This research was supported by NIH grant #SC1AI109055, the 2014 NMSU HHMI grant #52008103, and NSF PGR grant #1238731. We thank the participants of the NMSU Spring 2015 BIOL302 Molecular Methods class and Lavesh Bhatia for their technical support with the fat body culture experiments.

1. Preparing the Solutions and Reagents
<ol>
	<li>Amino acid stock solution
	<ol>
		<li>Prepare a 4X amino acid stock solution23,24 by weighing and adding the 20 different amino acids to an Erlenmeyer flask according to the concentrations given in Table 1. 
		NOTE: In some cases, amino acid(s) can be excluded from the solution and replaced by equal molar amounts of mannitol to maintain an equal concentration of osmolytes.</li>
		<li>Add the appropriate amount of ddH2O to produce the desired volume of solution.</li>
		<li>To ensure that the amino acids have completely dissolved, heat gently while continually stirring until the liquid is completely clear; it will take on a slight yellow hue. 
		NOTE: It may be necessary to reduce the pH to 6 by adding HCl to allow some of the more hydrophobic amino acids to dissolve.</li>
		<li>Aliquot the solution and sterile-filter using a syringe filter with a 0.2-&#181;m pore size.</li>
		<li>Store in a sealed container at -20 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>APS 
	Note: See25.
	<ol>
		<li>For the 20X salt stock solution, combine the salts listed in Table 2 in 50 mL of water. Stir until completely dissolved. 
		NOTE: It may be necessary to slightly heat the solution to completely dissolve the salts.</li>
		<li>Sterile-filter using a syringe filter with a 0.2-&#181;m pore size and store the solution in a 50-mL tube at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Calcium stock solution
	<ol>
		<li>For the 50X calcium stock solution, add 0.90 g of calcium chloride to 100 mL of water (Table 3); stir until completely dissolved.</li>
		<li>Sterile-filter using a syringe filter with a 0.2-&#181;m pore size, aliquot the solution into 50-mL tubes, and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Tris buffer (pH 7.4)
	<ol>
		<li>For the Tris buffer, combine the salt and solutions listed in Table 4 and add ddH2O up to 100 mL.</li>
		<li>Sterile-filter using a syringe filter with a 0.2-&#181;m pore size and aliquot the solution into 50-mL tubes; store at room temperature.</li>
	</ol>
	</li>
	<li>Fat body culture medium
	<ol>
		<li>To prepare the fat body culture medium, prepare all solutions listed above.</li>
		<li>Combine them as per the volumes in Table 5 to make 200 mL of fat body culture medium.</li>
		<li>Adjust the pH to 7.2 using NaOH or HCl.</li>
		<li>Sterile-filter the solution using a syringe filter with a 0.2-&#181;m pore size.</li>
		<li>Divide the solution into 15-mL aliquots and store at -20 &#176;C for up to 6 months.</li>
	</ol>
	</li>
</ol>
2. Preparing for Dissection
<ol>
	<li>Prepare a stereomicroscope (10-20x magnification) with illumination and lay out two ultra-fine tweezers and a pair of micro dissection scissors.</li>
	<li>Prepare all solutions necessary for the experiment. Thaw the fat body culture medium to room temperature. 
	NOTE: Precipitants can be dissolved by gently heating the solution to no higher than 30 &#176;C.</li>
	<li>With an aspirator, collect adult female mosquitoes (3-7 days after emergence) and anesthetize them using carbon dioxide or ice. 
	NOTE: Once anesthetized, the mosquitoes should be kept under CO2 for the shortest time possible, not exceeding 20 min. Alternatively, the mosquitoes can be anesthetized on ice and kept for approximately 30 min.</li>
	<li>Prepare a 6-well plate with 3 mL of APS per well.</li>
</ol>
3. Fat Body Dissection
<ol>
	<li>Focus the dissecting stereomicroscope and adjust the chair to a comfortable height.</li>
	<li>Place a concave microscope slide on the microscope surface and add two drops of APS to the center.</li>
	<li>Pick up a mosquito by a leg using a forceps and transfer it to the APS surface on the microscope slide.</li>
	<li>Carefully grip the mosquito by the thorax, with the forceps in the left hand, and rotate the mosquito so that the ventral side is upwards.</li>
	<li>While holding the body steady by the thorax, grasp the last two abdominal segments, with the forceps in the right hand, and gently pull. 
	NOTE: The last two abdominal segments will separate from the abdomen and the attached ovaries, Malpighian tubules, hindgut, and midgut; sometimes the crop will slide out of the abdomen. If they have not been removed, insert the closed tips of the forceps into the small hole created and remove the remaining tissues.</li>
	<li>Set the right forceps aside and pick up the spring scissors. Slide one blade into the hole in the abdomen up to the segment below the thorax. Gently and swiftly cut the abdomen lengthwise. 
	NOTE: Once the cut has been made, the abdomen should begin to expand outward, though this may not occur immediately.</li>
	<li>Proceed to make the second cut, which will be a lateral cut below the thorax and slightly below where the first cut ended. 
	NOTE: The abdomen should then open up and dissociate from the thorax, with the cuticle up and the fat bodies side-immersed within the APS. This abdominal body wall is the fat body preparation for in vitro culture.</li>
	<li>Lift the fat bodies from the APS solution using the tip of a pair of forceps and transfer them from the solution to the 6-well plate containing APS at room temperature. Allow the tissue to rest for approximately 0.5 h before advancing to the fat body culture.</li>
</ol>
4. Fat Body Culture
<ol>
	<li>Incubate the fat bodies on APS at room temperature for at least 0.5 h to equilibrate them.</li>
	<li>Transfer the fat bodies using the tip of the forceps and gently lift them out of the APS solution. Lower the tip into the fat body culture medium. 
	NOTE: The fat body should open on the surface of the medium and float freely. The fat body culture is typically performed in 96-well plates, with 150-200 &#181;L of medium in each well. One well can fit up to three individual fat bodies, and they are viable for more than 12 h (unpublished personal results).</li>
	<li>After the incubation period, transfer the fat bodies from the medium into 1.5-mL centrifuge tubes containing the appropriate reagents for downstream processing. 
	NOTE: Typical analyses include qPCR16,17,18,19, Western blotting18,20, transcriptomics26, proteomics21, or metabolomics22.</li>
</ol>

