This protocol can be used to understand the impact of toxicants on fitness at different developmental stages and examine the effect of exposure to pollutants across generations. Demonstrating the procedure will be two undergraduate students in my research laboratory that are a part of the communities to build active stem engagement at Colorado State University Pueblo, Hugh Long and Tyler Harvey. To begin working with an environmental contaminant of interest, first determine the 50%lethal dose of the compound Drosophilia.
Use established methods to create a dose response curve. Next, prepare the growth medium with the compound at the desired concentration. For instance, add the compound in the water aliquot when making instant medium.
In parallel, make control medium without the added compound. To set up the culture from which to collect experimental animals, transfer the reproductive adults to the growth medium using standard methods. Standardize the number of flies per vial and avoid overcrowding the adults.
Then, incubate the flies under standard conditions for one to four days. After the incubation, discard the adults, leaving behind fertilized eggs to mature for testing. Then, return the vials in the incubator.
The exposure can be set up in any developmental stage. In addition, experimental subjects can be collected in any developmental stage depending on the experimental goals. Check on the developing animals daily to identify the first sign of the stage of interest, such as wandering third instar larvae.
To test the effects of exposure on the wandering third instar larvae stage, collect the larvae within an hour of lights on, when they begin to emerge from the food substrate. Next, pour out the water and move the larvae to a lab tissue to gently remove any excess water. Once dried off, transfer the larvae to the appropriate tubes for collection.
To test the effects of exposure on adults, collect the newly eclosed adults;carbon dioxide anesthesia is okay. Be sure to collect the flies before they are reproductively mature and begin mating. Divide the flies into sex-specific groups and transfer them to vials loaded with the control or experimental-contaminated medium to match pre-existing exposure.
Later, 24 hours prior to measuring the contaminants in the adult flies, transfer the adults in both control and contaminated medium to a vial loaded with control medium to allow the adults to groom excess medium off their bodies. After 24 hours of grooming, the flies can be tested using the appropriate method. For example, behavioral changes can be tested using a mating choice assay.
Using these methods, studies of transgenerational effects of contaminant exposure are very easy. Simply set up crosses between exposed adults on the control medium and study the effects of Fnot exposure on their progeny. This protocol was used to evaluate the accumulation, elimination and sequestration of lead within the same generation of offspring.
Drosophila readily accumulated lead at different doses, developmental stages, and evolutionary scales. Using a multi-generational exposure, it was found that parental exposure is not transmitted to the first generation of adult offspring. Therefore, using this protocol, it is possible to test adaptive responses at different evolutionary scales, as well as transgenerational effects Fnot of exposure.
Next, flies were reared from egg stage to adulthood on control or leaded medium and tested after 24 hours of depuration, mate preference was tested. Females reared on leaded medium, preferentially mated with lead-exposed males over control-exposed males. On the other hand, males did not show the same bias.
After watching this video, you should have a good understanding of how to rear and collect Drosophila to answer various questions and toxicology. Once mastered, this protocol can be done in one to two months if it is performed properly. While attempting this procedure, it's important to remember to gently handle the flies, so not to induce unnecessary stress.
Following this procedure, other questions like behavioral, genetic and neurological questions can be tested. Don't forget that working with contaminants can be extremely hazardous and precautions, such as wearing personal protective equipment and preparing contaminants in a hood should always be taken while performing this procedure.
