Name: a bacterial oral feeding assay with antibiotic-treated mosquitoes
Uploaded: 2020-09-12T14:00:00
Description: Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

Dette arbejde blev støttet af National Natural Science Foundation of China (Grant No. 81902094, 81600497), og Science and Technology Plan Project of Hunan Province (2019RS1036).

1. Midgut dissektion og kultivable bakterier isolering
<ol><li>Forbered myg til dissektion.<ol>
<li>Saml myggene 7-9 dage efter fremkomsten med en aspirator. Bedøve de opsamles myg ved at udsætte dem for en temperatur på 4 °C i 3-5 min og holde myg bedøvet i en iskold petriskål indtil dissektion.</li>
	</ol></li>
	<li>Steriliser laboratorieinstrumenter og myggeoverfladen.<ol>
<li>Steriliser eksperimentbænken, dissekere mikroskop, saftræt og glasrutsjebanen ved at sprøjte 75% e...

<ol>
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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=Reciprocal+tripartite+interactions+between+the+Aedes+aegypti+midgut+microbiota,+innate+immune+system+and+dengue+virus+influences+vector+competence.">Reciprocal tripartite interactions between the Aedes aegypti midgut microbiota, innate immune system and dengue virus influences vector competence.</a> PLoS Neglected Tropical Diseases. 6 (3), 1561 (2012).</li><li>Dong, Y., Morton, J. C., Ramirez, J. L., Souza-Neto, J. A., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entomopathogenic+fungus+Beauveria+bassiana+activate+toll+and+JAK-STAT+pathway-controlled+effector+genes+and+anti-dengue+activity+in+Aedes+aegypti.">The entomopathogenic fungus Beauveria bassiana activate toll and JAK-STAT pathway-controlled effector genes and anti-dengue activity in Aedes aegypti.</a> Insect Biochemistry and Molecular Biology. 42 (2), 126-132 (2012).</li><li>Anglero-Rodriguez, Y. I., 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=An+Aedes+aegypti-associated+fungus+increases+susceptibility+to+dengue+virus+by+modulating+gut+trypsin+activity.">An Aedes aegypti-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity.</a> Elife. 6, 28844 (2017).</li><li>Wu, P., 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=A+gut+commensal+bacterium+promotes+mosquito+permissiveness+to+arboviruses.">A gut commensal bacterium promotes mosquito permissiveness to arboviruses.</a> Cell Host &amp; Microbe. 25 (1), 101-112 (2019).</li><li>M&#246;hlmann, T. 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Forskning i vært-mikrobe interaktioner har vist, at forskellige tarm mikrober påvirker deres vært fysiologi via divergerende mekanismer. Denne artikel introducerer metoden til at undersøge den respektive rolle myg gut mikrobe, herunder dissekere myg midgut, dyrkekultebare tarmbakterier, antibiotikabehandling, og genindføre bakterier af interesse.
For vellykket antibiotikabehandling skal følgende detaljer overvejes ved udførelsen af forsøget. I denne protokol blev myg behandlet med vatk...