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We have nothing to disclose.

Insect organ culture was used extensively to study insect endocrinology, development, and metabolism, as well as to investigate the interaction between specific organs and bacterial symbionts29,30,31,32,33,34. In vitro fat body organ culture was used specifically to study amino acid transport and the regulation of yolk protein production in mosquitoes and other Diptera16,17,35,36. During the process of vitellogenesis, the mosquito fat body uses an array of high-specificity amino acid transporters to import blood meal-derived amino acids from the hemolymph to synthesize large quantities of yolk proteins12,19,35,36. Fat body culture was instrumental to the delineation of the fat body nutritional requirements in this context18.
The quality of the starting material, female mosquitoes, is critical for the success of these experiments. Mosquito larvae raised in under-crowded conditions and fed on high-nutrient diets usually produce the best results. There are some important variables to consider when establishing mosquito fat body culture conditions in the laboratory in terms of experimental design. We showed in previous studies that fat body gene expression varies significantly depending on the individual life history and nutritional status of the mosquito11,22. The mosquito culture conditions should be uniform to reduce the variability in the size and nutritional reserves of the experimental mosquitoes. In addition, personnel performing the dissections should be trained to ensure rapid and accurate dissections with consistent results. Cell viability in isolated fat bodies can be checked using different staining methods37,38.
The experimental design of a fat body culture experiment should take into consideration the number of dissections possible in a given time period. When large quantities of fat bodies are required, multiple dissection sessions or multiple dissectors may be necessary. There is a wide range of future applications for in vitro fat body culture in mosquitoes and other insects. It will be especially useful for testing potential drug candidates for insect control. The use of transgenic techniques in insects to express specific reporter proteins in fat body trophocytes will open up new methods to develop powerful bioassays for the study of fat body physiology.

