The protocol represents an important step for establishing drosophila as a standard model for chemical safety testing and for developing new approach methodologies commonly called NAMs. The protocol describes an efficient and inexpensive method that utilizes liquid media to assess a chemical toxicants effects on the viability of adult drosophila melanogaster. Begin by stacking four sheets of grade one cellulose chromatography paper and cutting them into two inch wide strips.
Punch out flower shaped filter paper inserts using a 1.5 inch paper punch containing a flower shaped dye. Push the filter paper stack to the bottom of a 28.5 millimeter diameter polypropylene vial using a 22 by 220 millimeters unvarnished wooden dowel. Ensure the stack gives securely located at the bottom of the vial.
Store the prepared vials in plastic or cardboard trays and place the trays in large plastic bags until use. Open the glass vial containing the flies, place it on the mouth of the prepared plastic vial and transfer sorted sex matched flies to the starvation vial by tapping the bottom of the plastic vial against a bench top. Close the plastic vial with a cellulose acetate plug or flug and record any flies lost in this transfer.
Mark the starvation vials containing female flies with a stripe. To avoid accidental mixing, leave the vials with mail flies unmarked. Prepare the humidity chamber for overnight exposure by placing six standard paper towels at the bottom.
Then soak the paper towels with 100 milliliters of water and place the plastic grid over the wet towels to ensure the vials do not come in contact with the saturated paper towels. After placing the trays of starvation vials in a horizontal position within the humidity chamber transfer the humidity chamber to a 25 degrees Celsius incubator for overnight incubation. To prepare the chemical exposure vials, transfer four vials of starved female flies containing 20 flies per vial to four exposure vials of each chemical concentration.
Label these vials as female. Prepare chemical exposure vials containing blue dye by transferring a vial of starved female flies into a blue exposure vial for each chemical concentration. Label this vial as female.
Record the number of flies present in each vial after transfer and note the number that died or escaped. Normally, all 20 flies should survive overnight starvation and transfer. Place the exposure vials horizontally in a freshly prepared humidity chamber before placing the chamber in a 25 degrees Celsius incubator with approximately 60%humidity and a 12 is to 12 hours light is to dark cycle.
Examine the exposure vials 24 and 48 hours after the start of the chemical exposure. Count and record the dead flies in each vial at each time point. To determine if exposed flies consumed the blue exposure media in the blue exposure vials after 24 hours of the chemical exposure, examine the vial walls for the indications of blue feces which appear as small dots on the side of the exposure vial and the flug.
Next, anesthetize the flies using carbon dioxide before examining the abdomens for the presence of blue dye. Examine the flies for abnormal feeding behaviors such as regurgitation, crop distension and breakdown of the intestinal barrier function. Once done, discard all contaminated vials, filter paper, plugs and flies in the appropriate chemical waste containers.
If live flies remain in the vials, freeze the vials prior to discarding them in the proper waste container. Protocol efficacy was determined by exposing adult organ or male and female flies to the range of sodium arsonite concentrations from zero to two millimolar to score the lethality after 48 hours of exposure. In subsequent experiments, zero to five millimolar concentrations were selected that more precisely defined the sodium arsonite dose response curve.
Based on this model, the final lethal dose, or LD 10, LD 25 and LD 50 of male flies fed with sodium arsonite were 0.30 millimolar, 0.50 millimolar and 0.65 millimolar respectively. These values were slightly higher for female flies with an LD 10 of 0.30 millimolar, an LD 25 of 0.65 millimolar and an LD 50 of 0.90 millimolar. In the male and female flies fed with 1%F D and C blue containing sodium arsonite solutions, the ingested food was occasionally regurgitated at doses above 0.2 millimolar for females and 0.5 millimolar for males, suggesting that regurgitation could play the key role in the dross filia response to arsenic poisoning.
Following this exposure protocol that flies may be easily collected for further analysis, including genomic and metabolomic studies. This protocol is the first step in establishing the fruit fly drosophila melanogaster as a common genetic model for conducting large scale studies of precision toxicology.