Myg anses for at være de vigtigste vektorer af humane patogene sygdomme, overføre over hundrede patogener, herunder Zika virus, Dengue virus, og Plasmodium parasitter1. Når myg tage et blodmåltid at erhverve næringsstoffer til oviposition, de kan ved et uheld indtage patogener fra en inficeret vært via fordøjelseskanalen2. Vigtigere, myg midgut, som spiller en afgørende rolle i både blod måltid fordøjelse og patogen indgang, havne en meget dynamisk mikrobiom3.
Flere undersøgelser har karakteriseret lab-opdrættet og felt-indsamlet myg mikrobiota ved hjælp af enten en kultur-afhængig metode eller en bakterie sekventering assay4,5,6. Arter, herunder Pantoea, Serratia, Klebsiella, Elizabethkingia, og Enterococcus er almindeligvis isoleret fra myg i forskelligeundersøgelser 5,,7,,8,9. Interessant, myg gut mikrobiota svinger dynamisk i både samfundet mangfoldighed og mængden af bakterier arter, påvirket af udviklingsstadiet, arter, geografisk oprindelse, og fodring adfærd4. Undersøgelser viser, at blodfodring dramatisk øger den samlede bakteriebelastning med hurtig ekspansion af arter fra Enterobacteriaceae og en reduktion i den samledemangfoldighed 10,11. Desuden er mygge gut mikrobiota af larve fase normalt udryddet, når insektet gennemgår metamorfose under pupation og eclosion; således, nyligt opstået voksne myg nødt til at genbefolke deres mikrobiota4.
Gut mikrobiota modulerer insekt fysiologi i forskellige aspekter, herunder næringsstof absorption, immunitet, udvikling, reproduktion, og vektor kompetence12. Axenic myg larver undlader at udvikle sig ud over den første instar, mens en bakterier oral forsyning redder udvikling, hvilket indikerer, at myg tarmmikrobe er afgørende for larveudvikling 13,,14. Udover, udtynding af tarmbakterier forsinker blod måltid fordøjelse og næringsstof absorption, påvirker oocyt modning, og nedsætter oviposition15. Desuden fremkalder myg med tarmmikroflora højere immunrespons sammenlignet med antibiotikabehandlede myg, med konstant forhøjet antimikrobielt peptid udtryk mod andre patogener til at inficere16. Antibiotika er normalt oralt administreres for at fjerne pan tarmbakterier i disse undersøgelser, og derefter eksperimenter udføres for at sammenligne forskellen mellem axenic myg og myg med commensal mikrober. Men myg midgut havne et mangfoldigt fællesskab af mikrober, og hver bakterie arter kunne udøve en særskilt effekt mod værten fysiologi.
Mosquito microbiota regulerer vektor kompetence med divergerende virkninger. Kolonisering af Proteus isoleret fra felt-afledte myg af dengue-endemiske områder giver opreguleret antimikrobiel peptid udtryk og resistens over for dengue virus infektion16. Den entomopathogenic svamp Beauveria bassiana aktiverer Toll og JAK-STAT immun vej mod arbovirus infektion17. Derimod svampen Talaromyces isoleret fra Aedes aegypti midgut letter dengue virus infektion ved modulerende gut trypsin aktivitet18. Desuden, Serratia marcescens fremmer arbovirus transmission gennem en sekretorisk protein kaldet SmEnhancin, som fordøjer mucin lag på tarm epitel af myg19.
Denne procedure giver en systematisk og intuitiv metode til dissektion af myg midgut, isolering af kultivable bakterier kolonier, identifikation af bakteriearter, og genindførelse via oral fodring. Det giver repræsentative resultater af blod fodring med en commensal bakterie, Chryseobacterium meningosepticum, om myg æggesting udvikling og oviposition.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td >Sigma</td>
			<td >A2383</td>
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate has been used to prepare adenosine triphosphate (ATP) standard solutions</td>
		</tr>
		<tr >
			<td >Aedes aegypti</td>
			<td></td>
			<td></td>
			<td>Female mosquitoes</td>
		</tr>
		<tr >
			<td >Anticoagulant tube</td>
			<td>BD Vacutainer</td>
			<td>363095</td>
			<td>Collect fresh blood</td>
		</tr>
		<tr >
			<td >Centrifuge tube</td>
			<td>Sangon Biotech</td>
			<td>F601620-0010</td>
			<td>1.5 ml, Natural, Graduated, Sterile</td>
		</tr>
		<tr >
			<td >Cotton balls</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Disposable Tissue Grinding Pestle</td>
			<td>Sangon Biotech</td>
			<td>F619072-0001</td>
			<td>70 mm Long, Conical, Blue, Sterile</td>
		</tr>
		<tr >
			<td >Ethanol absolute</td>
			<td>Paini</td>
			<td></td>
			<td>Dilute it to 75% ethanol</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td>RWD</td>
			<td>F11029</td>
			<td>Dissection</td>
		</tr>
		<tr >
			<td >Hemotek Membrane Feeding System</td>
			<td>Hemotek</td>
			<td></td>
			<td>Components of the feeding system, including&#160; Hemotek temperature controller, feeder-housing assembly, metal feeder assembled.</td>
		</tr>
		<tr >
			<td >Incubator shaker</td>
			<td></td>
			<td>ZQZY-78AN</td>
			<td></td>
		</tr>
		<tr >
			<td >Inoculation Loops</td>
			<td>Sangon Biotech</td>
			<td>F619312-0001</td>
			<td>10 &#956;l, Yellow</td>
		</tr>
		<tr >
			<td >LB Agar Powder</td>
			<td>Sangon Biotech</td>
			<td>A507003</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g&#65307; Agar 15.0 g.</td>
		</tr>
		<tr >
			<td >LB Broth Powder</td>
			<td>Sangon Biotech</td>
			<td>A507002</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g.</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td>Zeiss</td>
			<td>Stemi508</td>
			<td></td>
		</tr>
		<tr >
			<td >Paper cup</td>
			<td></td>
			<td></td>
			<td>Place mosquito</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td>Sangon Biotech</td>
			<td>F104002</td>
			<td>4 inx 125 ft</td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td>Sangon Biotech</td>
			<td>F611203</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin G procaine salt hydrate</td>
			<td>Sangon Biotech</td>
			<td>A606248</td>
			<td>White powder. Soluble in water, soluble in methanol, slightly soluble in water, ethanol</td>
		</tr>
		<tr >
			<td >Single Channal Pipettor</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Streptomycin sulfate</td>
			<td>Sangon Biotech</td>
			<td>A610494</td>
			<td>Streptomycin sulfate is a glucosamine antibiotic that interferes with the synthesis of prokaryotic proteins.</td>
		</tr>
		<tr >
			<td >Sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792</td>
			<td>Soluble in water, ethanol and methanol, slightly soluble in glycerol and pyridine.</td>
		</tr>
		<tr >
			<td >TIANamp Bacteria DNA Kit</td>
			<td>TIANGEN</td>
			<td>DP302</td>
			<td>Extract DNA&#160;</td>
		</tr>
		<tr >
			<td >Utility Fabric-Mosquito Netting White</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td>Scintic Industries</td>
			<td>S1-0246</td>
			<td></td>
		</tr>
		<tr >
			<td >1.5ml EP tube</td>
			<td>Sangon Biotech</td>
			<td>F600620</td>
			<td></td>
		</tr>
		<tr >
			<td >10X PBS buffer</td>
			<td>Sangon Biotech</td>
			<td>E607016</td>
			<td>This product is a 10X solution. Please dilute it 10 times before use. The pH value is 7.4.</td>
		</tr>
	</tbody>
</table>

