Section II: WRBU Sand Fly Mounting Protocol
The cuticle must be cleared so that all characters are visible when the specimen is mounted. Sand flies can be cleared using a variety of chemicals and it is important to monitor temperature and timing of exposure. Because wings clear quickly and easily crumple as the length of time in NaOH increases, care should be taken to avoid over-clearing. If abdomen is difficult to clear, use a probe to create a small hole, at an angle, where the thorax and abdomen join. Because the head may clear more slowly than the body or wings, it may be necessary to leave the head in the NaOH for an extra 5 to 10 minutes until it is mostly clear and not dark pink. Record clear times for head and body in lab notebook.
1. Using a fine dropper, remove all ETH from crucible and cover specimen with 10% NaOH solution. Place covered crucible in oven at 45°C for at least 30 minutes. If more time is needed, use additional intervals of 15 minutes at a time, checking to see that all relevant taxonomic characters are visible. (mouth parts, wings and genitalia)
2. Remove NaOH solution and cover specimen with 5% acetic acid. Let sit for 20 minutes at room temperature.
3. Remove 5% acetic acid from dish and cover specimen with 95% ETH and let sit for 10 minutes. Repeat this step two additional times to make sure all acetic acid has been washed from specimen.