TITLE: Osmia lignaria laboratory rearing protocol
AUTHORS: Mary-Kate F. Williams, Natalie K. Boyle, Robert N. Schaeffer, Diana L. Cox-Foster

[DESCRIPTION]
Our protocol was designed to rear Osmia lignaria Say (Hymenoptera: Megachilidae) from immature stages to adult emergence following their natural phenology in northern Utah. Many aspects of the bee's biology can be analyzed using this protocol, including ecology, genetics, microbiome, pathology, toxicology, and pollinator management. An individual bee can be observed as a response to a treatment group, and its development and survival can be measured. The interaction of various stressors can help define synergistic, antagonistic, and additive effects, including acute, sublethal, and chronic treatment responses. Osmia lignaria was used as a model to rear solitary bees in a laboratory setting. 

[BEFORE_START]
Preliminary steps
Purchase or otherwise obtain Osmia lignaria Say (Hymenoptera: Megachilidae) (adults in cocoons) from a supplier near the researcher's location. The Orchard Bee Association provides guidelines for purchasing and obtaining O. lignaria (Orchard Bee Association [2021]. Guidelines for Sustainably Sourcing and Managing Mason Bees. Link: https://www.orchardbee.org/certification-guidelines-1).
Check bloom for the desired fruit or nut crop as often as needed to prepare for the release of O. lignaria using release boxes or tubes.
Osmia lignaria requires mud to complete nest construction. Ensure O. lignaria can access moist soil (mud) within the orchard during pollination (Bosch and Kemp 2001; full reference in protocol).
Obtain 3-D printed well plates (see 3-D print file) or create Smooth-CastTM 305 well plates using a silicon mold (see "Recipes" PDF file).
Sterilize glass and stainless steel items for the experiment (autoclave at 121 °C for 15 min). Repeat sterilization if more than one grafting date is needed.

[GUIDELINES]
Use sterilized (autoclaved or otherwise unopened or new) glass, plastic, and stainless steel items.
Check data loggers and incubators once a week while in use; ensure that programmed temperatures and RH do not deviate and safeguard against machine malfunction.

[STEPS]
SECTION: Propagation and acquiring solitary bee nests (Fig. 1)
1. Deploy corrugated plastic nesting boxes (Artz et al. 2013, 2014) into the orchard before bloom. Place nest blocks with straw inserts or bundles of cardboard tubes with straw inserts into nest boxes.
 

 

 

 


SECTION: Propagation and acquiring solitary bee nests (Fig. 1)
2. Once bloom has begun, place release boxes or release tubes containing cocoons into the orchard on trees that do not have nest boxes.
 

 

SECTION: Propagation and acquiring solitary bee nests (Fig. 1)
3. Once O. lignaria cocoons are placed in the orchard for release, check nest blocks or bundles of tubes daily for completed nests.

 

 

SECTION: Propagation and acquiring solitary bee nests (Fig. 1)
4. Carefully transport completed nests to the laboratory to retrieve provisions and eggs/first instars.

 

 

SECTION: Propagation and acquiring solitary bee nests (Fig. 1)
5. Continue checking for completed nests daily, and processing nests until the ideal amount of provisions and sample size (eggs/first instars) is obtained for the experiment.
 

 

SECTION: Separating eggs/first instars from provisions (Fig. 5)
6. Take a completed nest and shallowly cut along the straw laterally with a single-edge razor blade. Using size 7 insect pins, pin the nest open against an insect pinning board (or foamboard, or hard-backed item). This serves to secure the straw while retrieving provisions and eggs/first instars.
 

SECTION: Separating eggs/first instars from provisions (Fig. 5)
7. Using a stainless steel grafting tool, dip the flattened head into sterile Ringer’s Solution, then gently pick up the egg/first instar from the provision and deposit it onto a 2% agarose gel in a large Petri dish. Cover the large Petri dish with the dish lid to prevent egg/first instar desiccation. Keep the large Petri dish at room temperature. Set aside eggs/first instars for later.
 

