Abstract
The blue orchard bee (Osmia lignaria Say) is a solitary bee native to North America that is increasingly propagated, sold, and used for pollination of rosaceous orchard crops. While methods exist to rear blue orchard bees in a laboratory setting, present protocols vary in diet manipulation and fail to progress bees to adult emergence. Variability in published methods also makes standardized comparisons within or across species challenging. Here, we present a validation of a rearing protocol for O. lignaria in a laboratory setting, with the study employed over a 2-year period that mirrored the bees’ phenology in northern Utah (United States). Our protocol used 3D printed well plates with a well diameter that we recommend as appropriate for rearing O. lignaria in a laboratory. Each well permits the user to observe detailed life stages of individual O. lignaria bees without disturbing their development. To validate the protocol, we monitored O. lignaria development across 3 larval instars (first, second, and fifth), prepupal, pupal, and adult stages using a dissection scope and X-ray imaging. We confirm that diapause duration can be altered and affects the percent weight loss. Our data demonstrate that we can successfully rear bees to the adult stage (74%). Our protocol can be altered to fit any laboratory experiment and adapted to investigate other above-ground cavity-nesting bee species in a laboratory setting. Such investigations might include how multiple environmental conditions, nutritional factors, and stressors influence bee health.
Keywords: larvae, rearing, pollination, laboratory, survivorship
Introduction
The blue orchard bee (Osmia lignaria Say, Hymenoptera: Megachilidae) is a univoltine solitary bee native to North America and used in research and pollination in the United States, with spring emergence of adult bees. This species’ wide distribution, preference for foraging on important crops, and willingness to nest in artificial nests contribute to their utility as an alternative pollinator in spring-blooming orchards of almonds, apples, and cherries (Torchio 1991, 2003, Richards 1993, Bosch and Kemp 1999, 2001, Bosch et al. 2000, 2006, Brittain et al. 2013, Pitts-Singer et al. 2018). This has led to a robust industry for mason bee suppliers, who can supply managed populations of O. lignaria for commercial orchard pollination and field or laboratory-based trials; the utility of the bee as a pollinator has increased, given that adult emergence can be artificially manipulated by shortening diapause duration (Bosch et al. 2010, Andrikopoulos 2018). Many facets of O. lignaria’s biology have not been fully explored, including the influence of abiotic and biotic stressors on survival and development.
Here, we present data validating a rearing protocol for O. lignaria that allows individual bees to develop from eggs to adult emergence (Williams 2025). Success was achieved through a customizable rearing apparatus (ie 3D printed well plates, Fig. 1A) and a removable liner that closely mimics natural nesting conditions. With this protocol, researchers can effectively subject developing O. lignaria to various treatments (eg agrochemicals, pathogens, combinations of stressors, etc) and ask questions regarding bee health. Customized well diameters that mirror the diameter of nesting materials permit successful development from immature stages to adult emergence in a laboratory setting. Duration of adult diapause can be controlled to affect adult emergence. The replicability of our protocol can enable consistent and comparable data collection, thereby improving our understanding of health, survival, behavior, and development of solitary bees.
Fig. 1.
Images of the customized 3-D printed well plate, from different angles (A), and an example of a plate filled with grafted eggs/first instars on provisions (B). Dimensions of the plate include length (= 131.2 mm), width (= 55.6 mm), height (= 17.1 mm), well diameter (= 8.5 mm), and well depth (= 15.1 mm).
Materials and Methods
Our detailed protocol, including bee images, recipes, source and materials lists, recommendations, a 3D print file, and blank datasheets are available at Williams et al. (2024).
O. lignaria were purchased from a Utah supplier and allowed to create nests inside artificial materials placed at an apple orchard in Logan, Utah (2 ha) during May 2021 and 2022, using established practices (Bosch and Kemp 2001). Completed nests were identified by the presence of a mud plug at the nest entrance and were brought to the laboratory daily. First, eggs/first instars (nonfeeding stages; Eeraerts et al. 2020, Kopit et al. 2022) were separated from their original nest provision and retained on a 2% agarose gel until grafting (the manual transfer from an original nest cell to an artificial rearing plate and provision) on the same day onto homogenized provisions, encased in straw liners placed in 3D printed well plates (Fig. 1B). Ringer’s Solution was added to amassed provisions and mixed in a sterilized glass beaker. The batch of homogenized provisions was plated into a sterile glass petri dish, and glassine paper straws were used to obtain 0.35 g per straw. After each straw was placed into a well of the plate, eggs/first instars were grafted at random into individual wells using a sterilized stainless-steel honey bee queen-grafting tool dipped in Ringer’s solution.
