# Rearing painted lady butterflies

[EcoEvoDevoLab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)

Updated 24 March 2022


> Photos by [Dave Angelini](https://www.flickr.com/photos/142092478@N03/49113078266/in/album-72157670540258426/)

## General information

The painted lady butterfly, *Vanessa cardui*, is a model to study wing patterning and other topics. Caterpillars are commercially available throughout the year ([NatureGifts Caterpillar Refill Kit](https://www.nature-gifts.com/shop/grow-butterflies/caterpillar-refill-kit/)), and the entire life cycle can be completed in captivity. Newly laid eggs hatch and develop to adulthood in 5 weeks. This species is also native to New England, so (while we will not plan to release any animals) there is limited ecological risk from accidental release. Importantly, a [transcriptome is available](http://www.butterflygenome.org/?q=node/4) for this species and a [web portal has been constructed for BLAST search](http://www.butterflygenome.org/?q=node/5) of those sequences. The development of butterfly wings has also been studied in some detail.

## Larval food

This recipe is based on instructions from Frontier Agricultural Sciences and [Martin et al. (2020)](https://pubmed.ncbi.nlm.nih.gov/32034043/). [Item #F9698B from Frontier Agricultural](https://www.insectrearing.com/product/liter-11/) includes a dry mix (including soy flour, wheat germ, brewer's yeast, sugars, salts and perservatives), KOH solution, and dry agar. (Enough to make 10 L.) The instructions below are for preparation of 1 L. Ingredients can be mixed using a handheld kitchen mixer.

- In a 2-L beaker, mix 19 g dry agar with 710 ml water
- On a hot plate with a large stirring bar, bring the solution to a roiling boil
- Remove the mixture from the heat before it boilsnover (use a rubber mit)
- Remove the stir bar using tongs
- Add 246.1 g of dry diet and mix with hand-held mixer
- Add 6.84 ml of KOH solution (18% w/w, provided with the Frontier Agricultural diet) and mix for 10 s
- Add 300 µl of 25% (v/v) glacial acetic acid (or 75 µl of 100%) and mix for 10 s
- Optionally, add up to 30 g of fresh leaves from [mallow](https://en.wikipedia.org/wiki/Malva), [hollyhocks](https://en.wikipedia.org/wiki/Alcea) or [sunflower](https://en.wikipedia.org/wiki/Helianthus)
- Blend for 20 s or until thoroughly mixed
- Add 4 ml formaldehyde (37%)
- Add 8 ml canola oil
- Blend for 30 s or until thoroughly mixed
- Aliquot into 1.25 oz Plastic Souffle Cups (Frontier Agriculture Sciences, [item 9091](https://www.insectrearing.com/product/1-25-oz-plastic-cup/))
- Store in a sealed container at 4˚C

:::warning
[Martin et al. (2020)](https://pubmed.ncbi.nlm.nih.gov/32034043/) suggest stored larval food is good for no more than 2 weeks. However, in my experience larvae appear to grow well on it indefinitely, as much as 4 months after preparation.
:::


> Hollyhocks potted up for the winter.

## Rearing caterpillars

### Housing and equipment for raising caterpillars

- an incubator
- 1.25 oz Plastic Souffle Cups (Frontier Agriculture Sciences, [item 9091](https://insectrearing.com/product/1-25-oz-plastic-cup/))
- tray for cups (Frontier Agriculture Sciences, [item 9040](https://insectrearing.com/product/cup-tray-30-wells/))
- Squares of KimWipe or paper towel, roughly 4-cm on each side

### Care of hatchlings

- Embryos will develop at a temperature-dependent rate, hatching after about 3 days at 28°C to about 4 days at 22°C.
- In the morning of the hatching day, press small crumbs of larval food onto the side and lid of the cup. Hatchlings should be found feeding on the diet within 24 hrs.
- Leave the larvae at room temperature (22-25°C).

:::warning
Temperatures of 24-25°C (75°F) provide an optimal trade-off between developmental time and survival, with a developmental time of 30-36 days.
:::

- Examine the larvae every two days
- Add small amounts of fresh food as needed
- If there is excess moisture daily, add pinholes to the lid to release excess moisture
- Dispose of any dead larvae as biohazardous waste
- Remove individuals to seperate cups once they reach the third instar

:::warning
Handle caterpillars with a soft paint brush. They are harmless to humans, but they can be injured easily by rough handling.
:::

### Care of caterpillars

- Pack larval food into the bottom of the plastic souffle cups (8-10 ml of food per larva)
- Cover the cup with a small square of paper towel or KimWipe and snap on the lid

:::warning
The paper towel provides a place for caterpillars to attach themselves when they pupate. It also helps reduce condensation inside the cups. Sometimes the caterpillars will chew holes in the paper, in which case it can be replaced.
:::


> An older caterpillar in individual culture.

