Rearing the Cabbage White Butterfly (Pieris rapae) in Controlled Conditions: A Case Study with Heavy Metal Tolerance

In this study, we're interested in understanding how organisms deal with anthropogenic environments like cities, roadsides, and crop fields. We use cabbage white butterflies as a system because they're easy to manipulate experimentally. For instance, we're studying genetic variation intolerance to urban pollutants.

We collect samples from different locations and populations to study variation over species or space. Nets, coolers and envelopes are our main tools in the field. Butterfly traits are measured by microscopy and ICPMS is the best method to measure heavy metal concentrations in small samples.

One of the current experimental challenges is studying the variation across species in their developmental adjustments to human environments. The main difficulty lies in rearing diverse species under controlled experimental conditions. While cabbage white butterflies are easy to raise, finding suitable analogous diets for 10 or 20 different species that feed on various plants presents a significant challenge from an experimental perspective.

We are interested in mitigating exposure to urban toxins, the ecological interventions that could decrease the toxicity of heavy metals and other urban pollutants, such as microplastics are being explored. Additionally, we aim to investigate how to design pockets of habitat within city spaces to encourage the health of plants and animals living there, including humans. To begin, collect adult female butterflies with an aerial insect net and place them in the cage to harvest eggs.

Place the host plants in pots or in containers with water to maintain the leaf pressure. In case of direct egg collection and transfer to diet, attach a host plant leaf to the top of a plastic cup of water using a rubber band. Then stretch a piece of paraform around the cup's edge so that females touching the leaf will oviposit onto the paraform.

Spray the cage with water to maintain the relative humidity high in the cage. If potted plants are watered in the cage, place a towel under the pot to prevent water pooling and to protect butterflies from becoming trapped. Prepare 10%honey water for butterfly feeding.

Then to set up the feeder, rinse the yellow or orange sponges thoroughly with tap water and cut them into small squares. Place the sponges in 60 millimeter plastic petri dishes and add honey water to them. Change the feeders daily and clean the sponges in a mild bleach solution.

Followed by thorough rinsing with tap water to prevent mold growth. Print out a recipe containing weighing ingredients for artificial diet preparation where 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 forcer 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 paint brush, 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 glossing 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 the significance deviation from the control starting at 100 PPM. To measure wing traits on dead butterflies, hold the thorax in one hand, and using forceps remove each wing at its base.

Place the wings in a light box and take photographs for measurements. To measure aspects of reproduction, house the adult butterflies in mating cages, allowing one day for male reproductive maturation and one day for mating. To estimate either sacrifice females for egg counts through dissection at set time points, or collect eggs daily on host plants.

To estimate egg loads, remove the abdomen, place it in PBS, and make a cut along the ventral side. Using forceps, separate the from the cuticle and pull the ovaries from the gut, trachea and other abdomen contents. Carefully uncurl the four ovarioles within each ovary, and observe the transition from mature, yolked and shelled eggs to immature follicles.

Then use a counter to tally the total number of mature eggs. To assess the mating status of a dissected female, open the bursa copulatrix and separate the spermatophores.