Completed plates were incubated at temperatures and relative humidity (RH) required for O. lignaria development, reflecting what wild O. lignaria would experience in northern Utah. Larvae in the well plates were inspected daily to record life stage benchmarks (Fig. 2; Kopit et al. 2022, Scalici et al. 2023, Williams 2025). After O. lignaria completed their cocoons, the cocoons were removed from the straws, placed into gelatin capsules, and monitored daily for pupation using an enclosed X-ray imaging system. Cocoons were then transferred to winter incubation conditions (winter diapause). Three durations of winter diapause were examined (143, 150, and 178 d; the number of days bees spent in 4°C until the beginning of ramp-up conditions). In the spring, individual cocoons were placed into labeled glass scintillation vials plugged with cotton balls, transferred to warmer temperatures, and checked daily for adult emergence for up to 2 wk. Survival was recorded for each life stage, and pre- and post-winter diapause weights of adults in cocoons were recorded to evaluate physiological responses.
Fig. 2.
Life stages were recorded in the validation of our protocol. Details for proper identification of each life stage are provided in Williams et al. (2024).
Statistical Analyses
Statistical analyses were performed in JMP® Pro v.18. For validation, we determined if survival, development, and weight loss were consistent across 2 study years using our rearing protocol. We used the second instar stage to standardize survival and development throughout the experiment. Bees that died as first instars were excluded from all analyses. Bees that did not emerge as adults were excluded from development and weight analyses. Bees that did not emerge within the period of 2 wk after diapause were excluded from development timing analyses.
We defined protocol success as enabling O. lignaria to survive through each life stage and develop from newly emerged second instar to an emerged adult. A parametric survival analysis using a Weibull distribution evaluated survival from second instar to pupation and second instar to adult emergence by study year. Adults who died during diapause were assigned an arbitrary “death date” as halfway through winter diapause, and live adults were censored. A Fisher’s Exact test for count data was used to compare the number of bees that died and adults that successfully emerged in each study year. An analysis of means (ANOM) for proportions test was used to assess whether the proportion of final life stages differed between study years.
Number of days between the following development periods between study years was analyzed: second instar to fifth instar, fifth instar to cocoon initiation, cocoon initiation to cocoon completion, pupation to adult molt, and adult molt to adult emergence. To assess longer development times, second instar to pupation and second instar to emergence were analyzed. If the adult molt was not determined, it was estimated by adding 30 d after the pupation date, given that this molt occurs roughly 1 month after pupation (Bosch and Kemp 2001). Pre- and post-diapause weights of adults (inside cocoons) were used to calculate and analyze percent weight loss by study year.
Development time was analyzed using generalized linear models (GLMs) with a Poisson distribution, and the percent weight loss GLM was analyzed using a normal distribution. Male and female bees were analyzed separately, as development and weight are known to vary by sex (Rust 1991, Bosch and Kemp 2000, Bosch et al. 2000, Sgolastra et al. 2012, Pitts-Singer et al. 2014, McCabe et al. 2021, Scalici et al. 2023). Development time and percent weight loss were used as response variables in respective GLMs to compare averages between study years. Adult molt to emergence, second instar to emergence, and percent weight loss were analyzed using winter diapause duration as a predictor variable. Post hoc tests were performed for significant models using Student’s t-tests (for 2-level comparisons) or Tukey’s HSD test (for more than 2-level comparisons) where appropriate.
Results
Using our protocol, O. lignaria can be reared to the adult stage and through emergence. There was no significant variation between study years in the survival of bees going from second instar to pupae (P = 0.64, df = 1, χ2 = 0.22), nor the percent of bees surviving from second instar to adult emergence (P = 0.91, df = 1, χ2 = 0.01). However, we observed differences in pupal and adult mortality between study years (2-sided Fisher’s Exact test: P < 0.01) (Table 1).
Table 1.
Osmia lignaria that died over time according to the final life stage reached
| Life stage | 2021 (n = 95) | 2022 (n = 98) |
|---|---|---|
| Dead larvae | 5.3% (5) | 2.0% (2) |
| Dead prepupae | 9.5% (9) | 9.2% (9) |
| Dead pupaea | 20.0% (19) | 7.1% (7) |
| Dead adultsa | 14.7% (14) | 34.7% (34) |
| Alive adults | 50.5% (48) | 46.9% (46) |
The percentage (%) of O. lignaria in each life stage is followed by the number (n) of bees in that category. More bees failed at the pupal stage in 2021, and more bees failed at the adult stage in 2022 (2-sided Fisher’s Exact test: P < 0.01; ANOM proportions test).
