This week I spent most mornings and evenings looking for female virgins… don’t worry, I wasn’t looking for you! Instead I was trying to isolate adult female virgin flies from a vial containing both genders. I needed female flies that have not mated because I want to cross them with a desired male genotype and generate desired progeny.
So how can I tell they are virgins? I use our knowledge of the sex lives of flies! Ashburner et al. (1989) must have spent a lot of time watching flies ‘get it on’ to publish their Drosophila-A Laboratory Handbook which outlines environmental and genetic factors that can be altered to separate virgins and some of which will be briefly discussed here. One of the commonly applied techniques to select female virgins is to separate males from females before mating even occurs. Imagine separating kids by gender before they are sexually active, this way ensuring the females aren’t pregnant. Male flies will not mate for the first eight hours after they emerge from their pupal case giving us a perfect opportunity to pick out the virgins.
Let us first consider the general life cycle of flies to give us the background we need. Female flies will lay eggs (day 1) which take around 24 hours to become larvae (day 2). On day 4-5 pupae form and during days 5-9 metamorphosis occurs. Following this the adult fly will emerge from its pupa. Dr. Arne Christensen and Qilong Song captured some great footage which can give you a better understanding of these development stages.
I have vials of flies with a range of eggs, larvae and pupae in them. When selecting for virgins I check my vials for any adult flies that have emerged from their pupae every eight hours before they start mating. This requires the steps described below, with step one starting in the morning.
Female and male flies can be told apart by their phenotypic differences. Females tend to be larger and have regular narrow bands on their abdominal segments. Males are smaller and at the rear of their abdomen is evenly dark coloured. Another difference, which is less clear, is the appearance of sex combs on the front legs of just male flies. Check out step four above to see an example of a female and male fly.
During the day the flies are kept in 25°C incubators for 8 hours until they are sorted again. For ease of organizing this process around work hours the flies are left in 18°C incubators overnight. This slows down their development so that the next morning, 16 hours later, virgin flies can still be collected.
The rate at which adult flies emerge can also be affected by their genotypes. This happened with some of my fly stocks which each have a specific micro RNA they overexpress. Some genotypes went a few days without a single fly leaving its pupa whilst other genotypes had a dozen or so flies per vial every morning and evening. This could be due to the genotype affecting the natural development of the fly or its ability to grow at the two temperatures it is exposed to.
Nonetheless I was able to collect at least ten female virgins of each phenotype I was interested in within a week and a half. These will now be used to set up crosses with selected males with desired genotypes. More on that soon!
Stay too fly, for an MSci!