We thank Texas A&#38;M Agrilife Research Insect Vectored Diseases Grant Program for funding. We also thank the Adelman lab members for help on developing this method and suggestions when drafting the manuscript, as well as Kevin Myles lab members. We also thank the reviewers and editors for their help to make this manuscript better.

1. Plate preparation
<ol>
	<li>Drill holes in 24 well tissue culture plate lids (4&#8722;6 holes per well) using a household drill with a ~1.6 mm (1/16 in) bit (Figure 2). 
	NOTE: These holes prevent water condensation from agarose to accumulate on the lid, where mosquitoes may lay eggs. The standard size of female Ae. aegypti (&#34;Liverpool&#34; strain) is ~3.11 mm wingspan. Reducing the size of the holes is recommended when using smaller mosquitoes to avoid escape from the...

<ol>
	<li>Bond, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+irradiation+dose+to+Aedes+aegypti+and+Ae.+albopictus+in+a+sterile+insect+technique+program.">Optimization of irradiation dose to Aedes aegypti and Ae. albopictus in a sterile insect technique program.</a> PLoS One. 14 (2), 0212520 (2019).</li><li>Fernandes, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aedes+aegypti+larvae+treated+with+spinosad+produce+adults+with+damaged+midgut+and+reduced+fecundity.">Aedes aegypti larvae treated with spinosad produce adults with damaged midgut and reduced fecundity.</a> Chemosphere. 221, 464-470 (2019).</li><li>Inocente, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticidal+and+Antifeedant+Activities+of+Malagasy+Medicinal+Plant+(Cinnamosma+sp.)+Extracts+and+Drimane-Type+Sesquiterpenes+against+Aedes+aegypti+Mosquitoes.">Insecticidal and Antifeedant Activities of Malagasy Medicinal Plant (Cinnamosma sp.) Extracts and Drimane-Type Sesquiterpenes against Aedes aegypti Mosquitoes.</a> Insects. 10 (11), 373 (2019).</li><li>Marrelli, M. T., Moreira, C. K., Kelly, D., Alphey, L., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+transgenesis:+what+is+the+fitness+cost.">Mosquito transgenesis: what is the fitness cost.</a> Trends in Parasitology. 22 (5), 197-202 (2006).</li><li>da Silva Costa, G., Rodrigues, M. M. S., Silva, A. A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+blood-free+diet+for+Anopheles+darlingi+(Diptera:+Culicidae).">Toward a blood-free diet for Anopheles darlingi (Diptera: Culicidae).</a> Journal of Medical Entomology. , 217 (2019).</li><li>Gonzales, K. K., Tsujimoto, H., Hansen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+serum+and+BSA,+but+neither+red+blood+cells+nor+hemoglobin+can+support+vitellogenesis+and+egg+production+in+the+dengue+vector+Aedes+aegypti.">Blood serum and BSA, but neither red blood cells nor hemoglobin can support vitellogenesis and egg production in the dengue vector Aedes aegypti.</a> PeerJ. 3, 938 (2015).</li><li>Gonzales, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+SkitoSnack,+an+Artificial+Blood+Meal+Replacement,+on+Aedes+aegypti+Life+History+Traits+and+Gut+Microbiota.">The Effect of SkitoSnack, an Artificial Blood Meal Replacement, on Aedes aegypti Life History Traits and Gut Microbiota.</a> Scientific Reports. 8 (1), 11023 (2018).</li><li>Tsujimoto, H., Anderson, M. A. E., Myles, K. M., Adelman, Z. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Candidate+Iron+Transporters+From+the+ZIP/ZnT+Gene+Families+in+the+Mosquito+Aedes+aegypti.">Identification of Candidate Iron Transporters From the ZIP/ZnT Gene Families in the Mosquito Aedes aegypti.</a> Frontiers in Physiology. 9, 380 (2018).</li><li>Ioshino, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviplate:+A+Convenient+and+Space-Saving+Method+to+Perform+Individual+Oviposition+Assays+in+Aedes+aegypti.">Oviplate: A Convenient and Space-Saving Method to Perform Individual Oviposition Assays in Aedes aegypti.</a> Insects. 9 (3), 103 (2018).</li><li>Price, D. P., Schilkey, F. D., Ulanov, A., Hansen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+mosquitoes,+large+implications:+crowding+and+starvation+affects+gene+expression+and+nutrient+accumulation+in+Aedes+aegypti.">Small mosquitoes, large implications: crowding and starvation affects gene expression and nutrient accumulation in Aedes aegypti.</a> Parasites &amp; Vectors. 8, 252 (2015).</li><li>Valerio, L., Matilda Collins, C., Lees, R. S., Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+vector+arthropod+culture:+an+example+using+the+African+malaria+mosquito,+Anopheles+gambiae+(Diptera:+Culicidae).">Benchmarking vector arthropod culture: an example using the African malaria mosquito, Anopheles gambiae (Diptera: Culicidae).</a> Malaria Journal. 15 (1), 262 (2016).</li><li>Rueden, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ2:+ImageJ+for+the+next+generation+of+scientific+image+data.">ImageJ2: ImageJ for the next generation of scientific image data.</a> BMC Bioinformatics. 18 (1), 529 (2017).</li></ol>

