Name: rearing and injection of manduca sexta larvae to assess bacterial virulence
Uploaded: 2012-12-11T12:00:00
Description: Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Forfatterne vil gerne takke tidligere medlemmer af Goodrich-Blair lab: Samantha Orchard, Kimberly Cowles, Erin Herbert-Tran, Greg Richards, Megan Menard, og Youngjin Park for deres bidrag til udviklingen af ​​denne protokol. Dette arbejde blev finansieret af National Science Foundation tilskud IOS-0950873 og National Institutes of Health NRSA stipendium FAI084441Z.

1. Insekt Egg Sterilisation og opdræt <ol><li> Forbered kost ved først autoklavering 15 g af den ydede agar i 900-1.000 ml H 2 O. Umiddelbart efter autoklavering, blandes med 166 g hvedekim kost og blend godt i en laboratorieblender. Hældes i en skål (eller retter) til afkøling, derefter overføre kost til aluminiumsfolie, wrap stramt, og opbevar ved 4 ° C. </li><li> Ved ankomsten, sterilisere M. sexta æg med 250 ml 0,6% blegemiddelopløsning i 2-3 min i et glas filterholder og termokande ap...

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Den direkte injektion af M. sexta-larver med insektspecifikke bakterier, som beskrevet her, tjener som et enkelt og effektivt middel til at analysere bakteriel virulens. Fremgangsmåden er også meget fleksibel til forskellige forsøgspersoner og / eller betingelser. Bakterier kan fremstilles på forskellige måder før injektion. I tilfælde af X. nematophila, vildtypeceller dyrket i næringsrige Luria-Bertani (LB) medium til mid-log fase, er typisk de mest virulente, dræbe de fleste eller alle insek...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> 90 mm filterpapir </td><td> Whatman </td><td> 1001 090 </td><td></td></tr><tr><td> Glas filterholderen </td><td> Millipore </td><td> XX1004700 </td><td></td></tr><tr><td> Manduca sexta æg </td><td> Carolina Biological Supply </td><td> 143.880 </td><td></td></tr><tr><td> Gypsy Moth Kost + agar </td><td> MP Biomedicals </td><td> 0296029301 </td><td></td></tr><tr><td> 5,5 ounce plastbeholdere og låg </td><td> Solo Cup Company </td><td> URC55-0090 PL4-0090 </td><td></td></tr><tr><td> 1 ounce plastbeholdere og låg </td><td> DART Container Corporation </td><td> M.100 stk 100PCL25 </td><td></td></tr><tr><td> 1x PBS </td><td></td&gt; <td></td><td> 137 mm NaCl, 2,7 mM KCI, 8 mM Na 2 HPO 4, 1,46 mM KH 2 PO 4, pH 7,4 </td></tr><tr><td> Sprøjte </td><td> Hamilton </td><td> 80.208 </td><td> 30 gauge, 0,375 &quot;længde, punkt stil 2 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

Manduca sexta, almindeligvis kendt som tobak hornworm, betragtes som en betydelig landbrugs skadedyr, fodring på planter af natskyggefamilien, herunder tobak og tomat. Modtageligheden af M. sexta-larver til en række insektspecifikke bakteriearter 1-5, samt mangfoldighed af oplysninger om insekt immunsystem 6-8, og den verserende genomsekvens 9 gør det til en god model organisme til anvendelse ved undersøgelse host-mikrobe interaktioner under patogenese. Desuden M. sexta-larver er relativt store og nemme at manipulere og opretholde i laboratoriet i forhold til andre modtagelige insekter. Deres store størrelse også muliggør en effektiv væv / hæmolymfe ekstraktion til analyse af værtens respons på infektion. Fremgangsmåden præsenteres her beskriver direkte injektion af bakterier ind i hemocoel (blod hulrum) i M. sexta-larver. Denne fremgangsmådekan anvendes til at analysere og sammenligne de virulente egenskaber ved forskellige bakterielle arter, stammer, eller mutanter ved blot at overvåge den tid, at insektet dør efter injektion. Denne metode blev udviklet til at studere patogeniciteten af Xenorhabdus og Photorhabdus species, som typisk vedrører med nematode vektorer som et middel til at få adgang til det insekt. Insektspecifikke nematoder typisk inficere larver via naturlige fordøjelsesforstyrrelser eller respiratoriske åbninger, og frigive deres symbiotiske bakterielle indhold i insekt hemolymph (blod) kort efter 10. Injektionen her beskrevne fremgangsmåde omgår behovet for et nematode vektor, således frakobling af virkningerne af bakterier og nematode på insektet. Denne fremgangsmåde tillader nøjagtig tælling af infektiøst materiale (celler eller protein) i inokulum, hvilket ikke er muligt med andre eksisterende metoder til analyse entomopathogenesis, herunder nicking 11 og oral toksicitet assays 12 <em&gt;. Også oral toksicitet assays fat virulens udskilte giftstoffer indført i fordøjelsessystemet af larver, mens den direkte indsprøjtning metode omhandler virulens helcelle-podestoffer. Anvendeligheden af ​​den direkte indsprøjtning som beskrevet her er at analysere bakteriel patogenese ved at overvåge insekt dødelighed. Imidlertid kan denne fremgangsmåde let udvides til anvendelse ved undersøgelse af virkningerne af infektion på M. sexta immunsystem. Insektet reagerer på infektion via både humorale og cellulære responser. Den humorale reaktion indbefatter anerkendelse af bakteriel-associerede mønstre og efterfølgende produktion af forskellige antimikrobielle peptider 7, ekspressionen af gener kodende for disse peptider kan overvåges efter direkte infektion via RNA-ekstraktion og kvantitativ PCR 13. Den cellulære respons på infektion involverer rodknolddannelse, indkapsling, og fagocytose af infektiøse agenser fra hæmocytter 6 </sup&gt;. For at analysere disse svar, kan injiceres insekter blive dissekeret og visualiseres ved mikroskopi 13, 14.

Et repræsentativt eksempel på et insekt dødelighed assay er vist i figur 3.. I dette eksperiment blev insekter injiceret med cirka 50 kolonidannende enheder (CFU) af enten vildtype (ATCC19061) eller en attenueret mutant stamme (LRP 13) af Xenorhabdus nematophila dyrket til mid-log fase (n = 6 insekter pr stamme). Insekter blev observeret i ca 72 timer, og procentdelen af ​​injiceret insekter stadig i live ved hvert tidspunkt registreres. I dette tilfælde udviste den ...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Den her beskrevne metode anvender direkte injektion af insektspecifikke bakterier i hemocoel af<em&gt; Manduca sexta</em&gt; Insektlarver.<em&gt; M. sexta</em&gt; Er et kommercielt tilgængeligt og velundersøgte insekt. Således er denne fremgangsmåde er en enkel tilgang til analyse host-bakterielle interaktioner med udgangspunkt i en eller begge parter.

Opdræt og Injektion af<em&gt; Manduca sexta</em&gt; Larver at vurdere bakteriel virulens

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

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Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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