The overall goal of the following experiment is to test for differences in sperm competitive ability among drosophila, melanogaster males with distinct genotypes. This is achieved by tracking visible genetic markers that identify the paternity of the progeny from a female mated to two different males, genotypes in the assay, females are first allowed to mate with a reference male and then allowed to mate with an experimental male. The fraction of the offspring sied by the experimental male is inferred by examining the eye coloration of the progeny.
The results indirectly compare the competitive ability of the experimental male sperm by their relative performance to the reference male sperm. These methods can help answer key questions in the genetics and evolution in biology fields, such as assessing the impact of a genetic factor on the SPR competitive ability demonstrating this procedure will be S and undergraduates from our laboratory. The following protocol illustrates the analysis of a genetic factor called sic, which presumably encodes a protein involved in sperm motility.
Begin by setting up 15 to 20 vials to each vial. Lightly sprinkle the media with a few dried active yeast pellets. To facilitate ove position.
Always use fresh nutritive media such as a cornmeal yeast medium. Then to each vial, add eight to 10 females, but no more, and add five males. Sex identification can be achieved by visual inspection of a few morphological differences.
Store the vials at 25 degrees Celsius and transfer the adults to new vials at least every three to five days. Transfer the adults every other day for the best result. Retain the egg containing vials to harvest the progeny.
The number of vials required will depend on the number of experimental male types in the study and the estimated number of individuals needed for statistical power. Four or five days after parents are transferred out of a vial, increase the available surface for pupation in the vial by inserting a multi folded paper into the medium. Examine the vials for darkened pupa when they're visible.
Those flies will emerge within 24 hours, 11 to 12 days after the cross was first set up. Begin harvesting virgin females and naive males from these vials. First thing in the morning, discard flies that emerged overnight.
Then once or twice during the day on four to six hour intervals, collect flies from the vials, anesthetize the flies by introducing carbon dioxide into the vial. Then tap the flies down and sex them under the stereo microscope. Place the desired flies into different vials by sex and phenotype and label the vials appropriately.
Do not load the vials with more than 10 potentially. Virgin females set a 12 to 12 light dark cycle on the flies to regulate their time of occlusion to your advantage. The peak times of occlusion occur one to two hours after subjective dawn, and two hours before the lights are turned off.
There are two versions of this experiment. They're referred to as the offense assay and the defense assay. This section describes how to conduct the offense assay on the morning of the first day, set up the first mating using an aspirator.
An aspirator consists of a piece of tubing attached to a pipette tip with a barricade that keeps aspirated flies in the tip. The bore of the tip is widened. For the flies set up vials containing ten four or five day old virgin females.
With 10 naive reference males set up two to three such vials daily for five consecutive days. For each vial, allow the flies to mate for two hours, then discard the reference males and transfer each female into a new vial using an aspirator label each vial V one and leave the female in the vial for two days. On the third day, set up the second mating to naive males of the other genotype for each female.
Two hours before subjective nightfall, use an aspirator to transfer the female into a new vial with three, five, or six day old experimental males of the same genotype. Label the vials with the male genotype and label the new vial V two. The next morning is day four.
At two hours before the flies subjective, dawn discard the males by using the aspirator. On the sixth day, transfer each female into another new vial labeled V three. On the 10th day, discard the females on the 17th day.
Inspect the V one vials from the progeny. Determine whether the female was virgin or not when mated to the reference mail and whether it mated with the reference mail. On the same day, inspect the progeny from the V two vials by knocking the flies down with carbon.
Sort them by eye color and sex. Then record the progeny number fathered by the first and second males. In this experiment, only female progeny can be unambiguously assigned as cied by the reference or the experimental males.
In other experiments, different genetic markers could be utilized to determine the paternity of the male offspring as well. Discard the inspected progeny, but keep the V two vials for a second. Inspection on day 20, record the paternity of the progeny from the V two vials for a second time and make the first inspection of the emerged progeny from the V three vials.
Then three days later on day 23, inspect the emerged progeny in V three. Again, the recorded progeny counts should be organized appropriately for easy visual inspection and efficient analysis with a statistical package for each female and the counts from V two and V three. To calculate the total number of progeny cied by the reference male and the experimental male, calculate the P two score for each female as the proportion of progeny cied by the experimental male over the progeny from both the experimental and reference males.
Some females must be excluded from analysis. First, those with no progeny or very limited progeny, second females that died during the procedure and third females that were only successfully inseminated by one of the two males. Look for statistical differences in the P two values.
Among the experimental male types use parametric or non-parametric tests based on the skew of the P two value distribution and the dependency between the variance and the mean proportion. Data usually are not normally distributed. The arc sign transformation, also known as the angular transformation, may be applied to original P two values prior to a student's T-test or a NOVA test, or simply use non-parametric tests such as WIL Coxin two offense experiments with drosophila melanogaster.
Experimental males were conducted one with and another without a functional ESIC cluster. In all, 58%to 83%of the females were informative. Males without a functional ESIC cluster showed significantly lower P two scores compared to males with the intact esic cluster.
This indicates that the SIC cluster boosts the competitive ability of sperm. After watching this video, you should have a good understanding of how to perform double mating experiments, so you may detect the differences in sperm competitive ability between mutant and control males.