The EAgaL plate drastically reduces labor, time, and space to conduct individual fecundity and fertility assays in Aedes aegypti when compared to the FT method. Preliminary comparison between the FT method and the EAgaL plate resulted in shorter times for all steps (imaging technique was applied to the FT method) (Table 1). As a reference, an estimate of startup and&#160;per assay (one 24 well EAgaL plate versus 24 FTs) costs are provided in Table 2.
<p class="jove_...

The control of mosquitoes to protect humans from vector-borne pathogens is an important public health goal, mainly due to the lack of effective vaccines for most of the pathogens carried by mosquitoes. Many studies aim to reduce mosquito fitness in conjunction with a field-applicable population reduction strategy1,2,3. This includes extensive studies to create transgenic mosquitoes and/or CRISPR/Cas9 knockout lines. Such population modification approaches require a detailed assessment of individual fitness parameters4. Conventional laboratory techniques to assess the fitness of female mosquitoes includes the individual containment of mated, blood-fed female mosquitoes in 100 mL containers5, modified 50 mL conical tubes, or tubes for Drosophila rearing modified by providing moist surfaces using damp cotton and filter paper discs for oviposition (i.e., egg papers)1,2,6,7. Such methods require a relatively large space (e.g., 30 cm x 30 cm x 10 cm: W x L x H for up to 100 Drosophila tubes) (Figure 1), and the manipulation of individual egg papers for counting eggs and hatching larvae, which can be labor intensive. This manuscript presents a method to count mosquito eggs and determine hatch rates using 24 well plates and agarose as an oviposition surface to circumvent these issues8.
Concurrently, Ioshino et al.9 described a detailed method using 12 and 24 well plates to perform egg counting obtained from individual females. Their protocol represented a significant improvement from conventional methods in saving time and space9. However, the protocol they described continues to use wet filter paper as a surface for oviposition, which requires unfolding each individual paper to get counts, as eggs are often found underneath or in folds. Their protocol also did not include the use of imaging technologies or a method for larval counting.
Presented is an improved method to perform fitness assays for egg number (i.e., fecundity) and hatch rate (i.e., fertility) using agarose as an oviposition surface in a 24 well tissue culture plate format for Ae. aegypti that oviposit on moist surfaces. These plates were named &#8220;EAgaL&#8221; plates, from Egg, Agarose, and Larva. These 24 well plates provide individual mosquitoes with a minimal surface to lay eggs, thus simplifying and drastically reducing the time and effort needed to count and maintain eggs and hatched larvae for a few days. The EAgaL plate uses translucent agarose for the oviposition surface, which eliminates the need for handling egg papers and finding the eggs and larvae when hatched; photographing each well establishes a long-term archived record of the results and separates the counting process in both time and space from the rearing/handling process, where time is often limited.

<table>
	<tbody>
		<tr>
			<td>1.6 mm &#934; drill bit</td>
			<td></td>
			<td></td>
			<td>alternatively heated nails can be used</td>
		</tr>
		<tr>
			<td>1000 &#956;L pipette tips (long)</td>
			<td>Olympus plastics</td>
			<td>24-165RL</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Thermo Scientific</td>
			<td>930186</td>
			<td>clear, flat-bottom with ringed lid plates</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>VWR</td>
			<td>0710-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact digital camera</td>
			<td>Olympus</td>
			<td>TG-5/TG-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer (Windows, Mac or Linux)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji (imageJ) software</td>
			<td></td>
			<td></td>
			<td>download from: https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>sharp forceps may break mosquito&#39;s body</td>
		</tr>
		<tr>
			<td>Glass Petri dishes</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Household bleach</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Household electric drill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>illuminator for stereomicroscope (gooseneck)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P-1000 pipette</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>paint brushes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bands</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SD card</td>
			<td></td>
			<td></td>
			<td>to record digital camera images (DSHC, SDXC should be better)</td>
		</tr>
		<tr>
			<td>Spreadsheet software (Microsoft Excel)</td>
			<td>Microsoft</td>
			<td></td>
			<td>Any spreadsheet software works</td>
		</tr>
		<tr>
			<td>TetraMin fish food</td>
			<td>Tetra</td>
			<td></td>
			<td>ground with coffee grinder, blender or morter &#38; pestle</td>
		</tr>
		<tr>
			<td>Transfer pipetts</td>
			<td>VWR</td>
			<td>16011-188</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Mosquitoes were injected with dsRNA targeting a candidate iron transporter (FeT) or control gene (EGFP), blood-fed, and measured for fecundity and fertility output using the EAgaL plate method, following the procedure described above.
Mosquitoes in which FeT expression was silenced following dsRNA injection exhibited a significant reduction in both egg number and hatch rate (Figure 11A&#8722;C). All control and treatment mosquitoes were placed in ...

