# Beetle Care
[EcoEvoDevoLab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)
*Angelini Lab, Department of Biology, Colby College*
Updated June 5, 2019 - DMO
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This protocol describes how to keep live colonies of two beetles (Coleoptera), the red flour beetle Tribolium castaneum, and the broad horned flour beetle, Gnatocerus cornutus.
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## Tribolium Castaneum
Tribolium castaneum (red flour beetle, order Coleoptera; family Tenebrionidae).

* Stored goods pest (flour, grains, etc)
* Native to South America, but now has a cosmopolitan distribution. Spread through trade.
* Little known about their natural habitat, food source, or behavior.
* Holometabolous
* 6 larval instars under normal conditions (can be up to 9)

> Grow well in the lab.


## Gnatocerus Cornutus
G. cornutus (broad-horned flour beetle, order Coleoptera; family Tenebrionidae).

* Stored goods pest (flour, grains, etc)
* Native to South America, but now has a cosmopolitan distribution. Spread through trade.
* Little known about their natural habitat, food source, or behavior.
* Holometabolous (complete metamorphosis)
* Sexually dimorphic weapons (males have enlarged mandibles and horns. Horns are absent in females)

> Grow well in the lab.


## General Beetle Care

> Note: some sections of the rearing protocol are species specific, so be sure to take note of which species you are working with before you begin your experiments/care!

* **Label** all cultures!
* Ideally include:
* The species name (“TC” or “GC” is sufficient in our lab)
* Experimental line of origin (For *Tribolium*, e.g., “pu11” or “GA1”)
* Experimental manipulation and treatment ID number (if applicable)
* Your initials
* Date of set-up or last cleaning
* If you’re keeping your bugs in a shared space, like Arey 4th floor, it may help to add “Angelini Lab”.

### Rearing Beetles
* **Standard mass-rearing jars**
* ==*T. castaneum*==: ~100 beetles in a large mason jar, ⅓ filled with appropriate medium. Use a kimwipe or paper towel to cover the chamber, not the standard mason jar lid. This will allow adequate airflow. (See image - right).
* ==*G. cornutus*==: ~ 100 beetles in a large critter keeper, ⅓ filled with appropriate medium (below). Use large kimwipe to cover chamber.
* *G. cornutus* delays pupation and cannibalizes at high larval density, which is why we keep them in larger chambers at a lower population density.

* All beetles will be reared on our **Standard beetle food**, unless we are collecting eggs (see below) or the experiment requires a specialized medium.
* Standard beetle food consists of 95% whole wheat flour (organic, no pesticides!), 5% brewer’s yeast + 0.3% fumagillin by weight. Follow the steps below to prepare this food...
* Sift raw whole wheat flour with through no. 25 sieve.
* Discard any flour/bran that cannot be sifted through the sieve.
* Add sifted flour to a large critter keeper
* Weigh sifted flour
* Add 5% ground yeast and mix throughoully
* Place cap on container
* Bake flour + yeast mixtured at 60C for 24 hours
* Unbaked flour can be stored at room temperature in capped containers with a large tissue wipe between the cap and the container.
* Cool flour to room temperature
* You can use the refrigerator or freezer to expedite this process
* This must be done BEFORE adding fumagillin. Fumagillin is temperature sensitive and will denature at 60C.
* Add 0.03% fumagillin
* Wear a mask and gloves whenever working with fumagillin powder.
* Store at room temperature room temperature in capped containers with large tissue wipe between the cap and the container.

* **Rearing conditions**
* **Relative humidity** should be kept at 40-60%. This is important, since there is no water in the jars.
* **Temperature** can vary widely. Populations will survive and slowly reproduce at 25˚C, but 32˚C is ideal for reproduction and fast development. We will maintain populations at 25˚C and increase the temperature during experiments and when increasing stock.

* **Jar cleaning.** This should happen every month or when adult density becomes extremely high. To clean jars, empty the contents of the jar into No. 25 sieve with solid bottom attached. Sieve contents, separating adults, pupae, larvae, and molts from the medium. Dispose of old medium. Remove molts, sort beetles as needed, and return beetles to jar with fresh medium. We can split populations into multiple jars, or euthanize extra beetles, if the population is too dense to return to the same jar.

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> Note: **How many cages to keep?** In general, it is best to have **at least two cages** of each experimental line. If experiments are removing beetles from these cultures, having more jars on hand (before experiments begin) is a good way to prevent stock from becoming depleted to levels where experiments may be delayed by lack of beetles!
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* **Collecting eggs** is difficult in the standard flour medium. Eggs are small, and will not easily separate out of the stock flour medium using the No. 50 sieve. For this reason, an **egg collection flour medium** will be used when we want to collect beetle eggs.
* Egg collection medium consists of 95% pastry flour (organic, no pesticides!) and 5% brewer’s yeast + 0.3% fumagillin by weight.
* Pre-sift flour with the no. 50 sieve before beetles are added to remove all bran.
* Bake flour + yeast at 60C for 24 hours before adding fumagillin.
* Baking kills any macroparasites than may be in the medium.
* Make sure this is done BEFORE adding fumagillin. Fumagillin is temperature sensitive and will denature at 60C.
* When collecting eggs, first use the No. 25 sieve to separate beetles from flour medium, then use the No. 50 sieve to separate eggs from medium.

* **Individual cultures** can be set up in in fly vials with ~2cm of the appropriate flour medium.
* **Euthanize** beetles when cultures become too crowded or when you have more cages than needed for maintenance of the stock. You should also euthanize beetles used in experiments after you have collected all the data and specimens necessary.
* Beetles that you don’t need to keep should be killed by freezing them overnight at -20˚C.
* If you need to keep an entire beetle as voucher or specimen form an experiment, preserve it depending on the needs of the experient.
* 70% ethanol for preservation of anatomical structure (store at room temp.)
* 100% ethanol for preservation of DNA (store at 4˚C)
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* Never release lab insects into the wild!
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* **Dispose of flour waste** (collected when cleaning jars) by placing waste at -20˚C for 24 hours. This will ensure that no living beetle eggs or parasites are released into the wild when we place waste in the trash.