Name: physiological recordings and rna sequencing of the gustatory appendages of the yellow-fever mosquito aedes aegypti
Uploaded: 2014-12-30T14:00:00
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. Het grootbrengen Ae. aegypti volwassenen <ol><li> Hatch eieren in ongeveer ¾ inch water in ondiepe lade. Overbevolking zal de omvang van de volwassenen te verminderen. </li><li> Achterste larven bij 25 ° C (12-hl: 12-HD) en voeden met gemalen vis eten. OPMERKING: Overvoeren kan overlevingskansen verminderen. </li><li> Verwijder poppen individueel door Pasteur pipet dagelijks en over te dragen aan plastic schaaltjes (9 cm x 5,5 cm) in kleine insluiting emmers met fijn gaas deksels, dus tot oprichti...

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De meest uitdagende aspect van de opname actiepotentialen van smaak sensilla is te beslissen welke reacties zijn &quot;normaal&quot;. Bij toepassing van enkele smaak sensillum tip opnemen van de eerste keer voor een bepaalde insectensoorten, het totale aantal en de gevoeligheden van de smaak receptor neuronen (GRNs) zijn waarschijnlijk onbekend. Veel voorlopige opnames worden eerst nodig om het bereik en de concentraties van chemicaliën te testen beslissen. In dit geval, begonnen we met de waarneming dat één GRN reag...

Verbindingen zoals DEET, picaridin, Citronellal en IR3535 is aangetoond effectief af te weren muggen, waaronder de belangrijke ziekte vector Aedes aegypti 1,2. We nemen actiepotentialen van sensorische neuronen geassocieerd met specifieke smaak sensilla aan de betrokken met mug afstotende cellen te bepalen. Gekoppeld aan sequentie stroomafwaarts van uitgedrukte genen in deze weefsels kunnen we de genen waarschijnlijk medieert de respons van deze cellen om nieuwe verbindingen op verbeterde waterafstotendheid mogelijkheden screenen identificeren. RNA-seq is een krachtig hulpmiddel, in snel tempo standaard voor het bijhouden van ruimtelijke en temporele veranderingen in genexpressie. RNA-seq analyses van insecten chemosensory appendages en organen zijn gebruikt om moleculaire receptoren ontdekken in verschillende insectensoorten 3-5, sterk verbeteren van conventionele PCR zoekopdrachten gen door gen 6. Insecten vormen de meest diverse dier klasse, prewoordigd veel mogelijkheden om de verbinding tussen genen en unieke fenotypes bestuderen. RNA-seq technologie kan worden ingezet op een levend insect weefsel. Evenzo kan elektrofysiologische opname van zintuigcellen binnen uniporous gustatory sensilla worden bereikt in veel verschillende insectensoorten. De koppeling van deze twee technieken stelt onderzoekers in staat om de set genen die betrokken zijn in een waargenomen chemosensory fenotype versmallen. Verschillende soorten zullen een bijzondere uitdaging vormen, maar kan de verbinding tussen chemosensory receptor genen en een chemosensory aanpassing op de hoogte. De grootte en morfologie van chemosensory sensilla is variabel en kunnen uitgebreide probleemoplossing vereisen bij het opnemen van actiepotentialen om ruis te verminderen en te identificeren herhaalbare signalen. Dissectie van chemosensory organen kunnen onbeduidend of delicaat en tijdrovend, afhankelijk morfologie en grootte van de insecten. Herstel van hoge kwaliteit RNA kan wat het oplossen van problemen vereisen ook, zoals het vermijden van bepaalde pigmenten tijdensweefsel collectie. Demonstreert het effect van afstotende verbindingen door gedrags- beproevingen is direct en informatief, deze benadering is tijdsintensief en breed met betrekking tot werkingsmechanisme. Elektrofysiologie in combinatie met RNA-seq zorgt voor meer specifieke analyses van wat drijft vermijdingsgedrag bij insecten. Zodra de &quot;toolkit&quot; chemische discriminatie geïdentificeerd in een insectensoorten, specifieker pogingen verbeteren bekende insectenwerende mogelijk. Receptoren en geassocieerde eiwitten in sensorische cellen die verantwoordelijk zijn voor deze gedragingen kunnen heteroloog tot expressie worden gebracht voor directe chemische screening. Bovendien kunnen moleculaire modellering voorspellen welke chemicaliën sterke reacties zal uitlokken van deze receptoren 7. De momentopname van alle actieve genen in een smalle reeks van chemosensory weefsels kan ook nuttig zijn bij het identificeren van gelijkaardige genen in andere soorten. Met behulp van homologie en expressie similarities, onderzoekers kunnen sets van de moleculaire receptoren hoogstwaarschijnlijk bemiddelen reacties op insectenwerende middelen die zijn grotendeels effectief op insecten te vormen. We presenteren de volgende protocol om onderzoekers te helpen bij het deconstrueren insect chemosensory paden en om meer te overtuigen om zich te verdiepen in de neuroethology van niet-model en economisch belangrijke insecten.