Indicates bees that died at these life stages were significantly different between study years (2021 = 0.49; 2022 = 0.51)
Development time of female and male O. lignaria from second instar to fifth instar, fifth instar to cocoon initiation, cocoon initiation to completion, pupation to adult molt, and second instar to pupation did not differ between years (Table 2 and Supplementary Table S1).
Table 2.
Mean number of days for the development time of O. lignaria females and males for each study year
| Sex (n = 94) | Development period (days) | Study year |
|
|---|---|---|---|
| 2021 | 2022 | ||
| Females (23) | Second instar to fifth instar | 3.9 ± 0.1 | 3.8 ± 0.1 |
| 2021 (13) | Fifth instar to cocoon initiation | 6.2 ± 0.6 | 6.7 ± 0.4 |
| 2022 (10) | Cocoon initiation to completion | 6.6 ± 0.5 | 4.8 ± 0.3 |
| Second instar to pupation | 44.6 ± 1.4 | 48.3 ± 1.4 | |
| Pupation to adult molt | 29.9 ± 0.5 | 33.6 ± 1.1 | |
| Males (71) | Second instar to fifth instar | 4.0 ± 0.1 | 3.9 ± 0.1 |
| 2021 (35) | Fifth instar to cocoon initiation | 6.7 ± 0.2 | 6.7 ± 0.2 |
| 2022 (36) | Cocoon initiation to completion | 7.6 ± 0.3 | 7.8 ± 0.4 |
| Second instar to pupation | 44.7 ± 0.8 | 47.5 ± 1.0 | |
| Pupation to adult molt | 28.1 ± 0.5 | 29.5 ± 0.5 | |
| Sex (n = 94) | Development period (days) | Study year |
|
|---|---|---|---|
| 2021 | 2022 | ||
| Females (23) | Second instar to fifth instar | 3.9 ± 0.1 | 3.8 ± 0.1 |
| 2021 (13) | Fifth instar to cocoon initiation | 6.2 ± 0.6 | 6.7 ± 0.4 |
| 2022 (10) | Cocoon initiation to completion | 6.6 ± 0.5 | 4.8 ± 0.3 |
| Second instar to pupation | 44.6 ± 1.4 | 48.3 ± 1.4 | |
| Pupation to adult molt | 29.9 ± 0.5 | 33.6 ± 1.1 | |
| Males (71) | Second instar to fifth instar | 4.0 ± 0.1 | 3.9 ± 0.1 |
| 2021 (35) | Fifth instar to cocoon initiation | 6.7 ± 0.2 | 6.7 ± 0.2 |
| 2022 (36) | Cocoon initiation to completion | 7.6 ± 0.3 | 7.8 ± 0.4 |
| Second instar to pupation | 44.7 ± 0.8 | 47.5 ± 1.0 | |
| Pupation to adult molt | 28.1 ± 0.5 | 29.5 ± 0.5 | |
Means are for each study year ± standard error. The number of bees (n) is reported for each group by sex and study year. GLM results are reported in Supplementary Table S2.
As expected, variable winter diapause durations resulted in significant differences in the timing from adult molt to adult emergence and second instar to adult emergence (Supplementary Tables S2 and S3). This manipulation of diapause duration reflects the commercial management practices in which growers or bee managers can match emergence timing to crop bloom. Weight loss was significantly different between winter diapause durations, with more weight loss in adult bees that experienced a longer winter diapause duration (Supplementary Tables S4 and S5).
Given that we provided all larvae with the same amount of provision (0.35 g), we hypothesized that the weights for adults would be the same for males and females. In natural nests, the mother provisions haploid eggs (future males) with significantly less provision than diploid eggs (future females). Adult males weighed significantly less than females (Supplementary Tables S6 and S7), suggesting that weight and size are genetically determined and not based on the amount of diet provided. Percent weight loss during diapause was not significant between sexes (Supplementary Table S7).
Discussion
Our protocol improves upon prior efforts (Kopit et al. 2022) to rear O. lignaria by customizing 3D printed well plates with internal diameters that match the diameters of straws and reeds used in commercial operations.
Using our methods, we achieved 73.6% success for adult development in both study years, with 26.4% of our bees failing before the adult molt. In comparison, in 2 wild sites with trap nests, we observed 83.3% of nest cells develop into diapausing adults inside cocoons, while 16.7% failed before the adult molt (Williams 2025). Our survival is lower than reported studies using undisturbed nests (Scalici et al. 2023, Melone et al. 2024). Other factors, such as experimental manipulation, nutritional quality, and pathogen prevalence, may explain survival variation between the lab and field.
