# Rearing flour beetles [EcoEvoDevoLab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab) Updated 28 September 2021 Based on the protocol written for our lab by Devin O'Brien, June 5, 2019.  > Modified from a photo by [Salvador Vitanza, USDA-APHIS](https://extensionentomology.tamu.edu/insects/flour-beetle/) 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*. ## General information ### *Tribolium castaneum* *Tribolium castaneum*, the red flour beetle (order Coleoptera; family Tenebrionidae).  - Stored goods pest (flour, grains, etc) - 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 *Gnatocers cornutus*, the broad-horned flour beetle (order Coleoptera; family Tenebrionidae). Also sometimes spelled "*Gnathocerus*".  - Stored goods pest (flour, grains, etc) - Cosmopolitan distribution, spread through trade - Little known about their natural habitat, food source, or behavior. - Holometabolous - Sexually dimorphic weapons. (Males have enlarged mandibles and horns. Horns are absent in females) - Grow well in the lab. ## Beetle medium 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% organic, whole wheat flour (no pesticides!), 5% brewer’s yeast, and 0.03% fumagillin by weight. Follow the steps below to prepare this food.  - Sift whole wheat flour with through the No. 25 sieve - Discard any flour clumps or bran that cannot be sifted through the sieve - Add sifted flour to a large terrarium ("critter keeper") - Weigh the sifted flour - Add 5% (w/w) ground dried yeast (either brewer's yeast or nutritinal yeast) - Mix throughoully - Place a lid on the container (Unbaked flour can be stored at room temperature in capped containers with a large KimWipe between the lid and the container) - Bake the flour/yeast mixture at 60˚C for 24 hours (Baking kills any macroparasites, pests or rouge beetles that may be in the flour) - Cool flour to room temperature (You can use the refrigerator or freezer to expedite this process) :::warning **You must cool the medium before adding fumagillin**. Fumagillin is temperature sensitive and will denature at 60C. ::: - Add 0.03% (w/w) fumagillin :::warning **Wear a mask and gloves** when working with fumagillin powder. ::: :::info **Watch your math** when adding the fumagillin. If you have 95g flour, 5g yeast, add 0.03g (that's 30mg) stock fumagilin. Alternatively, mix up a 1:100 fumagillin-in-flour weight/weight "dilution". In that case, you'd add 3g in the example above. ::: - Store the medium at room temperature in containers with a large KimWipe between the lid and the container. ## Mass-rearing beetle stocks :::warning 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 or beetle care! ::: ### Labeling Labeling containers is essential! Ideally include: - The species name (“Tcst” or “Gcor” is sufficient in our lab) - Experimental line of origin (For *Tribolium*, e.g., “pu-11” or “GA1”) - Experimental manipulation and treatment ID number (if applicable) - Your initials - Date of set-up or last cleaning - If you’re keeping things in a shared space, it may help to add “Angelini Lab” ### New *Tribolium castaneum* stocks  - Place approximately 100 beetles in a large mason jar, 1-third filled with appropriate medium. Use a KimWipe or paper towel to cover the container, not the standard mason jar lid. This will allow adequate airflow. ### New *Gnatocerus cornutus* stocks - Place approximately 100 beetles in a large plastic terrarium ("critter keeper"), , 1-third filled with appropriate medium. Use large KimWipe to cover the container. :::danger Add a picture of a terrarium stock. ::: :::warning *G. cornutus* delays pupation and cannibalizes at high larval density, which is why we keep them in larger chambers at a lower population density. ::: ### General stock 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 typically maintain populations at 25˚C and increase the temperature during experiments and when increasing stock. :::info **How many containers to keep?** In general, it is best to have at least three large containers of each stock line. If experiments are removing beetles from these cultures, having more containers on hand (before experiments begin) is a good way to prevent stocks from becoming depleted to levels causing potential delays due to a shortage! ::: ### Maintaining stocks Containers should be exchanged every month or when adult density becomes too high to allow larval development. - Empty the contents of the jar into a No. 25 sieve with the solid bottom attached - Sieve the contents, separating adults, pupae, larvae, and molts from the medium. - Dispose of the old medium into a large plastic terraroium. Freeze it overnight, then dispose of it in the trash. - Remove the exuvia (shed cuticles) by gently blowing in the seive (Do this over the trash!) - Sort beetles as needed - Return beetles to a new, clean, dry jar with fresh medium - If needed split populations into multiple jars, or euthanize extra beetles, to maintain the optimal density ### Cleaning containers - Always **freeze old culture medium** at -20˚C overnight to ensure that no beetles are released from the lab - Then dispose of the medium in the trash - Clean used jars or terraria by rinsing them well with hot water (Be sure no visible flour remains) - Soak them in a dilute bleach solution for at least 1 hour - Rinse well in warm water - Air dry completely before using them again ## Small cultures Cultures for individuals, mating pairs, or other small numbers of beetles can be set up in the vials commonly used for fruit flies. Just add about 2 cm of the appropriate medium. :::danger Add a picture of a culture tube and tube flats ::: ## Beetle wrangling There are several simple ways to move beetles from container place to another! ### Paper strip transfer This method works well to move a large, indeterminant number of adult beetles. It will not move larvae or pupae. - Set-up the destination container for your beetles - Place the source and destination containers, side-by-side on the bench in front of you - Cut or fold a peice paper so that it is roughly 10 cm long and at least 4 cm wide - Fold it in half lengthwise, at least once - Insert one end of the paper into a container with adult beetles - Wait a few seconds for beetles to start climbing up the paper - Before they reach the top, move the paper into the new container - Gently flick the paper with your index finger to dislodge the beetles - Repeat as needed :::danger Add picture of the strip covered in beetles ::: ### Paper funnel transfer This method works to transfer adults and large larvae. - Set-up the destination container for your beetles on the bench - Lay a clean sheet of blank white paper onto the bench - Select a source container, with beetles and medium - Sift a small amount of medium from the source container into the No. 25 sieve - Return the sifted medium (which may contain small larvae and eggs) back into the source container - Tip the beetles and large larvae onto the center of the sheet of paper :::warning The beetles and larvae will quickly run in all directions! So you may want to practice the motions described below without any beetles, before trying it for real. ::: - Before they reach the edges of the paper, pick it up the paper and form it into a funnel, which you then insert into the destination container. - Flick the paper funnel with your finger to remove any lingering beetles :::danger Add pictures of the beetles on the flat sheet and the paper funnel. ::: ## Collecting beetle eggs Collecting eggs is difficult in the standard flour medium. Eggs are small and will not easily separate out of the stock medium using the No. 50 sieve. For this reason, an egg collection flour medium should be used to collect beetle eggs. - Sift organic pastry flour to remove any bran - As with the standard culture medium, bake the pastry flour (and yeast) at 60˚C for 24 hours and cool before preparing media - Prepare egg collection medium consisting of: - 95% sifted, organic pastry flour - 5% yeast - 0.03% (w/w) fumagillin - Move adult beetles (using paper strip transfer) onto the egg collection medium - Give them some time to lay eggs - To collect eggs, first use the No. 25 sieve to separate beetles, pupae and larvae from the medium - Next, sift using the No. 50 sieve, which will retain eggs from the egg-laying medium ## Euthanasia :::warning Never release lab insects into the wild! ::: 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 should be killed by freezing them overnight at -20˚C. If you need to keep an entire beetle as a 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) --- [EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)