Data from: Development and evaluation of a rearing protocol for laboratory assays utilizing the blue orchard bee (Osmia lignaria Say, Hymenoptera: Megachilidae)

The blue orchard bee (Osmia lignaria Say, Hymenoptera: Megachilidae) is a native solitary bee in North America. This pollinator species is crucial for orchard pollination, primarily apples, almonds, and cherries. Osmia lignaria is an increasingly managed commercial species sold to supplement honey bee pollination services or used alone. Consistent protocols to rear O. lignaria and other above-ground nesting solitary bee species are lacking. This makes comparisons to social species, such as honey bees and bumble bees, difficult, and data comparisons between solitary bee species are incompatible.

We collected data from two study years, comprising three studies, where we followed O. lignaria’s development from grafting of eggs or first instars in the summer to adult emergence the following spring. The data presented were control bees that were fed sterilized sham inoculate treatments in a larger pathogen-pesticide study.

Adult O. lignaria were first released into an apple orchard in Logan, Utah, USA, where they were allowed to pollinate and mate, and females created nests with provisioned offspring. We then took completed nests to the laboratory, where they were opened to obtain O. lignaria-collected provisions (pollen+nectar) and eggs/first instars. We retained these provisions and eggs/first instars in our rearing studies and to evaluate our protocol methods. We then grafted eggs/first instars onto homogenized provisions on a graft date inside 3-D printed well plates. 3-D printed well plates were desired for our protocol to provide enough space to allow O. lignaria to complete their life cycle to adult emergence the following spring.

Our research proves that O. lignaria can be reared in 3-D-printed well plates and emerge as adults the following spring. This can allow researchers to evaluate the chronic effects of pathogen and/or pesticide exposure on immature solitary bees. Alterations to the protocol, including different kinds of treatment types, can allow researchers to evaluate synergisms, antagonisms, or additive effects that may influence the survival, development, or weight metrics of O. lignaria. The protocol is replicable and could be adapted for other above-ground nesting solitary bee species.

Our O. lignaria rearing protocol is provided in full detail at: dx.doi.org/10.17504/protocols.io.eq2lyj4qplx9/v2

Key findings include: (1) Determination of development in fine detail and near-exact dates for individual developing O. lignaria inside 3-D printed well plates, (2) a 3-D print file that can be altered for other species if desired, (3) Number of days between life stages and the total number of days to each life stage, (4) Life status at various immature and mature life stages, (5) Sex determination for bees that reached adulthood (male or female), (6) Final life stage reached (larva, prepupa, pupa, or live/dead adult), (7) Suggested weight metrics evaluate for O. lignaria, and (8) Different winter diapause durations may produce variable emergence and weight loss results from year-to-year or study-to-study.

Additional variables in the dataset include: (1) Study year, (2) Study name, (3) Sample identifier), (4) Provision batch, (5) Graft date, and (6) Various dates that reflect temperature and relative humidity (RH) durations.

