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><tbody><tr><td>Scissors&amp;nbsp;</td><td>Fiskars</td><td>83872</td><td></td></tr><tr><td>Fly pad</td><td>Genesee Scientific</td><td>789060</td><td></td></tr><tr><td>Battery-powered aspirator w/ collection vial</td><td>Hausherrs Machine Works, Inc.</td><td>3740-01-210-2368</td><td></td></tr><tr><td>Fine tip forceps</td><td>World Precision Instruments, Inc.&amp;nbsp;</td><td>500085</td><td></td></tr><tr><td>Light microscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>96 well plate&amp;nbsp;</td><td>Sigma</td><td>CL S3383</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>Alanine</td><td>Sigma</td><td>A7627</td><td></td></tr><tr><td>Arginine</td><td>Sigma</td><td>A5006</td><td></td></tr><tr><td>Asparagine</td><td>Sigma</td><td>A0884</td><td></td></tr><tr><td>Aspartic Acid</td><td>Sigma</td><td>A9256</td><td></td></tr><tr><td>Cysteine</td><td>Sigma</td><td>W326305</td><td></td></tr><tr><td>Glutamic Acid</td><td>Sigma</td><td>G1251</td><td></td></tr><tr><td>Glutamine</td><td>Sigma</td><td>G3126</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G2879</td><td></td></tr><tr><td>Histidine</td><td>Sigma</td><td>H6034</td><td></td></tr><tr><td>Isoleucine</td><td>Sigma</td><td>I2752</td><td></td></tr><tr><td>Lysine</td><td>Sigma</td><td>L5501</td><td></td></tr><tr><td>Leucine</td><td>Sigma</td><td>L8000</td><td></td></tr><tr><td>Phenylalanine</td><td>Sigma</td><td>P2126</td><td></td></tr><tr><td>Proline</td><td>Sigma</td><td>P0380</td><td></td></tr><tr><td>Serine</td><td>Sigma</td><td>S4500</td><td></td></tr><tr><td>Threonine</td><td>Sigma</td><td>T8625</td><td></td></tr><tr><td>Tryptophan</td><td>Sigma</td><td>T0254</td><td></td></tr><tr><td>Tyrosine</td><td>Sigma</td><td>T3754</td><td></td></tr><tr><td>Valine</td><td>Sigma</td><td>V0500</td><td></td></tr><tr><td>Methionine</td><td>Sigma</td><td>M9625</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;-6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M2670</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>C8106</td><td></td></tr><tr><td>Tris pH8.0&amp;nbsp;</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>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

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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

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Immunology and Infection

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

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, ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

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 ...

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

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Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

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