Print out a recipe containing weighing ingredients for artificial diet preparation. Weigh all the dry ingredients, except agar, into one container. Place the dry ingredients mixture into a blender and add five milliliters of flaxseed oil to the ingredient mixture.
Mix 15 grams of fine mesh agar for each diet batch with 400 milliliters of distilled water in a one-liter beaker. Heat the mixture in a microwave until the agar is close to boiling, with fine bubbles throughout the mixture. After boiling, add 400 milliliters of faucet-temperature distilled water to bring the temperature down to mix with the dry ingredients.
Next, place 74-ounce diet cups on the counter with the edges touching. Add the agar mixture to the blender and thoroughly mix. After mixing, pour the mixtures into diet cups, ensuring the diet covers the bottom of each cup.
After the diet cools, place lids on the cups. Label the cups with diet type and stack them on trays. House host plant leaves with butterfly eggs in 30-ounce deli cups with a mesh cover inside a 24-degrees Celsius climate chamber.
After one week, inspect the cups for late first or early second instar larvae. Using a paintbrush, transfer the larvae to the artificial diet. Place three larvae into each four-ounce cup.
Place the cups on their sides in a plastic bin, allowing frass to fall to the bottom and away from the diet, reducing mold and disease risk. House the diet cups in controlled temperature conditions with low to moderate light levels. Monitor the cups for mold or disease every one to two days by inspecting through the clear cup lids.
Allow the larvae to pupate and emerge in the diet cups. Carefully remove adult butterflies from the cups with clean hands, gripping all four wings close to their body for a stable hold. To mark the butterflies, hold the dry individuals by the head and thorax.
Then, using a fine-tip sharpie, mark a number on the hind wing. Determine the sex of the butterflies by combining wing markings and genitalia examination. Next, grasp the adult by its head and thorax using one hand while opening a wax glassine envelope with the other hand.
Then, transfer the adult into the envelope and secure the wings through the envelope. Maintain the butterflies at five to six degrees Celsius for up to one week before experimentation. In this case study, the butterfly survival rate was not affected by increasing the concentration of nickel, but there was a significant effect for both copper and zinc.
Butterfly developmental rate was impacted by copper and zinc. As copper concentration increased, there was an increase in development time, with a significant deviation from the control starting at 50 ppm. As zinc concentration increased, there was an increase in development time, with a significant deviation from the control starting at 100 ppm.
