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 are often observed feeding on porous surfaces13.
<ol>
	<li>Order P. rapae as larvae from an insect supplier and rear them on artificial diet (see Table of Materials) until they pupate and emerge as adults in an environmental chamber set to 23 &#176;C and a 18L:6D photoperiod. Order M. domestica and C. vomitoria as pupae and rear them at the same environmental conditions as P. rapae. Do not feed adult butterflies and flies after they emerge from the pupae prior to the feeding experiments.</li>
	<li>Prepare a 20% sucrose solution (control) and a 20% sucrose nanoparticle solution to test for fluid uptake. Prepare the nanoparticle solution by adding fluorescent magnetic nanoparticles (iron oxide with a polyacrylic acid coating, approximately 20 nm in diameter)24 to a 20% sucrose solution (1 mg/mL dH2O nanoparticles with 20% sucrose solution, 1:1). Prepare a 1x solution of phosphate buffered saline (PBS) (10x diluted to 1x in dH2O, pH 7.4), which is used for dissections.</li>
	<li>Set up a feeding station that consists of a manual manipulator with a clamp and a separate feeding stage (a flat platform) (Figure 1). Place a concave slide on the feeding stage and have nylon net filters with pore size diameters of 11, 20, 30, 41, or 60 &#181;m and membrane filters with pores size diameters of 1, 5, 8, or 10 &#181;m nearby for the feeding experiments.</li>
</ol>
2. Feeding Protocol
<ol>
	<li>Wrap the insect&#39;s bodies, legs, and wings into a folded tissue paper. Position the insect so that only the head and mouthparts are exposed. Place the wings of the insect between two microscope slides, which are held together by the clamp on the manipulator so that the insect is suspended above the feeding stage (Figure 1).</li>
	<li>Administer a 30 &#181;L droplet of either the 20% sucrose solution or the 20% sucrose nanoparticle solution with a micropipette to the center of the concave slide. Place a single filter paper of a specific pore size onto the concave slide so that the center of the filter paper is aligned with the droplet of the nanoparticle solution. The contact between the droplet and the filter paper results in the solution spreading along the filter paper, filling the pores (Figure 1). 
	NOTE: The filter paper is placed on top of the droplet, rather than the other way around, to minimize the potential presence of nanoparticles on top of the filter paper where the insects feed.</li>
	<li>Position the insect with the manipulator so that only the distal regions of the mouthparts can contact the wetted filter paper on the feeding stage (Figure 1). Use an insect pin to extend the mouthparts onto the filter paper and allow the insect to feed for 45 s.</li>
	<li>To minimize the chance of insects feeding on liquid films that might be present on the surface of the filter paper, position the mouthparts so that they are in contact with a part of the filter paper that is touching the flat part of the slide (i.e., not directly above the concave part of the slide). If the insect does not express an interest in feeding, the mouthparts can be held to the filter paper with the insect pin for the duration of the feeding time.</li>
</ol>
3. Dissections
<ol>
	<li>Place the PBS solution in a 50 mm watch glass so that there is enough solution to cover the insect&#39;s body. Place the watch glass under a stereoscope and position insect dissecting equipment (spring micro dissecting scissors, insect pins, fine point dissecting forceps) next to the stereoscope.</li>
	<li>After feeding, remove the insect from the tissue paper and hold it with wings closed. Remove the head, legs, and wings of the insect with the spring micro dissecting scissors and place the insect in the PBS solution in the watch glass (Figure 2).</li>
	<li>If needed, anesthetize insects before dissections. Use forceps to hold the insect by the cuticle near the distal region of the abdomen. With the dominant hand, use the spring micro dissecting scissors to cut the cuticle in an anterior direction along the lateral side of the abdomen, starting at the posterior end, until the thorax is reached. Take special care to ensure that only the cuticle is cut and that the alimentary canal inside the insect is not damaged (Figure 2).</li>
	<li>Make additional cuts of the cuticle with the dissecting scissors to open the abdomen to reveal the alimentary canal inside (Figure 2). Remove the abdominal cuticle, fat body, and other structures with the assistance of insect pins and relocate them outside of the watch glass for subsequent disposal, leaving only the thorax and alimentary canal in the watch glass. 
	NOTE: The dissection will reveal the crop, which is a sac-like structure (an extension of the alimentary canal) located near the juncture of the thorax and abdomen.</li>
	<li>If the crop is not exposed, make additional incisions into the thorax with the scissors until the crop is revealed. Once the crop is visible, cut away the remaining parts of the insect leaving only the alimentary canal with the crop in the watch glass (Figure 2). 
	NOTE: The lepidopteran crop is nearly transparent and cellophane-like in nature, which might be difficult to recognize if it is not filled with fluids and expanded or if it is cut during the dissection.</li>
	<li>Use fine point dissecting forceps to place the crop onto a coverslip (24 mm x 24 mm) for subsequent imaging (Figure 2).</li>
</ol>
4. Determination of Ingested Nanoparticles
<ol>
	<li>Position the crop on the coverslip with the fine point dissecting forceps using care to prevent rupturing the crop. Use the CY3 channel (or phase contrast) on an inverted confocal microscope for imaging at 20X magnification. Image the crop immediately after dissection to prevent it from drying out.</li>
	<li>Hold a magnetic stir bar (41.3 mm in length and 8 mm in diameter) in the hand that is not in control of the operating stage of the microscope. Wave the magnetic stir bar back and forth near the crop (approximately 10 mm distance from crop) so that each back and forth motion takes approximately one second (Figure 2).</li>
	<li>While the magnetic stir bar is waved, inspect the crop for the nanoparticles. Slowly move the operating stage back and forth while looking through the ocular lens of the microscope for nanoparticle movement inside the nearly transparent crop. If the nanoparticles are present in the crop, indicating positive feeding, they will respond and wave in unison with the magnetic stir bar (Figure 2).</li>
</ol>

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H., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+for+functionalizable+photoluminescent+magnetic+nanoparticles.">A novel strategy for functionalizable photoluminescent magnetic nanoparticles.</a> Mater. Res. Express. 1, 045032 (2014).</li><li>Kingsolver, J. G., Daniel, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanics+and+energetics+of+nectar+feeding+in+butterflies.">On the mechanics and energetics of nectar feeding in butterflies.</a> J Theor Biol. 76, 167-179 (1979).</li><li>Krenn, H. 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The authors have nothing to disclose.

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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Research

JoVE Journal

Biology

6.3K Views.  Kent State University at Stark. 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.

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

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

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 study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

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

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

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

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

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 essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

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