ct tra i replicati biologici e di sradicare la scala Y in confronto di espressione relativa. </li><li> Valutare la somiglianza di RNA-Seq e dataset qRT-PCR usando la procedura di equivalenza bootstrap basato regressione-9, in modo iterativo applicato, per i dati di ogni tessuto. NOTA: Questa procedura identifica la più piccola &quot;regione di somiglianza&quot; che può essere costruito intorno alla dati RNA-Seq e qRT-PCR osservata, e consentire l&#39;espressione genica relativa come misurato da qRT-PCR da considerare statisticamente equivalente alla misurazione dell&#39;espressione genica relativa da RNA-seq. </li></ol>

<ol>
	<li>Klun, J. A., Khrimian, A., Debboun, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repellent+and+deterrent+effects+of+SS220,+picaridin,+and+DEET+suppress+human+blood+feeding+by+Aedes+aegypti,+Anopheles+stephensi,+and+Phlebotomus+papatasi.">Repellent and deterrent effects of SS220, picaridin, and DEET suppress human blood feeding by Aedes aegypti, Anopheles stephensi, and Phlebotomus papatasi.</a> J. Med. Entomol. 43 (1), 34-39 (2006).</li><li>Dickens, J. C., Bohbot, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini+review:+Mode+of+action+of+mosquito+repellents.Pestic.">Mini review: Mode of action of mosquito repellents.Pestic.</a> Biochem. Physiol. 106 (3), 149-155 (2013).</li><li>Pitts, R. J., Rinker, D. C., Jones, P. L., Rokas, A., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+chemosensory+appendages+in+the+malaria+vector+Anopheles+gambiae+reveals+tissue-+and+sex-specific+signatures+of+odor+coding.">Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue- and sex-specific signatures of odor coding.</a> BMC Genomics. 12 (271), (2011).</li><li>Zhou, X., Slone, J. D., Rokas, A., Berger, S. L., Liebig, , 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=Phylogenetic+and+transcriptomic+analysis+of+chemosensory+receptors+in+a+pair+of+divergent+ant+species+reveals+sex-specific+signatures+of+odor+coding.">Phylogenetic and transcriptomic analysis of chemosensory receptors in a pair of divergent ant species reveals sex-specific signatures of odor coding.</a> PLOS Genet. 8 (8), e1002930 (2012).</li><li>Shiao, M., Fan, W., Fang, S., Lu, M. J., Kondo, R., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+adult+Drosophila+antenna+by+high-throughput+sequencing.">Transcriptional profiling of adult Drosophila antenna by high-throughput sequencing.</a> Zoological Studies. 52 (42), (2013).</li><li>Bohbot, J., Pitts, R. J., Kwon, H. W., R&#252;tzler, M., Robertson, H. M., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+Aedes+aegypti+odorant+receptor+gene+family.">Molecular characterization of the Aedes aegypti odorant receptor gene family.</a> Insect Mol. Biol. 16 (5), 525-537 (2007).</li><li>Kain, P., Boyle, S. M., Tharadra, S. K., Guda, T., Pham, C., 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=Odour+receptors+and+neurons+for+DEET+and+new+insect+repellents.">Odour receptors and neurons for DEET and new insect repellents.</a> Nature. 502 (7472), 507-512 (2013).