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

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.