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 arbeidet ble støttet av National Natural Science Foundation of China (Grant No. 81902094, 81600497), og Science and Technology Plan Project of Hunan Province (2019RS1036).

1. Midgut disseksjon og dyrkbare bakterier isolasjon
<ol><li>Forbered myggen for disseksjon.<ol>
<li>Samle myggene 7-9 dager etter fremveksten med en aspirator. Bedøve de oppsamlede myggene ved å utsette dem for en temperatur på 4 °C i 3–5 min og holde myggene bedøvet i en iskald petriskål til disseksjon.</li>
	</ol></li>
	<li>Steriliser laboratorieinstrumenter og myggoverflaten.<ol>
<li>Steriliser eksperimentbenken, dissekere mikroskop, tang og glasssklie ved å sprøyte 75% e...

<ol>
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Z., McClure, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenome+and+culture-based+methods+reveal+candidate+bacterial+mutualists+in+the+Southern+house+mosquito+(Diptera:+Culicidae).">Metagenome and culture-based methods reveal candidate bacterial mutualists in the Southern house mosquito (Diptera: Culicidae).</a> Journal of Medical Entomology. 55 (5), 1170-1181 (2018).</li><li>Wang, Y., Gilbreath, T. M., Kukutla, P., Yan, G., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+gut+microbiome+across+life+history+of+the+malaria+mosquito+Anopheles+gambiae+in+Kenya.">Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya.</a> PloS One. 6 (9), (2011).</li><li>Xiao, X., 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+Mesh-Duox+pathway+regulates+homeostasis+in+the+insect+gut.">A Mesh-Duox pathway regulates homeostasis in the insect gut.</a> Nature Microbiology. 2 (5), 17020 (2017).</li><li>Gu&#233;gan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+impacts+of+anthropogenic+stressors+on+Aedes+albopictus+mosquito+vector+microbiota.">Short-term impacts of anthropogenic stressors on Aedes albopictus mosquito vector microbiota.</a> FEMS Microbiology Ecology. 94 (12), 188 (2018).</li><li>Valzania, L., Coon, K. L., Vogel, K. J., Brown, M. R., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-induced+transcription+factor+signaling+is+essential+for+larval+growth+of+the+mosquito+Aedes+aegypti.">Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito Aedes aegypti.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (3), 457-465 (2018).</li><li>Coon, K. L., Vogel, K. J., Brown, M. R., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquitoes+rely+on+their+gut+microbiota+for+development.">Mosquitoes rely on their gut microbiota for development.</a> Molecular Ecology. 23 (11), 2727-2739 (2014).</li><li>de O Gaio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+midgut+bacteria+to+blood+digestion+and+egg+production+in+Aedes+aegypti+(diptera:+culicidae)(L).">Contribution of midgut bacteria to blood digestion and egg production in Aedes aegypti (diptera: culicidae)(L).</a> Parasites &amp; Vectors. 4 (1), 105 (2011).</li><li>Ramirez, J. 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. W., 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=Impact+of+gut+bacteria+on+the+infection+and+transmission+of+pathogenic+arboviruses+by+biting+midges+and+mosquitoes.">Impact of gut bacteria on the infection and transmission of pathogenic arboviruses by biting midges and mosquitoes.</a> Microbial Ecology. , (2020).</li><li>Llorca, M., Gros, M., Rodr&#237;guez-Mozaz, S., Barcel&#243;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preservation+for+the+analysis+of+antibiotics+in+water.">Sample preservation for the analysis of antibiotics in water.</a> Journal of Chromatography. A. 1369, 43-51 (2014).</li><li>Berendsen, B., Elbers, I., Stolker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+stability+of+antibiotics+in+matrix+and+reference+solutions+using+a+straightforward+procedure+applying+mass+spectrometric+detection.">Determination of the stability of antibiotics in matrix and reference solutions using a straightforward procedure applying mass spectrometric detection.</a> Food Additives &amp; Contaminants: Part A, Chemistry, Analysis, Control, Exposure &amp; Risk Assessment. 28 (12), 1657-1666 (2011).</li><li>Hill, C. L., Sharma, A., Shouche, Y., Severson, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+midgut+microflora+and+dengue+virus+impact+on+life+history+traits+in+Aedes+aegypti.">Dynamics of midgut microflora and dengue virus impact on life history traits in Aedes aegypti.</a> Acta Tropica. 140, 151-157 (2014).</li><li>Eng, M. W., 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=Multifaceted+functional+implications+of+an+endogenously+expressed+tRNA+fragment+in+the+vector+mosquito+Aedes+aegypti.">Multifaceted functional implications of an endogenously expressed tRNA fragment in the vector mosquito Aedes aegypti.</a> PLoS Neglected Tropical Diseases. 12 (1), 0006186 (2018).</li><li>Kajla, M. K., Barrett-Wilt, G. A., Paskewitz, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria:+A+novel+source+for+potent+mosquito+feeding-deterrents.">Bacteria: A novel source for potent mosquito feeding-deterrents.</a> Science Advances. 5 (1), 6141 (2019).</li><li>Gon&#231;alves, G. G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+MALDI-TOF+MS+to+identify+the+culturable+midgut+microbiota+of+laboratory+and+wild+mosquitoes.">Use of MALDI-TOF MS to identify the culturable midgut microbiota of laboratory and wild mosquitoes.</a> Acta Tropica. 200, 105174 (2019).</li><li>Kuss, S. 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Forskning på host-mikrobe interaksjoner har funnet at ulike tarmmikrober påvirker vertens fysiologi via divergerende mekanismer. Denne artikkelen introduserer metoden for å undersøke den respektive rollen som myggtarmmikrobe, inkludert dissekering av myggmidgut, kultiverering av kultiverende tarmbakterier, antibiotikabehandling og gjeninnføring av bakteriene av interesse.
For vellykket antibiotikabehandling må følgende detaljer vurderes for å gjennomføre eksperimentet. I denne protoko...