Mosquitoes are vectors of devastating human diseases, including malaria, dengue fever, chikungunya, and Zika1,2,3. Despite intense international efforts to curb these diseases and to control disease-transmitting mosquito populations, epidemic outbreaks of mosquito-borne diseases are still common, especially in developing countries. Effective vaccines against many of these diseases are either unavailable or of limited efficacy4,5. The most effective way to prevent outbreaks is to control mosquito populations, mainly through the use of insecticide treatments. However, insecticide resistance has developed in many mosquito populations and become a common problem around the world6,7,8. The study of mosquito physiology is essential to the development of novel tools and strategies to control disease.
The mosquito fat body plays a central role in nutrient storage, metabolic homeostasis, reproduction, and xenobiotic catabolism9,10,11,12. It is the main storage organ for triglycerides, glycogen, and amino acids in form of storage proteins. It also functions as the location of synthesis for most hemolymph proteins and metabolites. In mosquitoes, the fat body is the sole source of yolk protein production that occurs in females after they take a blood meal13,14.
The principal cell type of the fat body is the large, polyploid trophocyte or adipocyte3,9,10,12. Fat body tissue is organized into lobes or sheaths and can be found in all body parts of the mosquito, with the largest portion located in the abdomen, where large lobes of fat body are attached to the abdominal body wall.
The mosquito fat body culture system presented here was developed in the &#39;70s and remains a powerful tool for studying fat body physiology10, especially in combination with current analysis technologies. The foundation of this technique is based on the isolation of the abdominal body walls and the associated fat body tissue. The hydrophobic nature of the abdominal cuticle causes it to float on the surface of the culture medium, with the attached lobes of abdominal fat body immersed. The spiracles and tracheolar structure are maintained, ensuring oxygenation of the cultured tissue. Henceforth, we will refer to these preparations as &#34;fat bodies.&#34; Isolated fat bodies remain viable for more than 12 h when incubated in the appropriate medium (unpublished results). Fat body culture is a valuable tool that has addressed a variety of questions regarding fat body endocrinology and physiology9,10,12,15,16,17.
Cultured fat bodies can be subjected to various experimental and control treatments, the timing of which can be decided upon by the investigator. At the end of the incubation period, the fat bodies can be collected and processed for downstream analyses, including qPCR16,17,18,19, Western blotting18,20, proteomics21, or metabolomics22. Experiments can be performed on different scales, from individual fat bodies to groups of hundreds that can be cultured together.
The representative results included here were derived from fat bodies cultured in the presence of amino acids and the steroid hormone 20-hydroxy ecdysone to simulate the blood meal activation of vitellogenesis16,17,23,24. We analyzed and compared the differential gene expression of not-activated versus activated fat bodies via next-generation sequencing analysis.

<table border="0" cellpadding="0" cellspacing="0" width="508">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">Scissors&#160;</td>
			<td style="width:192px;">Fiskars</td>
			<td style="width:143px;">83872</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">Fly pad</td>
			<td style="width:192px;">Genesee Scientific</td>
			<td style="width:143px;">789060</td>
			<td></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">Battery-powered aspirator w/ collection vial</td>
			<td style="width:192px;">Hausherrs Machine Works, Inc.</td>
			<td style="width:143px;">3740-01-210-2368</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Fine tip forceps</td>
			<td style="width:192px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:143px;">500085</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:64px;">Light microscope&#160;</td>
			<td style="width:192px;">Leica Microsystems</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">96 well plate&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:143px;">CL S3383</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">Sucrose</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:143px;">S9378</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Alanine</td>
			<td style="width:192px;">Sigma</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Arginine</td>
			<td style="width:192px;">Sigma</td>
			<td>A5006</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Asparagine</td>
			<td style="width:192px;">Sigma</td>
			<td>A0884</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aspartic Acid</td>
			<td style="width:192px;">Sigma</td>
			<td>A9256</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Cysteine</td>
			<td style="width:192px;">Sigma</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamic Acid</td>
			<td style="width:192px;">Sigma</td>
			<td>G1251</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamine</td>
			<td style="width:192px;">Sigma</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glycine</td>
			<td style="width:192px;">Sigma</td>
			<td>G2879</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Histidine</td>
			<td style="width:192px;">Sigma</td>
			<td>H6034</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Isoleucine</td>
			<td style="width:192px;">Sigma</td>
			<td>I2752</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Lysine</td>
			<td style="width:192px;">Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Leucine</td>
			<td style="width:192px;">Sigma</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Phenylalanine</td>
			<td style="width:192px;">Sigma</td>
			<td>P2126</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Proline</td>
			<td style="width:192px;">Sigma</td>
			<td>P0380</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Serine</td>
			<td style="width:192px;">Sigma</td>
			<td>S4500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Threonine</td>
			<td style="width:192px;">Sigma</td>
			<td>T8625</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tryptophan</td>
			<td style="width:192px;">Sigma</td>
			<td>T0254</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tyrosine</td>
			<td style="width:192px;">Sigma</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Valine</td>
			<td style="width:192px;">Sigma</td>
			<td>V0500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Methionine</td>
			<td style="width:192px;">Sigma</td>
			<td>M9625</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">NaCl</td>
			<td style="width:192px;">Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">KCl</td>
			<td style="width:192px;">Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:64px;">MgCl2-6H2O</td>
			<td style="width:192px;">Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:64px;">NaHCO3</td>
			<td style="width:192px;">Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:64px;">CaCl2-2H2O</td>
			<td style="width:192px;">Sigma</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:64px;">Tris pH8.0&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">EDTA</td>
			<td style="width:192px;">Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:64px;">ddH2O</td>
			<td style="width:192px;">Sigma</td>
			<td>W4502</td>
			<td></td>
		</tr>
	</tbody>
</table>

fat body organ culture system in aedes aegypti, a vector of zika virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