 

 

 

 

 

 

 

 

 

SECTION: Separating eggs/first instars from provisions (Fig. 5)
8. With the egg/first instar separated from the provision, use featherweight forceps to pool the O. lignaria-collected provisions into a weigh boat.
 

 

 

 

 

 

SECTION: Homogenization of provisions and treatment administration (Fig. 6)
9. Deposit all collected provisions into a sterilized large glass beaker. Mix the provisions thoroughly for 1 min to 2 min using a sterile stainless steel laboratory spatula.
 

SECTION: Homogenization of provisions and treatment administration (Fig. 6)
10. Distribute the homogenized provisions into a labeled 50 mL Falcon tube (one per treatment group). Ensure the same amount of homogenized provisions is distributed among the 50 mL  Falcon tubes before treatment administration.
 

 

SECTION: Homogenization of provisions and treatment administration (Fig. 6)
11. Add treatments to homogenized provisions. Homogenize provisions again by mixing the treated provisions in their respective tubes for 1 min to 2 min using a stainless steel laboratory spatula.
 

 

 

 

 

 

 

 

SECTION: Distribution of treated provisions into 3-D printed well plates (Fig. 7)
12. Carefully remove the homogenized treated provisions from their respective 50 mL Falcon tubes using each treatment group’s respective stainless steel laboratory spatula. Place the treated provisions into a small, labeled Petri dish.
 

SECTION: Distribution of treated provisions into 3-D printed well plates (Fig. 7)
13. Label the 3-D printed well plates with the plate number and orientation of treatments according to columns.
 

SECTION: Distribution of treated provisions into 3-D printed well plates (Fig. 7)
14. Cut straws, the same utilized during propagation, to the same length as the 3-D printed plate well depth ( ).
SECTION: Distribution of treated provisions into 3-D printed well plates (Fig. 7)
15. Using the cut straws as a cookie-cutter, weigh and punch 0.35 g of homogenized treated provisions. Use weigh paper cut into quarter sizes (4x4 in sheets cut to approximately 2x2 in sizes), to keep the scale clean.
 

 

 

 

 

SECTION: Distribution of treated provisions into 3-D printed well plates (Fig. 7)
16. Place the straw containing the homogenized treated provision into its designated place in the well plate.
SECTION: Distribution of treated provisions into 3-D printed well plates (Fig. 7)
17. Repeat these steps for each treatment group, plating one treatment at a time, until the target sample size is reached. Each treatment group should have an equal number of samples represented on the 3-D printed plates.

 

SECTION: Grafting eggs/first instars onto treated provisions (Fig. 9)
18. Using the stainless steel grafting tool dipped in Ringer’s Solution, gently pick up an egg/first instar from the 2% agarose gel inside the large Petri dish to transfer onto a treated provision inside a well. 
 

SECTION: Grafting eggs/first instars onto treated provisions (Fig. 9)
19. Gently deposit the eggs/first instars onto homogenized treated provisions using the stainless steel grafting tool.
 

SECTION: Grafting eggs/first instars onto treated provisions (Fig. 9)
20. Once eggs/first instars have been checked, document the bee’s graft date (considered as "Day 0") and starting development life stage (eggs or first instars only).
 

 

SECTION: Grafting eggs/first instars onto treated provisions (Fig. 9)
21. Cover the 3-D printed well plates with a piece of transparency film (sized to cover the surface of the plate) to prevent egg/first instar and provision desiccation.
 

 

SECTION: Grafting eggs/first instars onto treated provisions (Fig. 9)
22. When grafting is complete, place the 3-D printed well plates with transparency film covers onto a plastic tray and into an incubator set to 26 °C with  .
 

 

SECTION: Observing development and monitoring survival (Fig. 15)
23. Beginning on the graft date, monitor bees once a day for changes in development using a dissection microscope between 12 PM and nightfall. Document date of observed development life stage.
 