</li><li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+a+primary+chemoreceptor+unit.">The physiology of a primary chemoreceptor unit.</a> Science. 122 (3166), 417-418 (1955).</li><li>Robinson, A. P., Duursma, R. A., Marshall, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regression-based+equivalence+test+for+model+validation:+shifting+the+burden+of+proof.">A regression-based equivalence test for model validation: shifting the burden of proof.</a> Tree Physiol. 25 (7), 903-913 (2005).</li><li>Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-seq.">Mapping and quantifying mammalian transcriptomes by RNA-seq.</a> Nat. Methods. 5 (7), 621-628 (2008).</li><li>Trapnell, C., Williams, B. A., Pertea, G., Mortazavi, A., Kwan, 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=Transcript+assembly+and+quantification+by+RNA-seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nat. Biotechnol. 28 (5), 511-515 (2010).</li><li>Sanford, J. L., Shields, V. D. C., Dickens, 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=Gustatory+receptor+neuron+responds+to+DEET+and+other+insect+repellents+in+the+yellow-fever+mosquito,+Aedes+aegypti.">Gustatory receptor neuron responds to DEET and other insect repellents in the yellow-fever mosquito, Aedes aegypti.</a> Naturwiss. 100 (3), 269-273 (2013).</li><li>Sparks, J. T., Vinyard, B. T., Dickens, 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=Gustatory+receptor+expression+in+the+labella+and+tarsi+of+Aedes+aegypti.">Gustatory receptor expression in the labella and tarsi of Aedes aegypti.</a> Insect Biochem. Mol. Biol. 43 (12), 1161-1171 (2013).</li><li>Kent, L. B., Walden, K. K. O., Robertson, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Gr+family+of+candidate+gustatory+and+olfactory+receptors+in+the+yellow-fever+mosquito+Aedes+aegypti.">The Gr family of candidate gustatory and olfactory receptors in the yellow-fever mosquito Aedes aegypti.</a> Chem. Senses. 33 (1), 79-93 (2008).</li><li>Ramsk&#246;ld, D., Wang, E. T., Burge, C. B., Sandberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+abundance+of+ubiquitously+expressed+genes+revealed+by+tissue+transcriptome+sequence+data.">An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data.</a> PLoS Comput. Biol. 5 (12), e1000598 (2009).</li><li>Wagner, G. P., Kin, K., Lynch, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+based+criterion+for+gene+expression+calls+using+RNA-seq+data.">A model based criterion for gene expression calls using RNA-seq data.</a> Theory Biosci. 132 (3), 159-164 (2013).</li><li>Hart, T., Komori, H. K., LaMere, S., Podshivalova, K., Salomon, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+active+genes+in+deep+RNA-seq+gene+expression+studies.">Finding active genes in deep RNA-seq gene expression studies.</a> BMC Genomics. 14 (778), (2013).</li><li>Isono, K., Morita, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+designs+of+insect+taste+receptor+system.">Molecular and cellular designs of insect taste receptor system.</a> Front. Cellu. Neurosci. 4 (20), 1-16 (2010).</li></ol>