Abbreviations and acronyms in the “RAWdata” dataset

• Year = year data was collected
• Study_name = name of study data was collected
• Sample_ID = sample identifier (one for each individual bee, some IDs may be replicated for both years depending on plate replicate or position replicate)
• Provision_batch = homogenized O. lignaria-collected provisions provided to eggs and/or first instars on a given graft date
• Graft = date eggs and/or first instars were grafted onto homogenized provisions (provision batch)
• First = date first instar was observed
• Second = date second instar was observed
• Fifth = date fifth instar was observed
• Cocoon_initiation = date first signs of silk were observed
• Cocoon_completion = date cocoon was determined to complete
• Pupation = date pupation was observed, determined using X-ray imaging
• Adult_molt = date adult molt was observed, determined using X-ray imaging
• Arbitrary_Adult_molt = arbitrary adult molt date for bees where adult molt may not have been easily identifiable (+30 days since pupation date)
• Ramp_down = date bees were placed into temperature+RH ramp-down conditions
• Winter_diapause = date bees were placed in winter diapause conditions
• Arbitrary_Adult_death = arbitrary adult death date provided for adults that died before emergence conditions and did not emerge successfully the following spring
• Ramp_up = date bees were placed into temperature+RH ramp-up conditions
• Emergence_inc_start = date emergence incubation began
• Emergence = date emergence was observed (adults completely chewed out of cocoons)
• Date_pulled = date bees were pulled from the experiment and frozen at -80℃
• Graft_First = number of days between graft and first instar
• First_Second = number of days between first instar and second instar
• Second_Fifth = number of days between second instar and fifth instar
• Fifth_Cinitiation = number of days between fifth instar and cocoon initiation
• Cinitiation_Ccompletion = number of days between cocoon initiation and cocoon completion
• Ccompletion_Pupation = number of days between cocoon completion and pupation
• Pupation_Adult = number of days between pupation and adult molt
• Adult_ArbitraryDeath = number of days between adult molt and an arbitrarily assigned death date for adults
• ArbitraryDeath_Emergence = number of days between an arbitrarily assigned death date for adults and emergence
• Adult_Emergence = number of days between adult molt and emergence
• Second_Pupation = number of days between second instar and pupation
• Second_Emergence = number of days between second instar and emergence
• Total_Dev_Second = total number of days from graft to second instar
• Total_Dev_Fifth = total number of days from graft to fifth instar
• Total_Dev_Cinitiation = total number of days from graft to cocoon initiation
• Total_Dev_Ccompletion = total number of days from graft to cocoon completion
• Total_Dev_Pupation = total number of days from graft to pupation
• Total_Dev_Adult = total number of days from graft to adult molt
• Total_Dev_Emergence = total number of days from graft to emergence
• Total_Dev_Second_Pupation = total number of days from second instar to pupation
• Total_Dev_Second_Emergence = total number of days from second instar to emergence
• Alive_Dead_First = were bees alive or dead at the first instar stage?
• Alive_Dead_Second = were bees alive or dead at the second instar stage?
• Alive_Dead_Fifth = were bees alive or dead at the fifth instar stage?
• Alive_Dead_Cinitiation = were bees alive or dead at cocoon initiation?
• Alive_Dead_Ccompletion = were bees alive or dead at cocoon completion?
• Alive_Dead_Pupation = were bees alive or dead at pupation?
• Alive_Dead_Adult = were bees alive or dead at the adult molt stage?
• Alive_Dead_Emergence = were bees alive or dead at emergence?
• Status_First = 0 means bees were alive at the first instar stage and 1 means they were dead (used in survival analyses)
• Status_Second = 0 means bees were alive at the second instar stage and 1 means they were dead (used in survival analyses)
• Status_Fifth = 0 means bees were alive at the fifth instar stage and 1 means they were dead (used in survival analyses)
• Status_Cinitiation = 0 means bees were alive at cocoon initiation and 1 means they were dead (used in survival analyses)
• Status_Ccompletion Status_Pupation = 0 means bees were alive at pupation and 1 means they were dead (used in survival analyses)
• Status_Adult Status_Emergence = 0 means bees were alive at emergence and 1 means they were dead (used in survival analyses)
• Pre_diapause_weight = weight in milligrams obtained for adults in cocoons before being placed into winter diapause (only live adults at the end of emergence are provided weights)
• Post_diapause_weight = weight in milligrams obtained for adults in cocoons after winter diapause (only live adults at the end of emergence are provided weights)
• Percent_weight_loss_X100 = weight loss calculated for each adult (only live adults at the end of emergence are provided weight loss calculations); calculated as post-diapause weight subtracted from pre-diapause weight divided by pre-diapause weight then multiplied by 100 to obtain a percentage (%) of weight loss
• Sex = sex determined for bees that reached the adult stage (female or male)
• Final_life_stage = final life stage recorded for each bee
• Instar = if the bee’s final life stage was a larval stage, which larval stage was the final one recorded?
• Required_more_time = did adults require more time than the allowed emergence period to emerge successfully? (adults that were still alive when cut out of cocoons, they were considered live adults but not included in emergence analyses)
• Final_life_stage_2 = life stage for adults further categorized as live or dead
• Sex_2 = males and females further categorized into live or dead

Abbreviations and acronyms in the "Death_graft" dataset: The number of samples per year and study that did not survive past the first instar stage. The second instar stage was used to standardize survival and development since they begin to feed on provisions. Eggs and first instars were used as the initial grafting life stage because they are non-feeding stages of O. lignaria; therefore, they should be excluded from analyses unless desired.

• Year = year data was collected
• Study_name = name of study data was collected
• Sample_ID = sample identifier (one for each individual bee, some IDs may be replicated for both years depending on plate replicate or position replicate)
• Provision_batch = homogenized O. lignaria-collected provisions provided to eggs and/or first instars on a given graft date
• Graft = date eggs and/or first instars were grafted onto homogenized provisions (provision batch)
• First = date first instar was observed
• Death_graft = did sample experience death due to grafting? (likely via mechanical injury)
• Graft_First = number of days between graft and first instar
• Final_life_stage = final life stage recorded for each bee, for all these samples the final life stage reached was larva
• Instar = if the bee’s final life stage was a larval stage, which larval stage was the final one recorded? For all these samples, the final instar reached was first