Solitary bee species represent the most diverse group of bees on the planet but it can be quite difficult to successfully rear the larvae under highly controlled conditions. We have created a method for in vitro culturing of solitary bee species, allowing us to isolate and measure risk factors associated with their microbial symbionts. Among certain stem-nesting solitary bee species it is critical that the culturing techniques cater to the young larvae as well as their pollen-born microbial symbionts.
Here we have illuminated the main and interactive effects of multiple stressors, allowing us to discern whether the stressors are largely acting alone or in concert. Demonstrating the procedure will be Molly Bidwell and Prarthana Dharampal, both from my laboratory. Using a sterilized scalpel, dissect freshly plugged Osmia nesting reeds.
Split the reeds into two parts lengthwise to expose the individual chambers. Nests typically contain eight to 14 chambers with one egg per chamber. Identify the male eggs using the standard method.
Only male eggs are used in this experiment because of sex bias in pollen provision size. Male provisions are typically smaller than female provisions. Then with a sterilized instrument, remove each pollen provision along with the associated egg and place them in a clean weigh boat.
In the weigh boat, gently separate the egg from the provision. Then weigh the pollen provision and egg separately. Work quickly with the Osmia eggs to minimize their time off the pollen.
For host pollen provision preparations, first visually inspect the maternally collected host plant pollen excavated from the nesting chambers for parasites. Then to reduce any potential maternal bias, combine the pollen provisions into a single mass and mix them using a sterilized needle. Next, divide the host pollen into new provisions of standard mass.
To prepare non-host plant pollen provisions pulverize commercially purchased honey bee-collected pollen to a fine powder using a standard laboratory bone mill. Then, based on the moisture content of host pollen provisions, hydrate the pollen powder using sterilized 40%sugar solution. Mix this to a dough-like consistency.
Then make non-host pollen provisions that are the same weight as the maternally collected provisions. For the treatment plate, line individual wells of a 48-well culture plate with autoclaved tin cups. Prepare separate plates for the treatment and control groups.
Then using sterile forceps gently flare out the top rim of the cups so that they can accommodate the pollen provision. Next, load single pollen provisions into the tin cups. Now within each pollen mass make a centrally placed depression using a sterile wooden stick.
Use a new stick for each pollen provision. Into the depression, add the appropriate volumes of treatment solution such as fungicide and sterile water for controls. After each addition, pinch the depression closed using clean sterile forceps.
To begin, load each treatment plate well with a randomly selected male egg. Place it on top of the pollen provision using a clean fine paintbrush. Once the eggs have been placed, secure the lid onto the plate using tape on the corners.
Then, place each prepared plate into an incubator maintained at room temperature. Include one six-well plate loaded with sterile water to prevent desiccation. Leave the plates undisturbed inside the incubator.
Check the plate daily under a dissecting microscope without removing the lid. If the larvae are alive, they will be moving. If no movement is detected, discard the tin cup containing the dead larvae and the remaining pollen provision.
When a larva reaches the prepupal stage, remove it from the tin cup and use a brush to clean frass off the silk cocoon. Then using a dissecting microscope carefully cut through the silk cocoon and extract the prepupa using featherweight forceps. Handle the prepupa gently to prevent any damage to its body.
Now record the mass of the prepupa and the developmental time from the egg to the prepupal stage. Larval fitness was quantified using larval developmental time and prepupal biomass. There was a significant negative correlation between larval development time and prepupal biomass among fungicide treated and untreated groups.
Although there was a significant negative correlation for larvae raised on non-host pollen, no such relationship was observed for larvae raised on host pollen. Pollen source had a significant main effect on bee fitness. When raised on non-host pollen larval developmental time increased significantly, whereas prepupal biomass decreased significantly among both fungicide treated and untreated groups compared to larvae raised on host pollen.
Fungicide exposure did not have significant main effect on fitness. There was no statistically significant difference in developmental time and prepupal biomass for treatments raised on host and non-host pollen across fungicide treated and untreated groups. There was no significant interactive effect of fungicide exposure and pollen source on larval developmental time and prepupal biomass.
After watching this video you should have a good understanding of how to successfully rear solitary bee larvae under in vitro conditions. Once mastered, this technique can be used to raise bees under in vitro conditions from the egg to the pupal stage while causing minimal mortality from handling stress. While using this procedure, it is important to remember to use eggs or very young larvae.
This ensures that the larvae are raised almost entirely on the intended manipulation of diet treatments. This protocol can be extended to several other species of solitary bees and wasps and can be easily modified to test the main and interactive effects of multiple risk factors impacting pollinator health. By providing a means for in vitro culture for solitary bee larvae, this technique can help identify previously unknown risk factors among vulnerable bee populations.
For instance, some of their experiments based on this protocol can help ascertain the role of the pollen microbiome in conferring varying degrees of resistance to xenobiotic stressors. As an accessible and inexpensive technique, this protocol represents a versatile tool for hypothesis testing which can be easily modified by investigators to address their own research objectives.