Mygg anses å være de viktigste vektorene av menneskelige patogene sykdommer, overfører over hundre patogener, inkludert Zika-virus, Dengue-virus og Plasmodium parasitter1. Når mygg tar et blodmåltid for å skaffe næringsstoffer til oviposisjon, kan de ved et uhell innta patogener fra en smittet vert via fordøyelseskanalen2. Viktigere, myggmidgut, som spiller en avgjørende rolle i både blodmåltid fordøyelsen og patogen inngangen, havner en svært dynamisk mikrobiome3.
Flere studier har karakterisert lab-reared og feltsamlede myggmikrobiota ved hjelp av enten en kulturavhengig metode eller en bakteriesekvenseringsanalyse4,,5,,6. Arter som Pantoea, Serratia, Klebsiella, Elizabethkingiaog Enterococcus er vanligvis isolert fra mygg i ulikestudier 5,,7,,8,,9. Interessant, mygg tarm mikrobiota svinger dynamisk i både samfunnet mangfold og mengden av bakterier arter, påvirket av utviklingsstadiet, arter, geografisk opprinnelse, og fôring atferd4. Studier viser at blodfôring dramatisk øker den totale bakterielle belastningen med rask utvidelse av arter fra Enterobacteriaceae og en reduksjon i det totale mangfoldet10,11. I tillegg blir mygg tarmmikrobiota av larvalstadiet vanligvis utryddet når insektet gjennomgår metamorfose under pupering og eclosion; dermed må nylig dukket voksne mygg befolke mikrobiota4.
Gut microbiota modulerer insektfysiologi i ulike aspekter, inkludert næringsabsorpsjon, immunitet, utvikling, reproduksjon og vektorkompetanse12. Asniske mygglarver utvikler seg ikke utover den første instar mens en bakterie oral forsyning redder utvikling, noe som indikerer at myggtarmmikroben er avgjørende forlarvalutvikling 13,14. Dessuten, uttømming av tarmbakterier retards blod måltid fordøyelse og næringsabsorpsjon, påvirker oocyte modning, og reduserer oviposisjon15. I tillegg fremkaller mygg med tarmmikroflora høyere immunresponser sammenlignet med antibiotikabehandlede mygg, med stadig forhøyet antimikrobiell peptiduttrykk mot andre patogener for åinfisere 16. Antibiotika administreres vanligvis oralt for å fjerne pan tarmbakterier i disse studiene, og deretter utføres eksperimenter for å sammenligne forskjellen mellom asnic mygg og mygg med commensal mikrober. Myggmidgut har imidlertid et mangfoldig samfunn av mikrober, og hver bakterieart kan utøve en tydelig effekt mot vertens fysiologi.
Myggmikrobiota regulerer vektorkompetanse med divergerende effekter. Kolonisering av Proteus isolert fra feltavledede mygg av dengue-endemiske områder gir oppregulert antimikrobiell peptiduttrykk og motstand mot denguevirusinfeksjon16. Den entomopathogenic sopp Beauveria bassiana aktiverer Toll og JAK-STAT immunveien mot arbovirus infeksjon17. Derimot, sopp Talaromyces isolert fra Aedes aegypti midgut forenkler dengue virusinfeksjon ved å modulere gut trypsin aktivitet18. I tillegg fremmer Serratia marcescens arbovirusoverføring gjennom et sekretorisk protein kalt SmEnhancin, som fordøyer mucinlaget på tarmepitelet av mygg19.
Denne prosedyren gir en systematisk og intuitiv metode for disseksjon av myggmidgut, isolering av kultivable bakteriekolonier, identifisering av bakterieartene og gjeninnføring via oral fôring. Det gir representative resultater av blodfôring med en commensal bakterie, Chryseobacterium meningosepticum, på mygg eggstokkutvikling og oviposisjon.

