# Rearing milkweed bugs

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

Updated 26 October 2022


> Photo by Matthew Beziat, [Flickr](https://www.flickr.com/photos/109690096@N08/34382893874) ([CC BY-NC 2.0](https://creativecommons.org/licenses/by-nc/2.0/))

## General information

*Oncopeltus fasciatus* are seed bugs (order Heteroptera; family Lygaeidae). Species of *Oncopeltus* are common in the Caribbean, Central and northern South America. *Oncopeltus fasciatus* lives in the southern and temperate US, and individuals may colonize colder northern states in the summer. Some over-winter in diapause as adults. In the wild, they live and feed on milkweed (*[Asclepias](https://en.wikipedia.org/wiki/Asclepias)* sp.), and the bugs gain distasteful, toxic compounds called [cardenolides](https://en.wikipedia.org/wiki/Cardenolide) from the plant. Their bright red/orange and black colors are [aposematic](https://en.wikipedia.org/wiki/Aposematism), advertising their toxicity to would-be predators. To enhance this effect, the bugs congregate in semi-social groups.

Heteroptera, like *Oncopeltus*, are hemimetabolous, meaning that there is no dramatic metamorphosis, as in other insects like beetles, moths, bees and flies. Instead, juveniles (also called nymphs) resemble adults in their overall body plan, but lack wings or genitalia. There are five juvenile instars (stages separated by molts of the cuticle) before adulthood. Each instar can be distinguished by its relative size and pattern of pigmentation (see below).


*Oncopeltus* are a pale orange after molting ("teneral" or "callow"). Within hours, the cuticle hardens and the underlying cells produce new pigment. Milkweed bugs spend about 3-10 days in each juvenile instar, reaching adulthood about 5-6 weeks after hatching. The adult wings cover the abdomen and have a pattern of black and orange/red pigmentation. Adults will often be seen attached end to end. This is a "mate guarding" behavior by which males prevent females from seeking new mates after copulation.

All true bugs have a long beak (also referred to as a rostrum, proboscis or mouthparts), which is used to pierce seeds, inject saliva, and withdraw liquefied food. Bugs have a large number of symbiotic gut microbes that help them extract nutrients from the seed extract. In the wild they may also feed on vascular fluids of the host plant, especially at new growth.

Milkweed bugs grow very well in the lab. To maintain a large culture they typically require attention every 7-14 days. Experiments should be tended every 1-3 days. The lab strain of *Oncopeltus* has been bred since the 1970s to feed on shelled sunflower seeds and to lay their eggs in cotton balls.

These procedures are also the basis for the care of *Jadera*, which is described in more detail [elsewhere](https://hackmd.io/@EcoEvoDevoLab/rearingsoapberrybugs).

## Labeling

**Label** all bug containers! Ideally include:

- the species name ("Ofas" or "Jhae" are sufficient in our lab)
- the population of origin (for *Jadera*)
- the experimental manipulation and treatment ID number (if any)
- any hazard information (e.g. methoprene’s NFPA Health level is 1)
- your initials
- the date of set-up or last cleaning
- If you’re keeping your bugs in a shared space, like Olin 314, add "Angelini Lab".
- If all this doesn't fit on many small dishes, you can place them all into a larger container with more complete labeling.

## Bug stocks

A standard mass-rearing cage consists of a large (3.38 L) plastic aquarium containing a water flask, seed dish and cotton balls, covered by a large KimWipe and the aquarium lid. Bugs will be most healthy and productive is they are set-up in a cage as a batch of eggs. Juveniles do not foul their cage rapidly, but adults will quickly cover a cage in poo. So keeping cages with bugs of a similar age helps keep them healthy and makes it easier to clean them.


Each cage should contain:

- The **water flask** is a 50-ml Erlenmeyer flask filled with spring water or water from a potable water fountain (don't use tap water or distilled water!). Use a folded paper towel wick that is roughly 1-cm wide, extending at least 1 cm above the rim of the flask. The rim should be loosely packed with half a cotton ball. New, clean water flasks should be provided weekly.
- The **seed dish** is half a 4-cm Petri dish, containing a single layer of sunflower seeds. The seeds will grow mold, so adding extra seeds will not extend the useful time span of the seed dish. Seeds should be replaced when visibly moldy. Use only organic, unsalted, shelled sunflower seeds (e.g. from [Uncle Dean’s Natural Market](https://www.uncledeans.com/) in Waterville).
- **Cotton balls** are a place for *Oncopeltus* to lay eggs. The balls should be teased apart slightly to allow females easier access. Only adults will need cotton, and juveniles can be kept without it.
- The cage must be covered by two single-ply, extra-large **[KimWipe](https://www.thomassci.com/scientific-supplies/Kimwipes)**, folded in half. There are usually small holes in a KimWipe, so it’s important that two layers cover the cage. The lid of the aquarium just keeps the KimWipe in place!
- Temperature for *Oncopeltus* can vary widely. We generally keep them at room temperature, which is typically 21-24˚C.