SECTION: Observing development and monitoring survival (Fig. 15)
24. If a bee dies, excise the dead bee from the well and freeze in a 1.7 mL microcentrifuge tube at -80 °C.
 

 

SECTION: Observing development and monitoring survival (Fig. 15)
25. Once cocoons have darkened, and the nipple (apical end of the cocoon visible to the researcher) (Fig. 12) has become dark with white silk surrounding it, transfer the cocoon to a gelatin capsule and adhere to a sticky board.
 

 

SECTION: Observing development and monitoring survival (Fig. 15)
26. Transfer the sticky boards with developing bees to room temperature.
 

 

SECTION: Observing development and monitoring survival (Fig. 15)
27. Use an X-radiograph (hereby X-ray) imaging system and machine to document the prepupal, pupal, and adult molt stages. Perform X-rays 12 s at 24 kilovolts (kV) once a day, between 12 PM and nightfall, until ramp-down conditions.

 

 

 

 

 

 

 

 

SECTION: Winter diapause (Fig. 16)
28. On September 21 or 22 of the study year, perform a final X-ray then, if able, identify bees that are still at the pupal stage (have not molted into an adult).
 

 

SECTION: Winter diapause (Fig. 16)
29. After performing the X-ray in Step 28, weigh cocoons without frass to obtain a pre-winter diapause weight in grams (milligrams are also acceptable).
 

SECTION: Winter diapause (Fig. 16)
30. Once the pre-winter diapause weight is obtained in Step 29, place the cocoon it into a new gelatin capsule and back onto its original spot on the sticky board.
 

SECTION: Winter diapause (Fig. 16)
31. Ramp-down conditions for diapause induction: On September 23 of the study year, place box containing sticky boards and a data logger into an incubator programmed at 15 °C with   for one week.

 

SECTION: Winter diapause (Fig. 16)
32. Winter diapause conditions: On October 1 of the study year, in the early morning, place the box containing sticky boards and data logger into a walk-in cooler (or incubator) at 4 °C with   until the following spring. Osmia lignaria adults will enter diapause under these conditions.
 

 

 

SECTION: Winter diapause (Fig. 16)
33. On February 26 or 27, the following spring, perform an X-ray on the winter diapause sticky boards before obtaining a post-winter diapause weight.
SECTION: Winter diapause (Fig. 16)
34. After obtaining the post-winter diapause weight, place cocoons into sterile glass scintillation vials without lids (one cocoon per vial). Label vials with the sample identifier and plug with a cotton ball.
 

SECTION: Winter diapause (Fig. 16)
35. Place vials upright into a container and return to winter diapause conditions until ramp-up conditions.
 

SECTION: Emergence (Fig. 17)
37. On March 1, change the incubator temperature to 26 °C with  .
SECTION: Emergence (Fig. 17)
38. Check samples once a day between 12 PM and nightfall for two weeks (until March 15).
 

SECTION: Emergence (Fig. 17)
39. Once an adult emerges, record the emergence date and sex, then freeze the bee at -80 °C  in a labeled 1.7 mL microcentrifuge tube.
 

 

 

SECTION: Emergence (Fig. 17)
40. On March 15 (nighttime), cut bees that failed to emerge out of their cocoons using a single-edge razorblade, dissecting scissors, or scalpel blade (#5 or 7). Record the final stage of bee development and determine the sex of adults. Freeze bees in 1.7 mL microcentrifuge tubes at -80 °C.
 

 

SECTION: Emergence (Fig. 17)
41. Wash glass vials in 70% ethyl alcohol and water, and once dry autoclave vials (autoclave at 121 °C for 15 min) for future reuse.

 

 

 

SECTION: Winter diapause (Fig. 16)
36. Ramp-up conditions for emergence induction: On February 28, place samples in winter diapause and a data logger into an incubator set at 15 °C with   .