<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

Myggmidgut har et svært dynamisk mikrobiome som påvirker vertmetabolismen, reproduksjon, fitness og vektorkompetanse. Studier er utført for å undersøke effekten av tarmmikrober som helhet; Imidlertid kan forskjellige mikrober utøve tydelige effekter mot verten. Denne artikkelen gir metodikken for å studere effekten av hver enkelt myggtarmmibe og den potensielle mekanismen.
Denne protokollen inneholder to deler. Den første delen introduserer hvordan man dissekerer myggmidgut, isolerer kultivable bakteriekolonier og identifiserer bakteriearter. Den andre delen gir prosedyren for å generere antibiotikabehandlede mygg og gjeninnføre en bestemt bakterieart.

Midguts av mygg behandlet med antibiotika og uten antibiotika ble tatt ut for DNA ekstraksjon, og qPCR ble utført med universelle bakterielle primere. Figur 1 viser uttrykket for bakteriell 16S rRNA i kontrollgruppen og antibiotikabehandlingsgruppen. Resultatene viser at ca 98% av tarmbakteriene er fjernet, og tarmen sterilisering av penicillin og streptomycin var vellykket.
Med de beskrevne metodene ble bakteriestammer isolert og identifisert. C. meningosepticum er ...

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 bakteriell oral fôringsanalyse med antibiotikabehandlede mygg

Denne artikkelen presenterer en protokoll for å undersøke effekten av individuelle myggtarmet bakterier, inkludert isolasjon og identifisering av mygg midgut kulterbare mikrober, antibiotikauttømming av myggtarmbakterier og gjeninnføre en bestemt bakterieart.