- Keep cups of larvae in an incubator at 23-25°C with 40-60% relative humidity and a 12/12 light/dark cycle. Cover them from direct light to minimize condensation.
- Larvae can be kept together when they are small, but as they grow move them to individual cups of food

:::warning
As they grow, *Vanessa* caterpillars will spin silk. This can inhibit their movement and allow more condensation. Periodically, removing the silk from the cups may help growth.
:::

- Caterpillars will eventually molt to become pupae, typically hanging from the paper towel.


> A pupa attached to paper towel

- Hang pupae in a dedicated enclosure. Cut the paper towel into a rectangle about 2 cm by 4 cm, with the pupa attached at the center. Using a glue gun, glue the paper together onto a [T-pin](https://www.google.com/search?q=T-pin) so that the pupa hangs down.


- Pin the pupa to the top of a sytrofoam cooler, placed on its side, which will serve as an enclosure.
- If a pupa falls off the paper, it can be glued directly to a pin. First, place a drop of glue on a pin. Wait a few second for it to cool, then touch it to be the posterior of the pupa. Move it away about 1 mm, then wait in place until the glue cools enough to become rigid. Then pin the pupa up in the styrofoam container.


- Check the enclosure after a few days to move any eclosed butterflies to an adult enclosure.


- Keep humidity high during pupation. Line the bottom of the styrofoam cooler with paper towels and wet them daily.

:::warning
When they eclose, the butterflies will eliminate meconium, the feces they have been withholding during pupation. There's a lot of it, and they squirt it pretty far.
:::

## Care of adult butterflies

- House adult butterflies in a collapsible mesh enclosure (e.g. Carolina Biological Supplies [item #674291](https://www.carolina.com/butterfly-habitats/carolina-butterfly-sanctuary/674291.pr))
- Adults will consume a 1:1 solution of Gatorade and spring water
- Construct a feeder by cutting a hole in a small plastic container and create a wick out of a cotton ball
- Butterflies will also drink this solution from a folded paper towel placed in a the top of bottom half of a 4-cm diameter Petri dish
- Periodically remove any dead butterflies


> Enclosure for adult butterflies.

### Egg laying

Getting butterflies to lay eggs in captivity can be made easier with some planning. First, young adults are more fecund. So to maintain a stock, allow adults to lay eggs as soon as they appear and start the life cycle again. Second, butterflies can much more willing to lay eggs to live plants. During cold months, it is strategic to keep some of their prefered host plants ([mallow](https://en.wikipedia.org/wiki/Malva), [hollyhocks](https://en.wikipedia.org/wiki/Alcea) or [sunflower](https://en.wikipedia.org/wiki/Helianthus)) alive in the lab. Alternatively, florists can usually supply sunflowers year round.

- To induce egg laying, provide adults with fresh leaves of mallow, hollyhock, or sunflower in a beaker of water
- Place an incandescent light bulb outside the cage, but near the plant to gently warm it
- Females should immediately begin laying eggs


> Egg-laying

:::danger
**Update:** To encourage egg-laying, the Patel lab reccommends (1) keep the number of adults in a mesh cage low. It may help to have multiple small cages. (2) Place 5 plant leaves on a Petri dish on the bottom of the cage. A mild heat lamp may help, but not as much as simply placing the cage in a warm incubator. Aphid researchers extend the useful lifespan of leaves by cutting them off with a sharp blade and embedding the cut ends in a small block of agarose gel.
:::

## Clean up

- Dispose of any plastic cups in which larvae die
- Optionally, cups from successfully eclosed pupae can be reused. Scrape out any remaining food. Wash with warm water and lab soap. Allow them to dry completely before re-use.
- After a cohort of adults has died, spray the cage down with a dilute solution of bleach (10%). Wash the cage to remove any visible butterfly excrement.

:::info
The "boot room" in the basement of Olin has two large sinks, a stainless steel one in thr corner and a masonry sink on the floor. They are ideal for cleaning butterfly cages.
:::


## References

- **Martin A, Wolcott NS, O'Connell LA.** (2020.) Bringing immersive science to undergraduate laboratory courses using CRISPR gene knockouts in frogs and butterflies. *J. Evol. Biol.* **223**(Pt Suppl 1), jeb208793. [Link](https://pubmed.ncbi.nlm.nih.gov/32034043/)

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