As an example, we performed a fat body culture experiment and stimulated isolated fat bodies by incubating them on a solution containing a balanced mixture of all twenty naturally occurring amino acids and the insect steroid hormone 20-hydroxy ecdysone (10 &#181;M) for 6 h. As a control, fat bodies were incubated on APS for an equal amount of time.
After incubation, the total RNA was isolated using a tri-reagent27 following the manufacturer&#39;s instructions. The quality and quantity of extracted RNA samples were assessed using a spectrophotometer, fluorometric quantitation, and agarose gel electrophoresis. RNA sequencing libraries were generated using 4 &#181;g of total RNA and were quantified using two different techniques. Subsequently, the libraries were sent to a commercial provider for paired end-sequencing.
The results of this experiment are shown in Table 6. Genes showing the strongest transcriptional response to amino acid and 20-hydroxyecdysone were primarily yolk protein genes, which is in agreement with previous results11.
Figure 1 shows a heat map indicating the gene expression levels of 1,256 differentially expressed genes from cultured fat bodies after two different treatments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55508/55508fig1.jpg" /> 
Figure 1. Heat map of genes expressed in the fat body culture. The heat map was calculated based on the number of specific transcripts for each gene in the different libraries using the heatmap package28 that is part of the R software environment. The darker shade represents higher gene expression. 1,256 genes with statistically significant variation in expression (Q-values &#60; 0.05) are shown. The genes are ordered according to their averaged expression levels, indicated by the dendrogram on the left (not according to their phylogenetic relationships). Note the high number of genes with up- or down-regulated expression after stimulation with amino acids (AA) and 20-hydroxyecdysone (20E). APS = Aedes physiological saline. See Supplemental File 1 for a list of genes and their relative expression levels. <a href="https://cloudflare.jove.com/files/ftp_upload/55508/55508fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amino Acid</td>
			<td>Molecular Weight g/mol</td>
			<td>mM Concentration</td>
			<td>mg per Liter</td>
			<td>mg for 300 mL volume</td>
		</tr>
		<tr>
			<td>Alanine</td>
			<td>89.1</td>
			<td>26.68</td>
			<td>2377.19</td>
			<td>713.16</td>
		</tr>
		<tr>
			<td>Arginine</td>
			<td>174.2</td>
			<td>26.68</td>
			<td>4647.66</td>
			<td>1394.3</td>
		</tr>
		<tr>
			<td>Asparagine</td>
			<td>150.1</td>
			<td>26.68</td>
			<td>4004.67</td>
			<td>1201.4</td>
		</tr>
		<tr>
			<td>Aspartic Acid</td>
			<td>133</td>
			<td>26.68</td>
			<td>3548.44</td>
			<td>1064.53</td>
		</tr>
		<tr>
			<td>Cysteine</td>
			<td>121.16</td>
			<td>10.68</td>
			<td>1293.99</td>
			<td>388.2</td>
		</tr>
		<tr>
			<td>Glutamic Acid</td>
			<td>147.1</td>
			<td>26.68</td>
			<td>3924.63</td>
			<td>1177.39</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>146</td>
			<td>26.68</td>
			<td>3895.28</td>
			<td>1168.58</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>75</td>
			<td>53.32</td>
			<td>3999</td>
			<td>1199.7</td>
		</tr>
		<tr>
			<td>Histidine</td>
			<td>155.16</td>
			<td>80</td>
			<td>12412.8</td>
			<td>3723.84</td>
		</tr>
		<tr>
			<td>Isoleucine</td>
			<td>131</td>
			<td>10.68</td>
			<td>1399.08</td>
			<td>419.72</td>
		</tr>
		<tr>
			<td>Lycine</td>
			<td>183</td>
			<td>26.68</td>
			<td>4882.44</td>
			<td>1464.73</td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>131</td>
			<td>26.68</td>
			<td>3495.08</td>
			<td>1048.52</td>
		</tr>
		<tr>
			<td>Phenylalanine</td>
			<td>165</td>
			<td>10.68</td>
			<td>1762.2</td>
			<td>528.66</td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>115</td>
			<td>26.68</td>
			<td>3068.2</td>
			<td>920.46</td>
		</tr>
		<tr>
			<td>Serine</td>
			<td>105</td>
			<td>53.32</td>
			<td>5598.6</td>
			<td>1679.58</td>
		</tr>
		<tr>
			<td>Threonine</td>
			<td>119</td>
			<td>10.68</td>
			<td>1270.92</td>
			<td>381.28</td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>204</td>
			<td>10.68</td>
			<td>2178.72</td>
			<td>653.62</td>
		</tr>
		<tr>
			<td>Tyrosine</td>
			<td>181</td>
			<td>5.32</td>
			<td>962.92</td>
			<td>288.88</td>
