Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite that specifically infects Aedes aegypti mosquitoes. Rearing this parasite can be challenging for new researchers due to the complexity of the parasite's lifecycle. This protocol provides step-wise instructions for rearing E.aedis, including how to properly time the steps to the procedure, identify infectious spores, and propagate the next generation of parasites.

Accurate timing of this protocol can be challenging and may require some trial and error. Identifying spores can also be difficult. The visual aids in this protocol should provide assistance.

To begin, hatch A.Aegypti eggs infected with E.aedis by placing them in a larval rearing tray with one liter of deionized water and adding 50 milligrams of fish food. House the mosquitoes at 27 degrees Celsius and 80%relative humidity with a 14-hour light, 10-hour dark cycle. After hatching, reduce the density of larvae to approximately 100 larvae per tray, making new trays as necessary.

Add a piece of dry cat food to each tray. Replenish the food when depleted but do not provide any excess food. One 200 milligram piece of cat food every three days is sufficient.

When the infected larvae are third to fourth instars, hatch uninfected A.aegypti eggs into a new tray. Rear the mosquitoes at densities such that healthy A.aegypti reach second to third instar in 48 to 72 hours. Hatching batches of healthy eggs over multiple days can guarantee that larvae are at the correct stage when needed.

To harvest and quantify uninucleate spores, use a transfer pipette to move 10 infected fourth instar larvae to a 1.5 milliliter microcentrifuge tube. Remove the breeding water with a transfer pipette and wash the larvae once by adding approximately one milliliter of clean deionized water. Remove the wash water and add 500 microliters of clean deionized water to the 10 larvae and homogenize them using a pestle and a mechanical homogenizer.

Load 10 microliters of homogenate onto a hemocytometer and quantify the spores at 400X magnification. Make a fresh larval food slurry by mixing 1.2 grams of liver powder, 0.8 grams of Brewer's yeast, and 100 milliliters of water. Transfer 100 second to third instar healthy A.aegypti larvae into 150 milliliter beakers or specimen cups and add five times 10 to the fourth to one times 10 to the fifth spores to each cup with a pipette.

Add two milliliters of larval food slurry and deionized water for a final volume of 100 milliliters. After 12 to 24 hours of exposure, transfer the larvae into rearing trays. Add food and rear them to adulthood.

Monitor the dosed larvae for pupation and transfer pupae to an emergence cup in a cage. Sugar feed adults ad libitum. Blood feed adults according to standard protocols available through BEI Resources.

Collect eggs from blood-fed adults. Vertical transmission of E.aedis occurs at this step. When finished, clean all materials that came in contact with E.aedis with 10%bleach and autoclaving to prevent contamination.

This protocol demonstrates the full procedure necessary to propagate E.aedis in A.aegypti mosquitoes. A.aegypti eggs infected with E.aedis were hatched and larvae were reared to the fourth instar stage. In the fourth instar stage, visual signs of infection could be observed such as white sporocysts throughout the fat bodies of infected larvae.

Uninucleate spores were harvested from fourth instar larvae by homogenizing 10 larvae in 500 microliters of deionized water. These spores were piriform and readily visible at 400X magnification. A spore count of 4, 050 spores per microliter was calculated and these spores were used to infect healthy larvae via horizontal transmission.

Infection status and E.aedis loads of 25 filial generation larvae were assessed at seven days post-hatching. Vertical infection rate of E.aedis in the filial generation was found to be 96%and the mean spore load of infected individuals at seven days post-hatching was 3.31 times 10 to the fifth. To properly coordinate larval staging, it is advisable to account for some variability in rearing time.

Additionally, spore numbers increase rapidly so if you don't detect spores, just try again the next day. Good luck.