Methods to Test Endocrine Disruption in Drosophila melanogaster

Drosophila melanogaster is a powerful model to evaluate in vivo the potential toxic and endocrine effects of chemicals, such as endocrine disruptors, that represent a serious problem for human health and natural environments. Addressing the effect of EDCs on the hormonal life traits of the fly, such as fecundity, fertility, developmental time, and the lifespan. It's an easy, cheap, and fast way to investigate endocrine disruption in vivo.

The fertility, development, and lifespan in Drosophila can be affected by several factor that have to be carefully controlled to ensure the successful outcome of this protocol. Therefore, maximum attention in rearing and handling flies is required. To assay the taste of the selected EDC, after anesthetization, transfer 15 young flies from the fly bed to four parallel vials containing cornmeal medium mixed with a food coloring.

The control vial contains the solvent alone, and the three other vials are supplemented with different doses of the selected EDCs. Keep the vials in a humidified incubator for one day with a natural 12-hour light. After anesthetizing the flies again, transfer the immobilized flies to a white paper, and place it under a stereo microscope to observe.

Compare the abdominal coloring of each treatment group with respect to the control group. To assay the reproductive performance of the flies, prepare three vials of flies for each EDC as parental vials, with eight females and four males in 10 milliliters of cornmeal medium supplemented with EE2. For the control, prepare six vials of flies, each with eight females and four males in 10 milliliters of cornmeal medium food supplemented with EE2 solvent.

Rear flies in an incubator at 25 degrees Celsius. During their development, avoid overcrowding the larvae. After four days, invert each vial over an etherizer to remove the parents and return the vials to the incubator for five more days.

In the late afternoon of day nine, invert each vial over an etherizer to remove all newly-emerged flies from the vials and put the vials in another incubator at 18 degrees Celsius overnight. On the morning of day 10, on a Drosophila carbon dioxide work station, carefully invert the culture vial to prevent anesthetized flies from falling onto medium and insert a rubber tube with a needle into the vial between the cotton stopper and the vial wall. Open the valve to deliver carbon dioxide.

Within a matter of seconds, transfer the flies to a fly bed under a microscope. Collect virgin females and young males separately into two groups on the fly bed. Randomly subdivide each group of flies in small subgroups, with 10 females or 20 males per vial filled with fresh corresponding cornmeal medium.

Continue incubation and collect 30 virgin females and 30 males for each EDC, and 90 virgin females and 90 males for the control. House these groups of flies at 25 degrees Celsius until they are aged four days post-eclosion. Then transfer them into new vials containing fresh corresponding medium every two days.

Check there are no larvae in the vials of females. Then label vials with a different series of sequential numbers for the following treatment. After anesthetization, transfer one EE2 solvent-treated female into a small vial containing fresh cornmeal tomato medium without EE2 and add one EE2 solvent-treated male for the control across.

Pair an EE2-treated male with EE2 solvent-treated female or EE2-treated female with EE2 solvent-treated male for each treatment. House 20 single crosses at 25 degrees Celsius. Transfer each mating pair to fresh cornmeal tomato medium vials without EDC every day for the subsequent 10 days.

Visually inspect each vial every day for the eggs and report their number on the fertility spreadsheet. In each vial, check closely for newly-emerged flies and record the daily number of adult progenies over the 10-day period. After 10 days from the initial mating, remove the parents from the last series of vials.

In eclosion assay protocols, first, for each treatment group, set up 10 vials of young, healthy flies less than two days old, each with six females and three males in 10 milliliters of cornmeal food without EDC. Rear the flies on food for 24 hours and allow them to mate. Then prepare another 10 parallel vials per treatment group with 10 milliliters each of fresh cornmeal food supplemented with different EDC concentrations or the EE2 solvent alone for the control.

Transfer the mated flies to these new vials. Make a developmental spreadsheet to record the different series. Allow the flies to lay eggs for 16 hours.

Then, remove the parents from the vials. Incubate the vials at 25 degrees Celsius for three to four days, or until wandering larvae are visible. Every day, count the number of new pupae in each vial by writing a number in sequence by each pupa on the outside of the vial to avoid counting the same pupa twice.

Report the number of newly-emerged pupae for three to four days, or until no more pupae form. Starting from day nine, count the number of emerging adults daily, until no more adults emerge. Report it on the developmental spreadsheet and perform the rest of the eclosion assay according to the manuscript.

In the lifespan protocol, first set up 20 vials with eight females and four males, each filled with 10 milliliters of cornmeal food and house them at 25 degrees Celsius. After four days, discard the flies and place the vials back into the incubator. In the late afternoon of day nine, remove all newly-emerged flies from the vials and return the vials to the incubator.

After 16 to 24 hours, divide 250 one-day-old adult flies of both sexes into four groups under light carbon dioxide anesthetization and transfer them to 250-milliliter bottles containing adult medium food, three supplemented with different EDC concentrations and one with the EE2 solvent alone. Maintain the flies at 25 degrees Celsius for two to three days to allow them to mate. Then, sort each cohort of flies by sex into two groups.

Within each treatment condition, for both sexes, randomly subdivide each group into five parallel vials at a density of 20 individuals per vial. Prepare a lifespan spreadsheet in which the number of dead flies is subtracted from the number of surviving flies from the previous transfer, so that the number of survivors at each transfer is automatically obtained. After three days, transfer the flies without anesthesia to new vials containing the corresponding food at the same time and check for death.

Record the age of the flies and the number of dead flies. Repeat the transfer every three days until all of the flies die. In this experiment, lifespan curves were plotted as the cumulative survivorship versus the elapsed days for each EDC concentration and the control.

For the control, after a long initial period in which the survivorship curve remained relatively high, it declined exponentially after about 60 days. Following EDC exposure, the survivorship curve of the treated flies was affected significantly and showed dramatic decline after 36 days. It's advised about that the parallel vials under analysis should be very similar, otherwise it will be difficult to understand which to discard.

Therefore, we suggest taking great care, adopting a good experimental practice, using a large sample size. Following this protocols, it is also possible to evaluate the transgenerational impact of a selected endocrine disruptor as well as to test the potential additive effects of a combination of different endocrine disruptors.