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

This protocol measures male mosquito mating competitiveness, which is a prerequisite for male release-based mosquito population suppression programs. This fitness criterion is assessed as part of quality control and strain evaluation. This method reduces the experimental time by 90%and allows for the direct comparison of mating competitiveness between two sterile or Wolbachia-infected lines.

Performing this procedure will be Irene Li and Keng Wai Mak. To begin, sex the male and female mosquitoes at the pupal stage, according to their size differences. For each set of mosquitoes, transfer 100 male pupae into a pre-labeled cage for sucrose feeding or Rhodamine B sucrose feeding.

Place the female pupae in small batches of 40 to 50 per cage. Upon the emergence of the imagos, check the cages for the presence of male mosquitoes. To prepare the 0.2%Rhodamine B sucrose solution, dissolve 200 milligrams of Rhodamine B powder for every 100 milliliters of 10%sucrose solution.

Mix well to ensure that all the powder is dissolved. Prepare 20 sugar feeder bottles with a wick. Add 10 milliliters of 10%sucrose to 10 feeder bottles and 10 milliliters of 0.2%Rhodamine B sucrose solution for the other 10 feeder bottles.

Place five feeder bottles into every prepared male cage and allow the male mosquitoes to feed for three days before the mating experiment. Turn on the mercury burner lamp and the stereo microscope. Allow the light source to the mercury burner lamp to stabilize for 10 minutes.

Then set the fluorescence filters for red fluorescence protein one. Aspirate a small number of mosquitoes at a time into the glass tube of the oral aspirator. Through the glass tube, observe the body of the male mosquitoes under the fluorescent stereo microscope.

Use only male mosquitoes marked with Rhodamine B for the experiment. Transfer set A male mosquitoes into 12 paper cups secured with netting. Place 10 sucrose fed male mosquitoes per cup into six cups, and 10 Rhodamine B sucrose fed male mosquitoes per cup into the other six cups.

Repeat this step for set B male mosquitoes. Set up 12 cages for the mating assay. In each of the six cages, include 10 set A male mosquitoes which are Rhodamine B marked, 10 unmarked set B male mosquitoes, and 10 virgin wild-type female mosquitoes.

In the other six cages, include 10 unmarked set A male mosquitoes, 10 Rhodamine B marked set B male mosquitoes, and 10 virgin wild-type female mosquitoes. Label these cages to clearly distinguish between the two mating combinations. Place the respective cups of males in the mating cages according to the labels.

Remove the netting and gently tap the cup to jostle the males out of the cup. Carefully remove the paper cup and the netting from the cage to ensure no mosquitoes escape from the cage. Allow the male mosquitoes to acclimate in the mating cage for at least an hour.

Using an oral aspirator, transfer virgin wild-type female mosquitoes into 12 paper cups, each cup containing 10 mosquitoes. After the acclimation period for the male mosquitoes, transfer one cup of females into every mating cage and remove the netting. Gently jostle the cup to encourage any remaining female mosquitoes out of the cup, then carefully remove the paper cup and the netting from the cage to ensure no mosquitoes escape from the cage.

Allow mating to take place for three hours, and then terminate the mating experiment by removing all mosquitoes from each cage with a mechanical aspirator. Cold-anesthetize the mosquitoes on ice for at least five minutes. When the mosquitoes are fully anesthetized, gently pick up the female mosquitoes and house them in a separate paper cup secured with netting.

Label the paper cup by transferring the respective label from the mating cage onto the cup. To score female spermathecae, cold-anesthetize the female mosquitoes on ice for at least five minutes before dissection under a stereo microscope. Dissect the female mosquito under the stereo microscope.

Examine the spermathecae under microscope. If the female is not inseminated, all three spermathecae would be empty, and if inseminated, two or three of the spermathecae would be filled with motile sperm. For inseminated individuals, determine whether the spermathecae contain Rhodamine B marked seminal fluid by examining them under the fluorescent stereo microscope equipped with an RFP1 filter.

If the spermathecae is filled with seminal fluid which is not marked with Rhodamine B, no fluorescence is observed. On the other hand, if the spermatheca contains Rhodamine B marked seminal fluid, it would fluoresce orange. The objective of this mating competitiveness assay was to determine whether there could be a loss in male mating fitness after several generations of inbreeding.

Experimental triplicates of the mating competitiveness assay were conducted, and the insemination data are shown here. Wild-type females were inseminated by either the inbred or outcross males, with reciprocal marking. The results show that the proportion of females inseminated by the inbred or outcross males was not affected by the Rhodamine B marking.

A significantly higher percentage of females mated with the outcross males than with the inbred males in all three replicants, indicating that inbreeding could lead to loss in mating fitness. It's important to prepare extra cages of female mosquitoes. Do not use female mosquitoes from a cage that has been contaminated by male mosquitoes, as pre-inseminated females would render all data invalid.

Other than laboratory-based mating competitiveness assay, this procedure can be used in mark release-recapture experiments to study insect mating biology, population dynamics, and its dispersal.