a bacterial oral feeding assay with antibiotic-treated mosquitoes

Myg midgut havne en meget dynamisk mikrobiom, der påvirker værten stofskifte, reproduktion, fitness, og vektor kompetence. Der er gennemført undersøgelser for at undersøge virkningen af tarmmikrober som helhed; forskellige mikrober kan dog have forskellige virkninger over for værten. Denne artikel indeholder metoden til at undersøge effekten af hver enkelt myg gut mikrobe og den potentielle mekanisme.
Denne protokol indeholder to dele. Den første del introducerer, hvordan man dissekere myg midgut, isolere kultivable bakterier kolonier, og identificere bakterier arter. Den anden del giver mulighed for at generere antibiotika-behandlede myg og genindføre en bestemt bakterie arter.

Midguts af myg behandlet med antibiotika og uden antibiotika blev taget ud for DNA-ekstraktion, og qPCR blev udført med universelle bakterielle primere. Figur 1 viser ekspressionen af bakteriel 16S rRNA i kontrolgruppen og antibiotikabehandlingsgruppen. Resultaterne viser, at omkring 98% af tarmbakterierne er blevet fjernet, og tarmsterilisering af penicillin og streptomycin var en succes.
Med de beskrevne metoder blev bakteriestammer isoleret og identificeret. C. meningo...

Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

En bakteriel oral fodring assay med antibiotika-behandlede myg

Denne artikel præsenterer en protokol til at undersøge effekten af de enkelte myg tarmbakterier, herunder isolering og identifikation af myg midgut kultivable mikrober, antibiotika udtynding af myg tarmbakterier, og genindføre en bestemt bakterie arter.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Mosquito midgut havne et komplekst fællesskab af mikrober, der påvirker værten stofskifte, reproduktion, fitness, og dyrlægen kompetence. Med hver tarm mikrobe er en anden effekt. Denne video introducerer proceduren for at studere den respektive rolle hver bakterie stamme eller vært fysiologi.At erhverve en kultur af gut bakterielle kolonier, dissekere myg midgut under steril tilstand. Jord dissekere midgut, serotologisk, og spredt over agar plade. Vælg enkelte kolonier fra pladen, og identificer bakteriearten via deres 16 SRNA-gensekvens.Fodre myg med COD Prægning indeholdende antibiotika i træk på tre dage for at fjerne tarm mikrobiota. Effektiviteten af antibiotikabehandling kan bekræftes ved at dissekere midgut af aseptiske myg for koloni danner enhed analyse forud for efterfølgende ekstremister. Derefter genindføre specifikke bakterier arter, der tidligere isoleret fra myg tarmen via oral fodring assay.Effekten af bakterier på vært fysiologi kunne evalueres ved at sammenligne de ubehandlede myg, antibiotika-behandlede myg, og myg genindført specifik tilbageholdenhed. Midgut dissektion og dyrkes bakterier isolation. Til at begynde med, indsamle myg i bur med en aspirator og celle myg i indsamling boksen.Bedøve myg ved at underkaste en temperatur på 4 grader i tre til fem minutter. Hold myg bedøvet ved at placere dem i en iskold petriskål. Desinficere bænken, dissekere mikroskop, og snævring ved at sprøjte 75% ethanol for at undgå forurening med bakterier fra miljøet.Overflade sterilisere myg ved tape og ryste dem i 75% ethanol i tre minutter og skyl to gange med PBS buffer. Bisect myg separat på en steril glas dias, der indeholder din dråbe PBS. Fjern forsigtigt benene, vingerne og hovedet af myg ved hjælp af sniver.Clamp myg bryst og den sidste del af maven var stænde og trække fra hinanden for at isolere fordøjelseskanalen. Brug vices til at fjerne afgrøden og malpighian rør fra fordøjelseskanalen. Afgrøden er placeret ved pnterior af midgut og buler efter indtagelse af sukker vand.Malpighian rør er gruppe af slanke ekskrekulerende rør på krydset af midgut og hindgut. Placer den dissekerede makeup i EP-rør med 200 mikroliters sterile PBS buffer og male det med den sterile slibning præst i den rene bænk. Korn til at indlæse homogene til tre tempo vrangforestillinger på 50 mikroliter af hver vrangforestilling til LB agar plade og desperate plade.Inkuber pladen som 37 grader i en til to dage, indtil enkelte kolonier er mulige. Pluk enkelte kolonier i en 150 milliliter konisk kolbe, der indeholder 50 milliliter lb bh'er medium. ryste bakterierne ved 37 grader natten over.Identifikation af arter. Uddrag TOTO DNA ved bakteriel genomisk DNA konstruktion kit og forstærke 16 SRDNA ved PCR. Genopretning og rensning af DNA-fragmenter fra PCR-produkter af Ambrose gelelektrophorsis og det kørte recovery kit.Udført DNA sekventering på de rensede DNA fragmenter at have en opnået bakterier kæde sekvenser. Kør blast søgning til en forespørgsel de 16 SRNA gensekvens af identificere bakterier mod bakterier og archaea og database. Identifikationen af artsniveauet blev defineret som større end eller lig med 99%16 SRDNA-sekvensligheden til den nærmeste holdbankpost.Isolatet blev tildelt det til den tilsvarende, slægt, når er 16 SRDNA sekvens, lighed var mindre end 99% og større end eller lig med 95% antibiotika behandling og bakterie genintroduktion. den nødvendige mængde saccharose penicillin og streptomycin til at forberede os 10% saccharose opløsning, herunder forsøger 20 enheder af penicillin og 20 mikrogram streptomycin per milliliter. Infiltrere vatkugler i saccharose opløsning, der indeholder antibiotika og placere den over toppen af papiret