Mendelian Genetics and Raising Mosquitoes

Raising mosquitoes is one of the cornerstones of the research I am doing. Mosquitoes undergo several stages of development. The first is the egg stage. When the eggs hatch, they enter into the larvae stage. The larvae go through four stages, characterized by growth and molting. During the first stage the larvae are called first instar larvae. The first instar larvae are placed into rectangular, plastic containers and are given two pellets of cat food. The larvae continue to grow, and eventually molt again and enter into the second instar larval stage. After some time, the larvae molt agan and become instar three larvae. After a final molt, the larvae become instar four larvae. After a few days the instar four larvae grow into pupae. The pupae are extracted from the rectangular container and are placed into smaller, rounder containers. There are many places the puape might find themselves in the lab depending on if they carry the gene of interest or not. The larvae are offspring of regular female mosquitoes and male mosquitoes that are heterozygous for the gene of interest. This cross gives us a 50% chance of getting a heterozygous mosquito and a 50% chance of getting a homozygous recessive mosquito(as demonstrated by the punnett square).

The larvae are then scanned to see which ones have the gene of interest and which ones do not. If the gene is present, the larvaes' eyes will glow orange. Using a pipet, the larvae are removed from the round container and put on the cold plate of a microscope. The larvae are then separated into two groups: those that have glowing eyes and those that do not. The ones with glowing eyes are known as "Positives", while the ones withut glowing eyes are known as "Negatives". We can then examine all sorts of characteristics of the mosquitos. We can see how long they live compared to normal mosquitoes(remember that malaria can take weeks to manifest itself in the mosquito). The other major assay is the size and functionality of the organs of the transgenic mosquitos. These tests can give us insight into how the gene is affecting the mosquito. Of course the only way to tell the difference in size and functionality of the organs is to dissect the mosquitos.

The process of hatching, maturing, bloodfeeding, and hatching new eggs is repeated several times. Because a larger sample size reduces error, we want to maximize the number of mosquitos that can be used in organ assays, lifespan assays, etc. Sometimes we have to breed ten or more generations of mosquitos before we can begin the assays.

Even after rearing mosquitos for ten generations, performing dissections, extracting RNA and making complimentary DNA, running gels, recording data, running lifespan and organ assays, there is always the chance that the gene of interest didn't affect the mosquito in any way we care about. Research can take a very long time and not yield the desired results. If the gene doesn't significantly affect the mosquito, new mosquitos that have a different alteration are raised and experimented upon. With that said, it looks like I will have my hands full for the remainder of the project. Science. The most facsinating and frustrating concept ever.