Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

The scope of our research is to develop and share cost-effective and sustainable genetic strategies that aim at reducing the number of malaria transmitting mosquitoes in South-Saharian Africa. In the last few years, we have demonstrated the power of gene drive technologies in suppressing caged mosquito populations. We are currently running a number of experiments to challenge these technologies in more complex experimental setup and to answers the regulatory requirements that are absolutely necessary for the potential implementation of these technologies in the field.

FISH is a molecular cytogenetic technique that allows for the staining of a specific DNA sequence. It typically involves the squashing of the organ of interest, which can result in the loss of information about the spatial arrangement of cells. Whole-mount FISH allows for the preservation of the native cytological structure of the organs.

Whole-mount FISH can be used to study chromosome behavior in mosquito reproductive organs. Reproductive organs are the target of several genetic strategies for vector control. Whole-mount FISH has the potential to shed light on the mechanism that take place in the reproductive organs of well time mosquito, as well as in any genetics strain of interest.

Begin by using a commercial extraction kit to extract the genomic DNA from the pupae or from adult male anopheles mosquitoes. Next, prepare the PCR reaction mixture. Label the 18 SRDNA and satellite A GY53B by performing a PCR reaction.

For the preparation of the hybridization solution, add five microliters of labeled DNA probe to a 1.5 milliliter tube. Then add five microliters of salmon sperm DNA. Precipitate the DNA probe by adding two volumes of 100%ethanol and 0.1 volume of three molar sodium acetate.

Keep at minus 20 degrees Celsius for at least 2.5 hours. Centrifuge the tube at 17, 000 G for 20 minutes at four degrees Celsius. Then remove the ethanol and air, dry the pellet at room temperature in the dark for 20 minutes.

Next, prepare the hybridization buffer in a 1.5 milliliter tube. Vortex the solution for one minute and allow the dextran sulfate to dissolve at 37 degrees Celsius for 30 minutes. To obtain the hybridization solution, dissolve the DNA palette in 20 to 30 microliters of the hybridization buffer.

Vortex the solution for one minute, perform a quick spin, and store the tubes at 37 degrees Celsius in the dark. Begin by excising at least 20 testes from the pupae of anopheles mosquitoes in sterile PBS. Transfer the testes to a clean microscope slide containing a fresh drop of PBS.

Using P1000 boarded filtered tips or the tip of a dissection needle, transfer the testes into an embryo dish containing 3.7%formaldehyde in PBST. Incubate for 10 minutes at room temperature. Next, wash the testes in PBST for five minutes at room temperature.

Transfer the testes to the embryo dish containing RNAA and incubate it for a half hour at 37 degrees Celsius. Remove the RNA solution and add one milliliter of the penetrating solution. Incubate the testes in the penetrating solution at room temperature for 10 minutes.

Then wash the testes and PBST two times for five minutes each. Transfer the testes to a 1.5 milliliter tube containing 20 to 30 microliters of hybridization solution with the labeled DNA probe. Incubate the mixture overnight at 37 degrees Celsius in the dock to allow hybridization.

With the help of a P 1000 white bore filtered tip, transfer the testes into an embryo dish, then wash them in 2XSSC for five minutes. Remove the SSC, then mount the testes on a frosted glass slide using a commercially available mounting medium containing DAPI. Seal the slide with a cover slip sealant and incubate it at room temperature in the dark for two hours.

A good level of hybridization allowed for the visualization of the targeted chromosomes throughout spermatogenesis. The pairing of the labeled sex chromosomes could be seen in the pre-myotic and myotic stages. X or Y bearing spermitids could be followed throughout spermiogenesis marked by different levels of DNA condensation to the final step of arrow shaped mature spermatozoa.