kop, der indeholder myg.Dæk vat med en 10 centimeter petriskål for at forhindre en havnefront i at fordampe. Udskift vatkugler to gange om dagen og fodre myg i træk på tre dage. Myg er opdelt i to grupper.Den første gruppe blev placeret i en myg kop uden nogen behandling, enhver vatkugler læge med 10%Så cross blev givet dagligt. Den anden gruppe blev behandlet med antibiotika og tjene i tre dage. I hver gruppe blev Sergei myg dissekeret.Mika blev udvundet for total DNA-ekstraktion og QPCR blev udført ved hjælp af universelle bakterier primere, figur en viser udtrykket af 16 SRNA i kontrolgruppen og antibiotika behandling gruppe. Resultaterne viser, at omkostningerne eller realiseringen af penicillin og streptomycin var en succes. forberede bakteriel løsning, måler en bakterieopløsning i virus med et spektrofotometer, tilsæt en OT bakterier suspension i EP rør.Centrifuge suspensionen ved 5, 000 RCA i fem minutter ved fire graders Kassér supernatanten, vask bakteriepallen forsøger med steril PBS buffer. Re-suspendere bakteriel palle med 200 microliters sterile PBS buffer Tilføj 600 mikroliter, 10% saccharose opløsning, 200 mikroliter ATP og 200 microliter bakteriel suspension i en ny AP rør og bland godt. Alternativt kan bakterierne igen suspenderes i varme i aktivere dit blod.Forberedelse af opvarmning aktiveret blod. blodprop med antikoagulerende rør, skal der gå to til tre milliliter frisk blod. centrifuge ved 1000 i 10 minutter ved fire grader for at adskille plasma og blodlegemer.Opsaml plasmaet i et nyt EP-rør, og aktiver heri aktiveret ved en 56 grader i en time. Vask Blast hus gennem dine tider med sterile PBS buffer. Skyl suspenderet blast hus med varme aktiveret plasma.Saml membran- og fodringssystem. Træk filmsyge til det maksimale. fastgør med plastringen og arbejdsparafilmen for at undgå, at den lækkende langsomt tilsættes den forberedte bakterieopløsning til fødeenheden, til den maksimale tegnebog.For at undgå, at boblen faldt fra én retning, forsegles fødeenheden. Tilslut strømforsyningen. Vent, indtil temperaturen når 37 grader Installeret foderenheden med bakterieopløsning på fødeautomaten.Før fodring bakterier myg bør stoppes i 24 timer at metabolisere antibiotika. For foderenheden på papirkoppen med myg, så lad fodring i 90 minutter. Fuldt opmuntret myg kunne plukkes ud til yderligere undersøgelser.repræsentative resultater. Figur to viser os den gennemsnitlige anerkendelse pr myg. Efter plot fodring af antibiotika behandlet myg ser vi meningosepticum.Figur tre viser udtrykket af en variant udvikling relaterede gener 24 timer efter blodmåltid, der indeholder C meningosepticum. Resultaterne viser, at der ikke er nogen væsentlig ændring i kontrolgruppens og den falmende gruppes ægproduktion. Tak for din interesse i bakterier eller en fodring essay med antibiotika behandlet myg.

a-bacterial-oral-feeding-assay-with-antibiotic-treated-mosquitoes

Research

JoVE Journal

Biology

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

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

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

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

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-medieret Gene Knockdown og<em&gt; In Vivo</em&gt; Diurese Assay i Voksen Kvinde<em&gt; Aedes aegypti</em&gt; Myg

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

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

En Multi-påvisningsassay for Malaria Fremsendende Myg

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Højt fedtindhold Fodring Paradigme for Larver Zebrafisk: Fodring, Levende Imaging, og kvantificering af fødeindtagelse

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Opretholdelse af Aedes aegypti myg inficeret med Wolbachia

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

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Mundtlige bakterieinfektion og kaste i Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protokoller for afprøvning toksicitet af roman insektbekæmpelsesmidler kemi til myg

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.