The authors have nothing to disclose.

L&#39;aspetto più impegnativo di potenziali d&#39;azione di registrazione da sensilla gustativa è decidere quali risposte sono &quot;normali&quot;. Quando impiegando singolo sensillum gustativa punta registrando la prima volta per una determinata specie di insetti, il numero totale e sensibilità dei neuroni recettori gustativi (GRN) sono probabilmente sconosciuta. Molte registrazioni preliminari vengono prima tenuti a decidere la gamma e le concentrazioni di sostanze chimiche da testare. In questo caso, abbiamo comin...

Composti come DEET, Picaridin, Citronellale e IR3535 hanno dimostrato di respingere in modo efficace le zanzare, comprese le importanti vettori della malattia Aedes aegypti 1,2. Registriamo potenziali d&#39;azione dei neuroni sensoriali associati a specifici sensilla gustativa per determinare le cellule coinvolte con repellenza zanzare. Accoppiato con sequenziamento valle di geni espressi in questi tessuti, possiamo identificare i geni più probabile che mediano le risposte di queste cellule per lo screening di nuovi composti per le capacità repellenza migliorate. RNA-Seq è uno strumento potente, diventando rapidamente standard per il rilevamento delle modifiche temporali e spaziali in espressione genica. RNA-Seq analisi di appendici chemosensoriali insetti e gli organi sono stati utilizzati per scoprire recettori molecolari in diverse specie di insetti 3-5, migliorando notevolmente il convenzionale PCR-ricerche gene per gene 6. Gli insetti rappresentano la classe degli animali più diversi, presentano molte opportunità di studiare la connessione tra geni e fenotipi unici. Tecnologia RNA-seq può essere impiegato su qualsiasi tessuto vivente insetto. Allo stesso modo, la registrazione elettrofisiologica da cellule sensoriali all&#39;interno uniporous sensilli gustativi può essere raggiunto in molte diverse specie di insetti. L&#39;abbinamento di queste due tecniche permette ai ricercatori di restringere i geni coinvolti set in un fenotipo chemosensory osservato. Diverse specie presenteranno sfide specifiche, ma possono informare il collegamento tra i geni dei recettori chemosensoriali e un adattamento chemosensory. Le dimensioni e la morfologia dei chemosensory sensilli è variabile e rendere necessario l&#39;risoluzione dei problemi durante la registrazione di potenziali di azione per ridurre il rumore e identificare segnali ripetibili. Dissezioni di organi chemiosensoriali possono essere banale o delicati e richiede tempo, a seconda della morfologia e dimensioni dell&#39;insetto. Recupero di RNA di alta qualità può richiedere un po &#39;di risoluzione dei problemi e, come ad esempio evitare alcuni pigmenti durantecollezione tessuti. Mentre dimostrare gli effetti dei composti repellenti attraverso studi comportamentali è diretta e informativo, questo approccio è tempo intenso e largo rispetto al meccanismo d&#39;azione. Elettrofisiologia accoppiato con RNA-Seq consente analisi più specifiche su ciò che spinge a comportamenti di evitamento insetti. Una volta che il &quot;toolkit&quot; della discriminazione chimico è stato identificato in una specie di insetti, i tentativi più specifiche per migliorare il repellenti conosciute sono possibili. Recettori e proteine ​​associate a cellule sensoriali responsabili di questi comportamenti possono essere espressi heterologously per lo screening chimico diretta. Inoltre, modellistica molecolare in grado di prevedere quali sostanze chimiche si suscitare reazioni forti da questi recettori 7. L&#39;istantanea di tutti i geni attivi in ​​una serie limitata di tessuti chemosensoriali può anche essere utile per identificare geni simili in altre specie. Utilizzando omologia di sequenza e di espressione similarities, i ricercatori possono formare gruppi di recettori molecolari più probabile che mediano le risposte a repellenti che sono ampiamente efficaci sugli insetti. Vi presentiamo il seguente protocollo per aiutare i ricercatori a decostruire percorsi chemosensoriali insetti e di convincere di più per approfondire la neuroetologia di non-modello e insetti economicamente importanti.

<table><tbody><tr><td>Glass capillary</td><td>A-M Systems</td><td>628000</td><td>Standard, 1.5 mm X 0.86 mm, 4&quot;</td></tr><tr><td>Silver wire</td><td>A-M Systems</td><td>7875</td><td>.015&quot; bare</td></tr><tr><td>Tungsten wire</td><td>Alfa Products</td><td>369</td><td>0.127 mm diameter</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11252</td><td>#5SF Inox</td></tr><tr><td>Refridgerated stage</td><td>BioQuip Products</td><td>1429</td><td>Chill Table</td></tr><tr><td>Preamplifier</td><td>Syntech</td><td>Taste Probe</td><td>preamplifier</td></tr><tr><td>Software for electrophysiology</td><td>Syntech</td><td>Autospike</td><td>software for electrophysiology</td></tr><tr><td>TetraMin fish food</td><td>Tetra</td><td>Tropical fish food granules</td><td>fish food ground to fine powder</td></tr><tr><td>TRIzol</td><td>Life Technologies</td><td>15596-026</td><td>RNA isolation reagent</td></tr><tr><td>RNeasy Plus Mini Kit</td><td>Qiagen</td><td>74136</td><td>includes gDNA eliminator and RNeasy spin columns</td></tr><tr><td>Nanodrop spectrophotometer</td><td>Nano Drop Products</td><td>ND-1000</td><td>tabletop spectrometer</td></tr><tr><td>R statistics</td><td>freeware (created by Robert Gentleman and Ross Ihaka)</td><td>www.r-project.org</td><td>Use the lm function of the stats package and the equiv.boot function of the equivalence package in the R computing environment.</td></tr><tr><td>1.5 ml tube rack</td><td>Evergreen</td><td>240-6388-030</td><td>Pour liquid nitrogen into a few empty wells to freeze and grind tissue.</td></tr><tr><td>1.5 ml collection tubes with pestle</td><td>Grainger</td><td>6HAX6</td><td>RNase free</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>11178160</td><td>Spin down frozen tissue to keep at bottom of 1.5 ml tube.</td></tr><tr><td>Primer-BLAST Primer Designing tool</td><td>NCBI</td><td>n/a</td><td></td></tr></tbody></table>

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.