<table border="0" cellpadding="0" cellspacing="0" width="1272">
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			<td height="21" style="height:21px;width:251px;">Name of Material/ Equipment</td>
			<td style="width:367px;">Company</td>
			<td style="width:189px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glass capillary</td>
			<td style="width:367px;">A-M Systems</td>
			<td style="width:189px;">628000</td>
			<td>Standard, 1.5mm X 0.86mm, 4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Silver wire</td>
			<td style="width:367px;">A-M Systems</td>
			<td style="width:189px;">7875</td>
			<td>.015&#34; bare</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tungsten wire</td>
			<td style="width:367px;">Alfa Products</td>
			<td style="width:189px;">369</td>
			<td>0.127mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Fine forceps</td>
			<td style="width:367px;">Fine Science Tools</td>
			<td style="width:189px;">11252</td>
			<td>#5SF Inox</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Refridgerated stage</td>
			<td style="width:367px;">BioQuip Products</td>
			<td style="width:189px;">1429</td>
			<td>Chill Table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Preamplifier</td>
			<td style="width:367px;">Syntech</td>
			<td style="width:189px;">Taste Probe</td>
			<td style="width:467px;">preamplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Software for electrophysiology</td>
			<td style="width:367px;">Syntech</td>
			<td style="width:189px;">Autospike</td>
			<td style="width:467px;">software for electrophysiology</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">TetraMin fish food</td>
			<td style="width:367px;">Tetra</td>
			<td style="width:189px;">Tropical fish food granules</td>
			<td>fish food ground to fine powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy Plus Mini Kit</td>
			<td>Qiagen</td>
			<td style="width:189px;">74136</td>
			<td style="width:467px;">includes gDNA eliminator and RNeasy spin columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop spectrophotometer</td>
			<td>Nano Drop Products</td>
			<td style="width:189px;">ND-1000</td>
			<td>tabletop spectrometer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:251px;">R statistics</td>
			<td style="width:367px;">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 height="21">
			<td height="21" style="height:21px;width:251px;">1.5ml tube rack</td>
			<td style="width:367px;">Evergreen</td>
			<td style="width:189px;">240-6388-030</td>
			<td>Pour liquid nitrogen into a few empty wells to freeze and grind tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">1.5mL collection tubes with pestle</td>
			<td style="width:367px;">Grainger</td>
			<td style="width:189px;">6HAX6</td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Centrifuge</td>
			<td style="width:367px;">Thermo Scientific</td>
			<td style="width:189px;">11178160</td>
			<td>Spin down frozen tissue to keep at bottom of 1.5 mL tube.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primer-BLAST Primer Designing tool</td>
			<td style="width:367px;">NCBI</td>
			<td style="width:189px;">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.

Het spoor opnames van actiepotentialen van Ae. aegypti smaak sensilla (figuur 1) tonen de doeltreffendheid van directe stimulatie met diverse chemicaliën. Deze techniek kan worden gebruikt om reacties op elk stimuleren chemische gekwantificeerd door het tellen pieken van een gegeven amplitude en duur in een redelijke tijdspanne (over het algemeen minder dan 500 ms). Trace opnames moeten gemakkelijk reproduceerbaar onder een bepaalde set van experimentele omstandigheden zijn. Anders kan de waar...

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

Met twee methodologie om genexpressie in grote smaak aanhangsels van Aedes aegypti, hebben we de set genen vermoedelijk onderliggende neuronale reacties op bitter en afstotende verbindingen geïdentificeerd, zoals bepaald door elektrofysiologische onderzoeken.

Fysiologische Recordings en RNA Sequencing van de Smaak Appendages van de Gele-koorts Mosquito<em&gt; Aedes aegypti</em

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

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Biology

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

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RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

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This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

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Uitputting van ribosomaal RNA voor 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 ...

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One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

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

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

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Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

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

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Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...