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

This work was supported by National Science Foundation (NSF) grant no. IOS 1354956. We thank Dr. Andrew D. Warren (McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida) for permission to use the butterfly images.

1. Insect Species, Preparation of Solutions and Feeding Station Setup
NOTE: Cabbage butterflies (Pieris rapae L., Pieridae) are selected as the species of representative Lepidoptera because they have been used in previous studies of fluid-uptake abilities and mouthpart morphology22,23. House flies (Musca domestica L., Muscidae) and blue bottle flies (Calliphora vomitoria L., Calliphoridae) are used because they...

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Insect mouthpart functionality is historically inferred from studies of gross morphology (e.g., lepidopteran proboscis functionality related to a drinking straw25,26); however, recent studies that incorporate experimental evidence have resulted in a paradigm shift in our understanding of the complexities of insect mouthparts and structure-function relationships2,12,13...

Many insect groups have mouthparts (proboscises) adapted for feeding on fluids, such as nectar, rotting fruit, sap flows (e.g. Diptera1, Lepidoptera2, Hymenoptera3), xylem (Hemiptera4), tears (Lepidoptera5), and blood (Phthiraptera6, Siphonaptera7, Diptera7, Hemiptera8, Lepidoptera9). The ability of insects to feed on fluids is relevant to ecosystem health (e.g. pollination10), disease transmission4,11, biodiversification2,12, and studies of convergent evolution13. Despite the wide variety of food sources, a theme among some fluid-feeding insects is the ability to acquire small amounts of fluids, which could be confined to micro- or nano-sized droplets, liquid films, or porous surfaces.
Given the extensive diversity of fluid-feeding insects (more than 20% of all animal species14,15) and their ability to feed on a variety of food sources, understanding their feeding behaviors and fluid-uptake mechanisms is important in many fields. Insect mouthpart functionality, for instance, has played a role in the development of biomimetic technology, e.g., microfluidic devices that can perform tasks such as the acquisition of small amounts of fluids using methods similar to those employed by insects16. A fundamental problem in the studies of fluid uptake mechanisms, however, is determining not only how insects feed on fluids, but acquiring experimental evidence that supports the mechanism. Solely using behavior (e.g., probing with the proboscis12,17) as an indicator for feeding is insufficient because it does not confirm the successful uptake of fluids, nor does it provide a means to determine the route that fluids travel as they pass through the insect. In addition, performing experiments with small quantities of fluids better represents natural feeding scenarios where fluids are a limiting resource2,12.
X-ray phase contrast imaging was used with the Monarch butterfly (Danaus plexippus L.) to assess how butterflies feed on small amounts of fluids from porous surfaces12. Monarch butterflies use capillary action via spaces between cuticular projections (dorsal legulae) along the proboscis to bring fluids confined to small pores into the food canal. The incoming fluids form a film on the food canal wall that grows and collapses into a liquid bridge by Plateau instability12,18, which is then transported to the butterfly&#39;s gut by action of the sucking pump in the head. Although x-ray phase contrast imaging is an optimal tool for visualizing fluid flow inside insects12,19,20,21, the technique is not readily available and a more convenient method is needed for rapid assessment of an insect&#39;s ability to uptake fluids and ingest them.
To determine if the feeding mechanism for D. plexippus applies to other Lepidoptera and also to flies (Diptera) (both groups feed on liquids from porous surfaces), Lehnert et al.13 applied a technique for assessing an insect&#39;s ability to feed on small amounts of fluids from porous surfaces, which is reported in detail here. Although the protocol outlined here is for studies that use wetted and porous surfaces, the methodology can be altered for other studies, such as those addressing pool-feeding mechanisms. In addition, the applications extend to other fields, including microfluidics and bioinspired technology.

<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

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 acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

The study of patterns in fluid-uptake abilities among fluid-feeding insects requires determination of when feeding occurs. The protocol outlined here is used to test the limiting pore size hypothesis among Lepidoptera and Diptera13. The limiting pore size hypothesis states that fluid-feeding insects cannot feed from liquid-filled pores if the pore size diameter is smaller than the diameter of the feeding conduits12. Incoming fluids from the ...

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

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

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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Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...