Name: the ingestion of fluorescent, magnetic nanoparticles for determining fluid-uptake abilities in insects
Uploaded: 2017-12-20T15:00:00
Description: Watch this Scientific Journal Video about The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects at JoVE.com

Dette arbejde blev støttet af National Science Foundation (NSF) give nr. IOS 1354956. Vi takker Dr. Andrew D. Warren (McGuire Center for Lepidoptera og biodiversitet, Florida Museum of Natural History, University of Florida) for tilladelse til at bruge sommerfugl billeder.

1. insektarter, forberedelse af løsninger og fodring Station Setup
Bemærk: kål sommerfugle (Pieris rapae L., Pieridae) er valgt som arter af repræsentant Lepidoptera, fordi de har været anvendt i tidligere undersøgelser af væske-uptake evner og mouthpart morfologi22,23. Stuefluer (Musca domestica L., Muscidae) og blå flaske fluer (Calliphora vomitoria L., Calliphoridae) er anvendt, fordi de er ofte obse...

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Insekt mouthpart funktionalitet er historisk udledes fra undersøgelser af brutto morfologi (fx., Lepidoptera Snabel funktionalitet relateret til en drikke halm25,26); men nyere undersøgelser, der omfatter eksperimentelle bevismateriale har resulteret i et paradigmeskift i vores forståelse af kompleksiteten af insekt mouthparts og struktur-funktion relationer2,12,13...

Mange insekt grupper har mouthparts (Snabel) tilpasset fodring på væsker, såsom nektar, rådnende frugt, sap strømme (f.eks. Diptera1, Lepidoptera2, Hymenoptera3), vedvævet (Hemiptera4), tårer (Lepidoptera 5), og blod (Phthiraptera6, Siphonaptera7, Diptera7, Hemiptera8, Lepidoptera9). Evnen af insekter til foder på væsker er relevante at økosystemers sundhed (fx bestøvning10), sygdom transmission4,11, biodiversification2,12, og undersøgelser konvergent evolution13. Trods den brede vifte af fødekilder er et tema blandt nogle væske-fodring insekter evnen til at få små mængder af væsker, som kunne være begrænset til mikro - eller nano-størrelse dråber, flydende film eller porøse overflader.
I betragtning af den omfattende mangfoldighed af væske-fodring insekter (mere end 20% af alle dyrearter14,15) og deres evne til at fodre på en række fødevarer kilder, forstå deres fodring adfærd og væske-uptake mekanismer er vigtigt inden for mange områder. Insekt mouthpart funktionalitet, for eksempel, har spillet en rolle i udviklingen af biomimetiske teknologi, fx, mikrofluid enheder, der kan udføre opgaver såsom erhvervelse af små mængder af væsker ved hjælp af metoder ligner dem ansat af insekter16. Et grundlæggende problem i undersøgelser af væske optagelse mekanismer, dog bestemmelse af ikke blot hvordan insekter lever af væsker, men erhverve eksperimentelle bevismateriale, der understøtter mekanismen. Udelukkende ved hjælp af adfærd (fx sondering med Snabel12,17) som en indikator for fodring er utilstrækkelig, fordi det ikke bekræfte den succesfulde optagelse af væsker, og heller ikke det giver et middel til at fastlægge, hvilken rute der væsker rejse som de passerer gennem insektet. Derudover repræsenterer udfører eksperimenter med små mængder af væsker bedre naturlig fodring scenarier hvor væsker er en begrænsende ressource2,12.
X-ray kontrast imaging blev brugt med Monarch butterfly (Danaus plexippus L.) til at vurdere hvordan sommerfugle lever af små mængder af væsker fra porøse overflader12fase. Monarch sommerfugle bruge kapillaritet via mellemrum mellem cuticular fremskrivninger (dorsal legulae) langs Snabel for at medbringe væsker begrænset til små porer i mad-kanalen. De indgående væsker danner en film på mad canal væggen, der vokser og kollapser i en flydende bro af Plateau ustabilitet12,18, som derefter transporteres til den sommerfugl gut af handling af sutter pumpen i hovedet. Selv om fase kontrast røntgenoptagelser er en optimal værktøj til at visualisere væskestrøm inde insekter12,19,20,21, teknikken er ikke let tilgængelige og en mere bekvem metoden er behov for hurtig vurdering af et insekt evne til optagelse væsker og indtage dem.
Til at bestemme, hvis fodring mekanismen for D. plexippus gælder andre Lepidoptera og også fluer (Diptera) (begge grupper feed på væsker fra porøse overflader), Lehnert et al. 13 anvendt en teknik til at vurdere et insekt evne at fodre med små mængder af væsker fra porøse overflader, som er rapporteret i detaljer her. Selv om den protokol, der er skitseret her til undersøgelser, der bruger chloroformvædet og porøse overflader, kan metoden, der ændres til andre undersøgelser, som dem adressering pool-fodring mekanismer. Derudover udvider applikationer til andre områder, herunder mikrofluidik og bioinspired teknologi.