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

Mygg midgut har et komplekst fellesskap av mikrober som påvirker vertens metabolisme, reproduksjon, fitness og veterinærkompetanse. Med hver tarmmibe er en annen effekt. Denne videoen introduserer prosedyren for å studere den respektive rollen til hver bakteriestamme eller vertsfysiologi.For å skaffe seg en kultur av tarmbakterielle kolonier, dissekere myggmidgut under steril tilstand. Jord den dissekere midgut, serotologinate, og spredt over agar plate. Velg enkeltkolonier fra platen, og identifiser bakteriearten via deres 16 SRNA gensekvens.Mate myggene med COD Emboss som inneholder antibiotika i påfølgende tre dager for å fjerne tarmmikrobiota. Effektiviteten av antibiotikabehandling kan bekreftes ved å dissekere midgut av aseptisk mygg for kolonidannende enhetsanalyse før påfølgende ekstremister. Deretter gjeninnfører spesifikke bakteriearter som tidligere isolert fra myggtarm via oral fôringsanalyse.Effekten av bakterier på vertsfysiologi kan evalueres ved å sammenligne de ubehandlede myggene, antibiotikabehandlede mygg og myggene gjeninnført spesifikk tilbakeholdenhet. Midgut disseksjon og dyrkingbare bakterier isolasjon. Til å begynne med, samle myggene i buret med en aspirator og celle myggene i samlingsboksen.Bedøve myggene ved å utsette en temperatur på 4 grad i tre til fem minutter. Hold myggene bedøvet ved å plassere dem i en iskald petriskål. Desinfiser benken, dissekere mikroskop og tang ved å sprøyte 75% etanol for å unngå forurensning av bakterier fra miljøet.Overflaten steriliserer myggen ved å teipe og riste dem i 75% etanol i tre minutter og skyll to ganger med PBS-buffer. Bisect myggen separat på en steril glasssklie som inneholder din dråpe PBS. Fjern forsiktig bena, vingene og myggens hode ved hjelp av tang.Klem myggens bryst og den siste delen av magen var tang og trekk fra hverandre for å isolere fordøyelseskanalen. Bruk tang for å fjerne avlingen og mispighianrøret fra fordøyelseskanalen. Avlingen er plassert på pnterior av midgut og buler etter inntak av sukkervann.Malpighian rør er gruppe av slanke ekskresjonsrør i krysset mellom midgut og bakgut. Plasser den dissekerte sminken i EP-røret med 200 mikroliter steril PBS-buffer og slip den med den sterile slipepastoren i den rene benken. Korn for å laste homogenatet til tre tempo vrangforestillinger på 50 mikroliter av hver vrangforestilling til LB agar plate og desperat plate.Inkuber platen som 37 grader i en til to dager til enkeltkolonier er mulige. Plukk enkeltkolonier i en 150 milliliter konisk kolbe som inneholder 50 milliliter lb BH-er medium. riste bakteriene på 37 grader over natten.Identifikasjon av arter. Trekk ut TOTO DNA med bakteriell genomisk DNA-konstruksjonssett og forsterke 16 SRDNA av PCR. Gjenoppretting og rensing av DNA-fragmenter fra PCR-produkter av Ambrose gel elektroforese og kjørte utvinning kit.Utførte DNA-sekvensering på de rensede DNA-fragmentene for å ha en oppnådd bakteriekjedesekvenser. Kjør blast søk til en spørring 16 SRNA gensekvens av identifisere bakterier mot bakterier og arkaea og database. Identifikasjon til artsnivået ble definert som større enn eller lik 99%16 SRDNA sekvens likhet med nærmeste team bank oppføring.Isolatet ble tildelt den til den tilsvarende slekten når det er 16 SRDNA-sekvens, likheten var mindre enn 99%og større enn eller lik 95% antibiotikabehandling og bakteriereintroduksjon. veie den nødvendige mengden sukrose penicillin og streptomycin for å forberede oss 10% sukrose løsning, inkludert å prøve 20 enheter penicillin og 20 mikrogram streptomycin per milliliter. Infiltrer bomullsballene i sukroseløsningen som inneholder antibiotika og legg den over toppen av papirkoppen som inneholder mygg.Dekk bomullsdotten med en 10 centimeter petriskål for å hindre at en vannkanten fordamper. Erstatt bomullsballene to ganger om dagen og mat myggen i påfølgende tre dager. Mygg er delt inn i to grupper.Den første gruppen ble plassert i en myggkopp uten behandling, noen bomullballer lege med 10%Så kors ble gitt daglig. Den andre gruppen ble behandlet med antibiotika og tjener i tre dager. I hver gruppe ble Sergei mygg dissekert.Mika ble ekstrahert for total DNA-ekstraksjon og QPCR ble utført ved hjelp av universelle bakterier primere, figur en viser uttrykket av 16 SRNA i kontrollgruppen og antibiotika behandlingsgruppen. Resultatene viser at kostnaden eller realiseringen av penicillin og streptomycin var vellykket. forberede bakteriell løsning, måler en bakterieløsning i viruset med et spektrofotometer, tilsett en OT bakterier suspensjon i EP-rør.Sentrifuger suspensjonen ved 5.000 RCA i fem minutter ved fire grader Kast supernatanten, vask bakteriepallforsøkene med steril PBS-buffer. Re-suspender bakteriepallen med 200 mikroliter steril PBS buffer Tilsett 600 mikroliter, 10% sukroseløsning, 200 mikroliter ATP og 200 mikroliter bakteriell suspensjon i et nytt AP-rør og bland godt. Alternativt kan bakteriene henges på nytt i varme for å aktivere blodet ditt.Fremstilling av oppvarming aktivert blod. koagulerende friskt blod med antikoagulantrør, ta to til tre milliliter friskt blod. sentrifuge ved 1000 i 10 minutter ved fire grader for å skille plasma og blodceller.Samle plasmaet i et nytt EP-rør og heraktiver aktivert i en 56 graders i en time. Vask Blast huset gjennom tidene dine med steril PBS buffer. Skyll suspendert blast hus med oppvarming aktivert plasma.Monter membran og fôringssystem. Trekk filmsyken til det maksimale. fikse med plastringen og arbeidsplassen parafilm for å unngå lekkasje sakte legge forberedt bakteriell løsning til materenheten, til maksimal lommebok.For å unngå at boblen falt fra en retning og forsegler materenheten. Koble til strømforsyningen. Vent til temperaturen når 37 grader Installert materenheten med bakterieløsning på materen.Før fôring bakterier mygg bør stoppes i 24 timer for å metabolisere antibiotika. For mateenheten på papirkoppen med mygg, la det mate i 90 minutter. Fullt oppmuntret mygg kunne plukkes ut for videre studier.Representative resultater. Figur to viser oss den gjennomsnittlige anerkjennelsen per mygg. Etter plottfôring av antibiotika behandlet myggene ser vi meningosepticum.Figur tre viser uttrykket for en variant utviklingsrelaterte gener 24 timer etter blodmåltid som inneholder C meningosepticum. Resultatene viser at det ikke er noen vesentlig endring i eggproduksjonen til kontrollgruppen og falmingsgruppen. Takk for din interesse for bakterier eller et fôringsessay med antibiotikabehandlet mygg.

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-mediert Gene knockdown og<em&gt; In Vivo</em&gt; Diurese Assay i voksen hunn<em&gt; Aedes aegypti</em&gt; Mygg

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-deteksjon analysen for Malaria Sending Mygg

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.

Høy fett Fôring Paradigm for Larve Sebrafisk: Fôring, Live Imaging, og Kvantifisering av matinntak

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.

Opprettholde Aedes aegypti mygg infisert 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.

Muntlig bakteriell infeksjon og Shedding 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 Testing toksisitet av romanen Insecticidal kjemikalier til mygg

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.