**How many** cages to keep? In general, it is best to have at least three cages of each unique genotype. If experiments are removing bugs from these cultures, having more cages on hand (before experiments begin) is a good way to prevent the stock from becoming depleted to levels where experiments may be delayed by lack of bugs.

**Mold** is bad. It competes with the bugs for food. Juveniles may become tangled in hyphae and loose limbs when molting or die. Some molds will infect and kill bugs posing a serious potential threat to the health of the entire lab population. Therefore, cages with any dead moldy bugs should be cleaned with bleach.

## Bug wrangling

Bug wrangling is an art that you will master with practice!

It is easiest to start a new cage with unhatched eggs. Simply set up a cage as described above and move the cotton from an adult cage, which should be full of yellow-to-red eggs, into a new clean cage. Cages with lots of tiny juveniles can be very hard to wrangle. Therefore, it’s best to avoid allowing a large number of eggs to hatch in a filthy cage. Avoid this by letting eggs hatch in a new clean cage.

Cages with adults and many older juveniles may start to get covered in poo. these conditions can promote mold growth, reduce the life span of bugs and reduce their production of eggs. It's also just unpleasant. Therefore, it will be necessary to move adults and large juveniles into one or more new cages.

When moving bugs to a new cage:
1. Set up the new cage with seeds, water and cotton.
2. Place the old and new cages besides one another.
3. Move the old KimWipe, with any bugs on it, to the new cage. Give the surface of the wipe a sharp flick with you index finger to kick off any bugs into the new cage.
4. **Defense**! Watch out for bugs climbing up and out of the cage. Using a fan-shaped paint brush, knock climbers back, or sweep them from the old cage to the new one. - Sometimes people work in a pair: one on defense, one on offense. -

:::warning
**Be gentle!** An occasional squashed bug is expected, but be especially careful with bugs that are part of an experiment. Never use metal forceps on a live bug. Use a brush to sweep the body in brief contact. Don’t drag a bug along a surface. You can flip a bug over and it will flail to grab onto anything you offer it. This can be a safe way to get a single bug.
:::

5. Lift up the old water and seed dish, and sweep bugs from them into the new cage.
6. Hold the old cage side ways. As bugs “run away” towards the bottom edge, sweep them into the new cage.
7. When the new cage contains several dozen bugs, any stragglers can be euthanized by placing the old cage in a -20˚C freezer overnight. (Please don’t put dirty bug cages in any freezer with reagents for molecular biology.) Milkweed bugs are prolific enough that it usually isn’t necessary to save every last one. (Although, for *Jadera* we typically want to save every bug.)

:::warning
CO~2~ can be used to anaesthetize bugs. It may be tempting to knock out an entire cage of bugs in order to move them to a new cage. However, **CO~2~ is not advisible here** for several reasons: It can be hard to distinguish live bugs from dead ones. The CO~2~ can also be retained in thenew cage, causing health problems for the bugs later. It also uses a lot of CO~2~! Overall, it's just better to hone your skills at wrangling and work without CO~2~.
:::

## Keeping bugs in Petri dishes

Small numbers of bugs can be kept in 6- or 10-cm Petri dishes. This set-up can be used to observe the development of 1-8 bugs or for mating pairs.

- **Water**: fold a paper towel and soak it with spring water.
- **Seeds**: Add 5-6 sunflower seeds. In 6-cm dishes, try to prevent the seeds and water from touching, as this will cause germination and/or mold growth. Larger deep dishes (10 x 2.5 cm), can allow the seeds to be placed in a lid or the base of a small (4-cm) dish which will slow mold growth.
- For very hatchlings, place a small KimWipe with two layers under the lid. This will prevent hatchlings from crawling out under the lid and escaping.
- Don’t tape the culture dishes shut. Peeling the tape off is a pain.
- Store multiple dishes in a larger container or aquarium with a snap-on lid. Be sure it is not air-tight. The larger container will prevent mice from disturbing dishes to eat the sunflower seeds. (It has happened!)


## Clean-up

**Euthanize** bugs when cultures become too crowded or when you have more cages than needed for maintenance of the stock population. You should also euthanize bugs used in experiments after you have collected all the data and specimens necessary.

Bugs that you don’t need to keep should be killed by freezing them overnight at -20˚C. (Remember, in the wild they would either die slowly from disease, be crushed in the jaws of a predator, or freeze.)

If you need to keep an entire bug as a voucher or specimen from an experiment, preserve it depending on the needs of the experiment:
- 70% ethanol for preservation of anatomical structure (store at room temp.)
- 100% ethanol for preservation of DNA (store at 4˚C)

:::warning
Never release lab insects into the wild!
:::

**To clean used cages**, filled them with water and a shot of bleach (roughly 30 ml) and allowed to soak for at least 1 hour. Then wash them with lab soap. Be sure to remove any visible bug feces from the cages. Cage lids can simply be rinsed in tap water.

Allow water flasks and petri dishes to soak in a similar dilute bleach solution overnight. Then wash them with lab soap, rinse and allow them to air dry before reusing.

Wash brushes or forceps used in bug care with lab soap and allow them to air dry.

:::warning
To protect your cloths, wear a lab coat or lab apron when working with bleach.
:::

---

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