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. Opdræt Ae. aegypti voksne <ol><li> Hatch æg i ca. ¾ tommer vand i lavvandede bakke. Overbelægning vil reducere størrelsen af ​​voksne. </li><li> Rear larver ved 25 ° C (12-hL: 12-HD) og foder med jorden fiskefoder. BEMÆRK: overfodring kan reducere overlevelsesrater. </li><li> Fjern pupper individuelt ved Pasteur-pipette dagligt og overføre til plast retter (9 cm × 5,5 cm) inde små indeslutning spande med fine mesh låg og dermed skaber 24 timers alder grupperinger. </li><li> Feed voksn...

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Det mest udfordrende aspekt af optagelse aktionspotentialer fra gustatory sensilla er at beslutte hvilke svar er &quot;normal&quot;. Når ansætte enkelt gustatory sensillum tip optagelse første gang for en given insektarter, det samlede antal og følsomhed smagssansen receptor neuroner (GRNs) er sandsynligvis ukendt. Mange indledende optagelser først skal tage stilling området og koncentrationer af kemikalier for at teste. I dette tilfælde, vi begyndte med observationen af, at en enkelt GRN reageret for lave koncen...

Forbindelser som DEET, Picaridin, Citronellal og IR3535 har vist til effektivt at frastøde myg, herunder vigtig sygdom vektor Aedes aegypti 1,2. Vi registrerer aktionspotentialer fra sensoriske neuroner, der er forbundet med særlig gustatoriske sensilla at bestemme de involverede med myg frastødning celler. Kombineret med nedstrøms sekventering af udtrykte gener i disse væv, kan vi identificere de gener sandsynligvis mediere responser i disse celler for at screene nye forbindelser for forbedrede skyende kapaciteter. RNA-seq er et kraftfuldt værktøj, hurtigt at blive standard for sporing tidsmæssige og rumlige ændringer i genekspression. RNA-seq analyser af insekt chemosensory lemmer og organer er blevet anvendt til at afdække molekylære receptorer i flere insektarter 3-5, i høj grad forbedre på konventionel PCR-baserede søgninger genet ved gen 6. Insekter repræsenterer de mest forskelligartede dyr klasse, presenterer mange muligheder for at studere sammenhængen mellem gener og unikke fænotyper. RNA-seq teknologi kan anvendes på et hvilket som helst levende insekt væv. På samme måde kan elektrofysiologiske optagelse fra sanseceller inden uniporous gustatory sensilla opnås på mange forskellige insektarter. Parringen af ​​disse to teknikker giver forskerne at indsnævre de opstillede gener involveret i en observeret chemosensory fænotype. Forskellige arter vil indebære særlige udfordringer, men kan oplyse sammenhængen mellem chemosensory receptor gener og en chemosensory tilpasning. Størrelsen og morfologi chemosensory sensilla er variabel og kan kræve omfattende fejlfinding, når du optager aktionspotentialer at reducere støj og identificere gentagne signaler. Dissektioner af chemosensory organer kan være triviel eller delikat og tidskrævende afhængigt af morfologi og størrelse af insekt. Inddrivelse af høj kvalitet RNA kan kræve en vis fejlfinding samt, såsom at undgå visse pigmenter undervæv samling. Mens demonstrere virkningerne af frastødende forbindelser gennem adfærdsmæssige forsøg er direkte og informativ, denne metode er tidskrævende og bredt med hensyn til virkningsmekanisme. Elektrofysiologi kombineret med RNA-seq giver mulighed for mere specifikke analyser af, hvad der driver undgåelse adfærd i insekter. Når &quot;værktøjskasse&quot; af kemisk forskelsbehandling er blevet identificeret i en insektarter, at mere specifikke forsøg forbedre kendte afskrækningsmidler er mulige. Receptorer og tilhørende proteiner i sanseceller, der er ansvarlige for disse adfærdsmønstre kan udtrykkes heterologt til direkte kemisk screening. Desuden kan molekylær modellering forudsige, hvilke kemikalier vil fremkalde stærke reaktioner fra disse receptorer 7. Et snapshot af alle aktive gener i en smal sæt chemosensory væv kan også være nyttige til at identificere lignende gener i andre arter. Brug sekvenshomologi og ekspression similarities, kan forskerne danne sæt molekylære receptorer sandsynligvis medierende svar på afskrækningsmidler, der stort set effektive på insekter. Vi præsenterer følgende protokol for at hjælpe forskere med at dekonstruere insekt chemosensory veje og overtale mere at dykke ned i Neuroetologi af ikke-model og økonomisk vigtige insekter.

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			<td height="21" style="height:21px;width:251px;">Glass capillary</td>
			<td style="width:367px;">A-M Systems</td>
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			<td>Standard, 1.5mm X 0.86mm, 4&#34;</td>
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			<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:251px;">Refridgerated stage</td>
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			<td style="width:189px;">1429</td>
			<td>Chill Table</td>
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			<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>
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			<td height="21" style="height:21px;width:251px;">Software for electrophysiology</td>
			<td style="width:367px;">Syntech</td>
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			<td style="width:467px;">software for electrophysiology</td>
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			<td height="41" style="height:41px;">TetraMin fish food</td>
			<td style="width:367px;">Tetra</td>
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			<td>fish food ground to fine powder</td>
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			<td height="21" style="height:21px;">TRIzol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>RNA isolation reagent</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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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.

De spor optagelser af aktionspotentialer fra Ae. aegypti gustatory sensilla (figur 1) demonstrere effektiviteten af direkte stimulering med en række kemikalier. Denne teknik kan anvendes til at kvantificere responser til de stimulerende kemisk ved at tælle pigge af en given amplitude og varighed over et rimeligt tidsrum (generelt mindre end 500 ms). Trace optagelser skal være let at reproducere under et bestemt sæt af eksperimentelle betingelser. Ellers kan de observerede fysiologiske reakt...

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

Anvendelse af to metoder til at estimere genekspression i de store smagsmæssige vedhæng af Aedes aegypti, har vi identificeret sæt gener putativt underliggende neuronale reaktioner på bitre og frastødende forbindelser, som bestemt ved elektrofysiologiske undersøgelse.

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

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