Le registrazioni in tracce di potenziali d&#39;azione da Ae. aegypti sensilla gustativa (Figura 1) dimostrare l&#39;efficacia della stimolazione diretta con una gamma di prodotti chimici. Questa tecnica può essere usata per quantificare risposte a qualsiasi sostanza chimica stimolante contando punte di una data ampiezza e durata in un intervallo di tempo ragionevole (generalmente inferiore a 500 ms). Registrazioni Trace devono essere facilmente riproducibili sotto un dato insieme di condizioni...

Watch this Scientific Journal Video about Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti at JoVE.com

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

Utilizzando due metodi per stimare l&#39;espressione genica nei principali appendici gustative di Aedes aegypti, abbiamo individuato il set di geni putativamente sottostanti le risposte neuronali di composti amari e ripugnanti, come determinato da un esame elettrofisiologico.

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 ...

physiological-recordings-rna-sequencing-gustatory-appendages-yellow

Research

JoVE Journal

Biology

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

8.0K Views.  United States Department of Agriculture.  Utilizzando due metodi per stimare l'espressione genica nei principali appendici gustative di Aedes aegypti, abbiamo individuato il set di geni putativamente sottostanti le risposte neuronali di composti amari e ripugnanti, come determinato da un esame elettrofisiologico. 

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

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Registrazioni Sensillum singolo in Insetti Drosophila melanogaster E Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Ricerca

Neuroscienze

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

Dissezione delle ghiandole salivari e midgut da Ae. aegypti zanzare

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

Biologia

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

Un protocollo per la raccolta e la colorazione ematociti concentrati dal Mosquito febbre gialla Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Immunologia e infezioni

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

Editing del genoma nella zanzara della febbre gialla Aedes aegypti utilizzando CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

Genetica

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Alimentazione e quantificazione del sangue di origine animale e dei pasti artificiali nelle zanzare Aedes aegypti

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong preference for human hosts over other warm-blooded animals for blood meals. Olfactory cues play a critical role for mosquitoes as they explore their environment and seek a human host to obtain blood meals, thus transmitting human diseases. Odorant receptors (ORs) expressed in the olfactory sensory neurons are known to be responsible for the interaction of mosquito vectors with human odors. To gain deeper insights into Ae. aegypti&#8217;s olfactory physiology and investigate their interactions with humans at the molecular level, we used an optimized protocol of Xenopus Oocytes heterologous expression to functionally analyze Ae. aegypti odorant receptors in response to human odors. Three example experiments are presented: 1) Cloning and synthesizing cRNAs of ORs and odorant receptor co-receptor&#160;(Orco) from four to six days old Ae. aegypti antennae; 2) Microinjection and expression of ORs and Orco in Xenopus oocytes; and 3) Whole-cell current recording from Xenopus oocytes expressing mosquito ORs/Orco with a two-electrode voltage-clamp. These optimized procedures provide a new way for researchers to investigate human odor reception in Aedes mosquitoes and reveal the underlying mechanisms governing their host-seeking activity at a molecular level.

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong ...

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.

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, ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

Sistema di coltura di organo del grasso corporeo in Aedes Aegypti, vettore di Virus di Zika

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Un protocollo di estrazione del DNA delle zanzare a base di perline magnetiche per il sequenziamento di nuova generazione

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

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

Riepilogo

Esplora Altri Video

Capitoli in questo video

Registrazioni Sensillum singolo in Insetti Drosophila melanogaster E Anopheles gambiae

Dissezione delle ghiandole salivari e midgut da Ae. aegypti zanzare

Un protocollo per la raccolta e la colorazione ematociti concentrati dal Mosquito febbre gialla Aedes aegypti

Editing del genoma nella zanzara della febbre gialla Aedes aegypti utilizzando CRISPR-Cas9

Alimentazione e quantificazione del sangue di origine animale e dei pasti artificiali nelle zanzare Aedes aegypti

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

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

Sistema di coltura di organo del grasso corporeo in Aedes Aegypti, vettore di Virus di Zika

Un protocollo di estrazione del DNA delle zanzare a base di perline magnetiche per il sequenziamento di nuova generazione

Somministrazione efficace di interferenza orale dell'RNA (RNAi) alle zanzare Anopheles gambiae adulte