Daily beetle colony maintenance
I. New females
1. Check black tubes for emergent females. Set aside emergent females. Check collection dates on black tubes. Discard any sticks older than 45 days. Clean emptied tubes with bleach, dry completely, and store in zip lock bags.
2. Thaw frozen sticks for newly emerged females. Seal stick ends with wax (paraffin or parafilm). Place in new sterile petri dishes.
3. Add emergent females, 1 per stick, per petri dish. Label petri dish with date. Place in Starter Box
II. Starter Box
1. Check starter box for excavating beetles. Label dishes with excavating females with the “bored – in” date and unique ID number. Move dish to Working Colony and enter ID and “bored – in” date in spreadsheet.
2. Discard any dishes with dead females, signs of mites, or females that have failed to bore in for longer than 5 days.
III. Working colony
1. Check Working Colony spreadsheet for ripe galleries. Harvest galleries that have reached the “ripe date”. Date will depend on current demand for experiments (ask James). If eggs are needed, this will be 4 – 6 days after “bored – in date”. If pre-pupae are needed, this will be 14 – 16 days after “bored – in” date.
i. Harvesting
1. Place sterile #40 Whitman filter paper in sterile petri dish and moisten with 500 ul sterile PBS.
2. Use sterile pipet tips to transfer eggs/pre-pupae to moistened filter paper, being as fast and sanitary as possible. Add no more than 12 eggs or 6 pre-pupae per filter paper. Wear gloves and clean gloves with EtOH between every stick.
3. If gallery is in good shape and may produce more eggs/pre-pupae, carefully reclose gallery, taking care not to crush beetles. Use small strips of parafilm to bind the ends of the stick back together. Record “opened” date on petri dish and spreadsheet. Return petri dish to working colony. Check 2-3 days later.
4. Discard spent galleries and remove from spreadsheet.
ii. Specimen treatments
1. If current experimental protocol requires axenic larvae/pre-pupae, apply chemical treatment per protocol to eggs/pre-pupae on filter paper (ask James for protocol).
2. Transfer cleaned eggs/pre-pupae to fresh sterile filter paper in new sterile petri dish. Label dish with “harvested” date and chemical treatment, then seal petri dish with parafilm, and move it to the small incubator (27° C).
Weekly beetle colony maintenance
Monday: Clean Starter Box and re-supply stocks.
1. Remove all petri dishes
2. Pour perlite into autoclave bag using large funnel and autoclave. Leave
perlite in closed autoclaved bag for use next week.
3. Wipe down container with bleach solution. Dry well.
4. Replace with autoclaved and cooled perlite from previous week.
5. Check and replenish stocks of sterile filter papers, sterile tubes of PBS, media, etc.
Tuesday: Collect fresh galleries.
1. Collect as many occupied X. compactus galleries as possible. Effort will have to be adjusted to match need/loss. Only collect galleries with female visible at entrance.
2. Cut twigs to fit in black tubes. Seal cut twig ends with wax.
3. Place 4-6 galleries per clean black tube. Put tubes in walk-in hot freezer.
Wednesday: Clean Working Colony.
6. Remove all petri dishes/ tubes
7. Pour perlite into autoclave bag and autoclave.
8. Wipe down container with bleach solution. Dry well.
9. Replace autoclaved perlite, petri dishes, and tubes.
Thursday: Collect fresh sticks.
1. Find sweet gum stems 2-6 mm in diameter. Cut sticks ~5 cm length, with no nodes!
2. Collect enough sticks to replace what was used in previous week.
3. Store in zip lock bag in freezer