		</tr>
		<tr>
			<td>Valine</td>
			<td>117</td>
			<td>10.68</td>
			<td>1249.56</td>
			<td>374.87</td>
		</tr>
		<tr>
			<td>Methionine</td>
			<td>149</td>
			<td>10.68</td>
			<td>1591.32</td>
			<td>477.4</td>
		</tr>
	</tbody>
</table>
Table 1. 4X Amino acid stock solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Weight in grams added to 50 ml ddH2O</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>8.0 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.074 g</td>
		</tr>
		<tr>
			<td>MgCl2-6H2O</td>
			<td>0.120 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>0.0250 g</td>
		</tr>
	</tbody>
</table>
Table 2.20X salt stock solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Weight in grams added to 100 ml ddH2O</td>
		</tr>
		<tr>
			<td>CaCl2-2H2O</td>
			<td>0.90 g</td>
		</tr>
	</tbody>
</table>
Table 3.50X calcium stock solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Concentration of stock solution</td>
			<td>Volume stock for 100 ml buffer</td>
		</tr>
		<tr>
			<td>Tris pH8.0&#160;</td>
			<td>1 M</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>0.25 M</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>NA</td>
			<td>0.3 g</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>NA</td>
			<td>to 100 mL (~93 mL)</td>
		</tr>
	</tbody>
</table>
Table 4.&#160;Tris buffer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume stock for 200 ml&#160;</td>
		</tr>
		<tr>
			<td>Amino Acid Stock Solution</td>
			<td>150 mL</td>
		</tr>
		<tr>
			<td>Salt Stock Solution</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Calcium Stock Solution</td>
			<td>4 mL</td>
		</tr>
		<tr>
			<td>TES Buffer</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>26 mL</td>
		</tr>
	</tbody>
</table>
Table 5. Fat body culture medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Annotation</td>
			<td>Gene description</td>
			<td>Fold change</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td>AAEL006138</td>
			<td>Vitellogenin-B</td>
			<td>3443</td>
			<td>2.52E-112</td>
		</tr>
		<tr>
			<td>AAEL006126</td>
			<td>Vitellogenin-C</td>
			<td>2795</td>
			<td>8.64E-91</td>
		</tr>
		<tr>
			<td>AAEL006563</td>
			<td>vitellogenic carboxypeptidase</td>
			<td>1002</td>
			<td>2.17E-119</td>
		</tr>
		<tr>
			<td>AAEL010434</td>
			<td>Vitellogenin-A</td>
			<td>220</td>
			<td>1.14E-27</td>
		</tr>
		<tr>
			<td>AAEL006542</td>
			<td>vitellogenic carboxypeptidase</td>
			<td>185</td>
			<td>2.14E-65</td>
		</tr>
		<tr>
			<td>AAEL012678</td>
			<td>AAEL003006-PA [Aedes aegypti](65%)</td>
			<td>96</td>
			<td>4.00E-70</td>
		</tr>
		<tr>
			<td>AAEL000080</td>
			<td>hypothetical protein&#160;</td>
			<td>82</td>
			<td>6.69E-188</td>
		</tr>
		<tr>
			<td>AAEL015312</td>
			<td>Vitellogenic cathepsin B</td>
			<td>77</td>
			<td>1.27E-15</td>
		</tr>
		<tr>
			<td>AAEL009588</td>
			<td>nuclear receptor 3</td>
			<td>75</td>
			<td>4.58E-56</td>
		</tr>
		<tr>
			<td>AAEL010529</td>
			<td>hypothetical protein</td>
			<td>66</td>
			<td>1.32E-29</td>
		</tr>
	</tbody>
</table>
Table 6.Experimental results.
Supplemental File 1. <a href="https://cloudflare.jove.com/files/ftp_upload/55508/Table_of_genes_for_figure_1.xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus at JoVE.com

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

fat-body-organ-culture-system-in-aedes-aegypti-a-vector-of-zika-virus

Research

JoVE Journal

Biology

9.4K Views.  New Mexico State University. The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

Video: Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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.

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

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.

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain&#8217;s fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.&#160;
Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.&#160;

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Here, we present a protocol for studying mating competitiveness of male Aedes aegypti using fluorescent dye as a marker. Female mosquitoes are exposed to both marked and unmarked males for copulation. Post mating, their spermathecae are examined under a fluorescence microscope to determine their mating partner.

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.