# Rearing milkweed bugs

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

## *Oncopeltus* general information
*Oncopeltus fasciatus* are seed bugs (order Heteroptera; family Lygaeidae). Species of the genus 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 may over-wintered in diapause as adults. In the wild, they live and feed on milkweed (*Asclepias* sp.), and the bugs gain distasteful, toxic compounds called cardenolides from the plant. Their bright red/orange and black colors are aposematic, 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. 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 in [Rearing soapberry bugs](https://hackmd.io/@zli23/Jadera).

## Oncopeltus care
:::danger
Label all cultures!
:::

Ideally include:
- the species name (“Of” or “Jh” 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 the Arey 4th floor, it may help to add “Angelini Lab”.
If all this doesn’t fit on many small dishes, you can label the larger container they are in.

A standard mass-rearing cage consists of a large plastic aquarium containing a water flask, seed dish and cotton balls, covered by a large KimWipe and the aquarium lid. Cage cleaning should happen when the bugs cover the walls with bug poo. Dense cultures should be moved to a clean aquarium and receive new seeds, water and cotton.

==The water flask== is a 50-ml Erlenmeyer flask filled with spring water (not tap or distilled!) and a folded paper towel wick that is roughly 1 cm wide, at least 0.25 cm thick, and 5 cm long (from the bottom of the flask to about 2 cm above the rim). The rim should be loosely packed with half a cotton ball. New, clean water flasks should be provided weekly.
Use the Poland Spring water outside the Biology Department office.

==The seed dish== is half a Petri dish of 3-10 cm diameter 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 organic, unsalted, shelled sunflower seeds (Uncle Dean’s Good Groceries).

==Cotton balls== are a place for Oncopeltus to lay eggs. The balls should be teased apart slightly to allow females easier access. A good size for the cotton ball is about the size of a chicken egg. Only adults will need cotton, and juveniles can be kept without it.

==The cage== must be covered by two single-ply, extra-large KimWipe, 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, 20-24˚C.
How many cages to keep? In general, it is best to have at least two 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 is an art that you will master with practice! When moving bugs to a new cage:

* Set up the new cage with seeds, water and cotton.
* Place the old and new cages besides one another.
* To start a “new” cage, just move the cotton and its eggs from the old cage to the new one. However, if you need to transfer juveniles or adults, continue…
* Move the old KimWipes 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.
* Sometimes people work in a pair: one on defense, one on offense.
* Using a brush or paper towel piece, sweep the body in brief contact.
* 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.
* Lift up the old water and seed dish, and sweep bugs from them into the new cage.
* Hold the old cage side ways. As bugs “run away” towards the bottom edge, sweep them into the new cage.
* 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. (But for Jadera this is more critical. See below.)

:::danger
* Be gentle! An occasional squashed bug is not terrible, but be especially careful with bugs that are part of an experiment.
* Never use metal forceps on a live bug.
* Defense! Watch out for bugs climbing up and out of the cage. Using a tortilla chip-sized piece of paper towel or a fan-brush, knock climbers back, or sweep them from the old cage to the new one.
* Cages with lots of tiny juveniles can be very hard to manage. Therefore, it’s best to avoid allowing a large number of eggs to hatch in a filthy cage.
* Don’t drag a bug along a surface.
:::

## individual cultures

Individual cultures can be set up 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.

1. Water: start with a paper towel folded up like the wick for a water flask. Cut off a roughly 1 cm2 piece and add it to the Petri dish. Soak this with spring water.

2. 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.

3. Egg carton: very young juveniles may benefit from a small piece (2 cm2) of paper egg carton folded diagonally. This gives them a place to shelter, which may reduce stress, and provides a good place to molt. (This is crucial for Jadera.)

:::danger
Don’t tape the culture dishes shut. Peeling the tape off is a pain.
:::

4. Store multiple dishes in a Tupperware container or aquarium with a snap-on lid. Be sure it is not airtight. This will prevent mice from disturbing dishes to eat the sunflower seeds. (It has happened!)

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

## After care
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)

Never release lab insects into the wild!