May 19, 2026
Transfer Method of Ambrosia Beetle Sterilization
- M. Elizabeth Moore1,
- Thomas Johnson1,
- Brian Lovett1
- 1USDA-ARS, Cornell University
- Moore Entomology Developed Methods
Protocol Citation: M. Elizabeth Moore, Thomas Johnson, Brian Lovett 2026. Transfer Method of Ambrosia Beetle Sterilization. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxjrzwl8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 19, 2026
Last Modified: May 19, 2026
Protocol Integer ID: 317480
Keywords: ambrosia beetle, fungal mutualist, rearing, colony, lab, contaminating fungi, entomology, mycology, transfer method of ambrosia beetle sterilization, ambrosia beetle sterilization, contaminating fungal spore, fungal spore, obligate fungal mutualist, conducive to fungal growth, dry sterile filter paper, host of other fungi, fungi, contaminating, fungal growth, other fungi, transfer method, beetle, problematic in beetle, substrate
Abstract
Ambrosia beetles have an obligate fungal mutualist, but also are associated with a host of other fungi. When rearing, the substrate (wood or artificial media), must be conducive to fungal growth to propagate the fungal mutualist. This means that “contaminating” fungi will also thrive. Contaminating fungi prove problematic in beetle rearing due to difficulty handling bolts, increased time in caring for the colony, and could potentially be a health hazard to the humans caring for the beetles. The method described in this protocol uses a series of transfers between wet and dry sterile filter paper to mechanically and/or abiotically reduce the number of contaminating fungal spores that make it into the growth medium. This protocol is modified from Grosmann, 1956.
Attachments
BF00430256.pdf
2.2MB
Materials
• Healthy, adult ambrosia beetles
• 3-4 sterile petri dishes per batch of beetles (over 2-3 days)
• 3-4 pieces of sterile filter paper (autoclaved) that fit easily in petri dish (may need to be cut to size) per batch of beetles (over 2-3 days)
• Autoclaved DI water
• 1000uL pipette and tips
• Soft forceps
• Fine forceps
• Lighter
• Biosafety cabinet
Transfer order and beetle density:
Alternate beetles between wet and dry petri dishes, starting and ending with a wet dish to prevent desiccation. The standard number of transfers is 3 (wet -- dry -- wet), but more steps can be added if needed.
We have observed that beetle digging success is strongly correlated with undamaged tarsites, and that leg damage increases with high beetle density. For a standard size petri dish, we recommend a beetle density of 100 beetles or less.
Prepare before starting
Turn BSC on for at least 4 minutes
Sterilize surfaces of BSC with 70% Ethanol
Label petri dish with the beetle information (species, source, date collected, approximate number, etc), transfer number (first dish is T0, second dish is T1, etc), and the details of the next transfer (wet or dry, time of day)
Transfer method of sterilization
12h
Place all materials, including beetles (should be held in a collecting flask with the opening covered), into the BSC
Flame sterilize fine forceps using the lighter
Use the sterilized fine forceps to remove sterile filter paper from container and place into each sterile petri dish
For wet transfers, add 1000uL of autoclaved DI water to the filter paper in each petri dish (omit this step for dry transfers)
Add beetles to petri dish, using flat forceps as needed (beetles can be dumped into the petri dish directly from the flask, and flat forceps used for stragglers).
Parafilm petri dish and return to growth chamber
WAIT ~8-12 HOURS
12h
Example: If beetles are placed in their first wet dish in the afternoon of day 1, transfer to a dry dish the morning of day 2, then transfer to a new wet dish in the afternoon of day 2.
Repeat steps 5-11
Always alternating wet and dry dishes
WAIT ~8-12 HOURS
Repeat steps 1-6
Beetles should end on a wet transfer so they don’t desiccate while waiting to set up on bolts or artificial media
At the end of the transfers, beetles can be held in a wet petri dish for up to a week before establishing on wood or artificial media, however beetles do better if transferred to substrate within 3-4 days. We recommend following the Ambrosia Beetle Surface Sterilization Protocol (https://dx.doi.org/10.17504/protocols.io.x54v9bk44l3e/v1) immediately before setting up on wood or media.
Protocol references
Francke-Grosmann, H. (1956) Hautdrüsen als träger der pilzsymbiose bei ambrosiakäfern. Zeitschrift für Morphologie und Ökologie der Tiere, 45, 275–308.