Watch this Scientific Journal Video about Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates at JoVE.com

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.

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 ...

This procedure allows mosquito researchers to easily address their mosquitoes'reproductive fitness at the individual level. Compared to conventional methods, this protocol significantly reduces the time, space and labor required to test mosquitoes'fecundity and fertility at the individual level. Begin by preparing plates for the oviposition experiment.Drill four to six holes per well in the lid of a 24-well culture plate using a household drill with a 1.6 millimeter bit. One day before the experiment, wash and rinse the plates and soak them in 1%to 5%bleach for 30 to 60 minutes at room temperature. Then, rinse the plates thoroughly under running deionized water and dry them.Melt 2%agarose in deionized water and immediately add 500 microliters of the molten agarose to each well of a 24-well plate. Leave the plates on the lab bench overnight to dry out any condensation. At least 16 hours before the blood feeding, remove any source of water or sugar from the female mosquitoes.On the day of the feeding, heat a water circulator to 37 degrees Celsius and feed the mosquitoes with vertebrate blood placed in artificial feeders for 15 to 30 minutes. Then, transfer the mosquitoes to a glass dish on ice. Select the ones that are engorged with blood and place them into a container with 30%sucrose water.Allow at least 72 hours for the females to finish excretion and egg development. About one hour before transferring mosquitoes to the 24-well plate, use a transfer pipette to add two to three drops of water into each well. Knock down mosquitoes with carbon dioxide and transfer them to a glass dish on ice.Then, individually place each mosquito on an inverted lid of the 24-well plate. Once all 24 mosquitoes have been placed, cover the lid with an inverted plate bottom, secure it with a fresh new rubber band and place it in an environmental chamber until mosquitoes recover. Then, turn the plate right side up.Allow the female mosquitoes to oviposit for 24 to 48 hours. Then, remove the females by releasing them from the plates into a large cage. Before counting the eggs, check each well for eggs that are on the well walls and at the margin of the agarose and plastic surface where they are difficult to resolve in photographs.Use a wet paintbrush to move these eggs to the flat surface so that all eggs are in a uniform plane and do not overlap each other. Use forceps to remove any broken legs, wings, and other particles in the wells that may interfere with imaging eggs. Set a compact digital camera in microscope mode, which allows the user to focus on objects as close as one centimeter and take an image of each well.After photographing each well of a plate, take an image of the entire plate. Use an imaging order label to distinguish each plate later. Add about five drops of water to each well to prevent its agarose plug and the eggs from drying and to induce embryo development and hatching.Transfer the images to a computer and rename the files with the plate and well IDs for easier organization. Open the images with ImageJ and use the Multi-point Tool to mark each egg, zooming in or out to count the egg clumps. After marking all the eggs, double click the Multi-point icon to bring up the number of marks and record the results in a spreadsheet.Start adding food to the wells that contain hatched larvae as soon as they appear. Approximately five to eight days later, anesthetize the larvae by covering the plate with crushed ice for 15 to 20 minutes. Insert a black material underneath the plate to enhance the contrast and take images of each well while keeping the plates on ice.After photographing each well, take an image of the entire plate with an imaging order label. Open images with ImageJ and use the Multi-point Tool to count the larvae. Larvae can vary in shape, angle and focus.Exclude the shed cuticles, which look like head-only larvae with a little bit of body, or shrunken larvae. Record the results in the spreadsheet. Mosquitoes were injected with double-stranded RNA targeting a candidate iron transporter, FeT, or a control gene, EGFP.Then, they were blood-fed and measured for fecundity and fertility output using the Eagle plate method. Mosquitoes in which iron transporter expression was silenced exhibited a significant reduction in both egg number and hatch rate. FeT-silenced mosquitoes also exhibited delayed excretion and small and light-colored eggs.Taking good pictures is a critical step for this method because the quality of the pictures and quantity of individual eggs or larvae directly affect the quality of the results.

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

Abstract