<table>
	<tbody>
		<tr>
			<td>20% sucrose solution</td>
			<td>Domino Sugar</td>
			<td></td>
			<td>Sugar needed to produce the sucrose solution with dH2O</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10X concentration diluted to 1X in dH2O for insect dissections</td>
		</tr>
		<tr>
			<td>Single depression concave slide</td>
			<td>AmScope</td>
			<td>BS-C6</td>
			<td>Slide is necessary for feeding stage setup</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY6004700 (60 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY4104700 (41 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY3004700 (30 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY2004700 (20 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY1104700 (11 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TCTP04700 (10 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TETP04700 (8 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TMTP04700 (5 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>RTTP04700 (1 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Iris microdissecting scissors</td>
			<td>Carolina Biological Supply Company</td>
			<td>623555</td>
			<td>Scissors used for dissections</td>
		</tr>
		<tr>
			<td>Insect pins (#1)</td>
			<td>Bioquip Products</td>
			<td>1208B1</td>
			<td>Pins used during dissections and feeding trials</td>
		</tr>
		<tr>
			<td>Extra-fine point dissecting forceps</td>
			<td>Carolina Biological Supply Company</td>
			<td>624684</td>
			<td>Dissecting equipment</td>
		</tr>
		<tr>
			<td>Leica M205 C Stereoscope</td>
			<td>Leica Microsystems</td>
			<td>M205 C</td>
			<td>Stereoscope used for dissections</td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td>Fluorescent microscope used to detect magnetic nanoparticles</td>
		</tr>
		<tr>
			<td>Fisherbrand PTFE Disposable Stir Bar</td>
			<td>Fisherscientific</td>
			<td>S68067</td>
			<td>Magnet used to detect nanoparticles</td>
		</tr>
		<tr>
			<td>Kimtech Science Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34155</td>
			<td>Tissues used to secure insects during feeding trials</td>
		</tr>
		<tr>
			<td>House fly (Musca domestica) pupae</td>
			<td>Mantisplace.com</td>
			<td></td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Blue bottle fly (Calliphora vomitoria) pupae</td>
			<td>Mantisplace.com</td>
			<td></td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Cabbage butterfly (Pieris rapae) larvae</td>
			<td>Carolina Biological Supply Company</td>
			<td>144102</td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Finnpipette F1&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>4641080N</td>
			<td>micropipette for dispensing liquids</td>
		</tr>
		<tr>
			<td>Finntip 250 pipette tips</td>
			<td>ThermoFisher Scientific</td>
			<td>9400250</td>
			<td>micropipette tips</td>
		</tr>
		<tr>
			<td>Microscope Glass cover slides (=coverslips) (24 x 24 mm)</td>
			<td>AmScope</td>
			<td>CS-S24-100</td>
			<td>coverslips for viewing the insect&#39;s crop on confocal microscope</td>
		</tr>
	</tbody>
</table>

the ingestion of fluorescent, magnetic nanoparticles for determining fluid-uptake abilities in insects

Væske-fodring insekter indtager en bred vifte af væsker, som er til stede i miljøet som pools, film, eller begrænset til små porer. Undersøgelser af flydende erhvervelse kræver vurdering af mouthpart struktur og funktion relationer; dog er væske optagelse mekanismer historisk udledes fra observationer af strukturelle arkitektur, undertiden uledsagede med eksperimentelle bevismateriale. Her rapporterer vi en roman metode til vurdering af væske-uptake evner med sommerfugle (Lepidoptera) og fluer (Diptera) ved hjælp af små mængder af væsker. Insekter er fodret med en 20% rørsukkeropløsning blandet med fluorescerende, magnetiske nanopartikler fra papirfiltre af specifikke pore størrelser. Afgrøde (indre struktur, der bruges til opbevaring af væsker) er fjernet fra insektet og placeret på en Konfokal mikroskop. En magnet er vinkede af afgrøde til at bestemme tilstedeværelsen af nanopartikler, der angiver, hvis insekter er stand til at indtage væske. Denne metode bruges til at afsløre en udbredt fodring mekanisme (kapillaritet og flydende bro dannelse), der potentielt deles blandt Lepidoptera og Diptera, når fodring fra porøse overflader. Desuden, denne metode kan bruges til undersøgelser af fodring mekanismer blandt en bred vifte af væske-fodring insekter, herunder dem, der er vigtigt i smitteoverførsel og bioefterligning og potentielt andre undersøgelser, der involverer nano - eller mikro-størrelse ledningsanlæg hvor flydende transport kræver kontrol.

Undersøgelse af mønstre i væske-uptake evner blandt væske-fodring insekter kræver bestemmelse af når fodring opstår. Den protokol, der er skitseret her bruges til at teste den begrænsende pore størrelse hypotese blandt Lepidoptera og Diptera13. Den begrænsende pore størrelse hypotese hedder det, at væske-fodring insekter ikke kan foder fra væskefyldte porer hvis pore størrelse diameter er mindre end diameteren af den opfodring ledningsanlæg<sup class...

Watch this Scientific Journal Video about The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects at JoVE.com

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Væske-fodring insekter har mulighed for at erhverve minut mængder af væsker fra porøse overflader. Denne protokol beskriver en metode til at bestemme direkte mulighed for insekter at indtage væske fra porøse overflader ved hjælp af fodring løsninger med fluorescerende, magnetiske nanopartikler.

Indtagelse af fluorescerende, magnetiske nanopartikler til bestemmelse af væske-uptake evner i insekter

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

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