Our development data were comparable to published data (Bosch and Kemp 2000, Scalici et al. 2023), although some variation could be due to differences in provision quality (Kopit et al. 2022) and varying rearing conditions. We could not compare development to adult emergence since the winter diapause duration and emergence conditions differed from published studies. Our protocol utilizes a constant temperature and RH rather than the temperature fluctuations bees experience naturally. Scalici et al. (2023) found that continuous temperatures resulted in higher survival and shorter egg-to-adult development periods for O. lignaria. Further investigation is required to understand how programmed fluctuations in temperature and RH impact solitary bee development using our protocol.
Pre- and post-diapause weights for adults fell within an expected range and were comparable to those reported by Kemp et al. (2004). Percent weight loss of O. lignaria is linked to winter diapause duration (Andrikopoulos 2018); likewise, we observed bees that experienced longer winter diapause lost more weight.
To utilize our protocol effectively, we encourage researchers to heavily stock their nesting site with emerging adults in the spring to maximize the number of eggs and first instars available for grafts. Grafting from the original provision onto an agarose gel, then onto a homogenized provision, risks the survival of eggs or first instars via mechanical injury. When planning sample sizes, we advise overestimating deaths of eggs/first instars following grafting, which may not be visible until one to two days post-graft.
Our protocol provides a foundation for experiments to study above-ground cavity-nesting solitary bees. Improving the consistency and compatibility of rearing methods can standardize laboratory rearing of solitary bees and will lead to a better understanding of responses to abiotic and biotic stressors. Our protocol can be modified to suit other solitary bee species that nest in different-sized cavities by adjusting the well diameter and depth using the STL file provided in Williams et al. (2024). We encourage users of our protocol to continue refining this protocol for their respective studies and target species. Future research using our methods can inform conservation and management strategies for these crucial pollinators.
Supplementary Material
Acknowledgements
We thank Mountain West Mason Bees for supplying O. lignaria for bee propagation, Zollinger’s Fruit and Tree Farm for utilizing their apple orchard, T. Pitts-Singer, and the Behavior Lab at the USDA-ARS-PWA Pollinating Insect Research Unit for their help in setting up nesting boxes and advice during the propagation season. We also thank M. Johnson Bird, T. Burt, C. Huntzinger, and T. Olsen for their help in opening nests to obtain provisions and eggs/first instars. Additionally, C. Huntzinger also helped immensely with grafting. We also thank K. Hageman and K. Kapheim for reviewing our manuscript before submission.
Contributor Information
Mary-Kate F Williams, Department of Biology, Utah State University, Logan, UT, USA; Pollinating Insect Research Unit, Pacific West Area, Agricultural Research Service, United States Department of Agriculture, Logan, UT, USA.
Natalie K Boyle, Department of Entomology, The Pennsylvania State University, University Park, PA, USA.
Robert N Schaeffer, Department of Biology, Utah State University, Logan, UT, USA.
Diana L Cox-Foster, Pollinating Insect Research Unit, Pacific West Area, Agricultural Research Service, United States Department of Agriculture, Logan, UT, USA.
Supplementary material
Supplementary material is available at Journal of Insect Science online.
Funding
This work was supported by United States Department of Agriculture, Agricultural Research Service base funds from project 2080-21000-019-000D (NP 305) to the United States Department of Agriculture, Agricultural Research Service, Pacific West Area, Pollinating Insect Research Unit in Logan, Utah, USA. RS acknowledges support from National Science Foundation (DEB-2211232).
Conflicts of Interest
The authors declare they have no potential conflict of interest regarding the study in this paper and no relevant financial or non-financial interests to disclose. Because experimental work was conducted with an unregulated invertebrate species, no approval of research ethics committees was required to accomplish the goals of this study.
Author Contributions
Mary-Kate Williams (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Validation, Visualization, Writing—original draft, Writing—review & editing [equal]), Natalie K. Boyle (Conceptualization, Investigation, Methodology, Project administration, Supervision, Validation, Writing—review & editing [equal]), Robert N. Schaeffer (Data curation, Formal analysis, Methodology, Project administration, Resources, Supervision, Validation, Writing—review & editing [equal]), and Diana Cox-Foster (Conceptualization [equal], Formal analysis [equal], Funding acquisition [equal], Methodology [equal], Resources [equal], Supervision [equal], Validation [equal], Writing—review & editing [equal])
Data Availability
Our dataset has been submitted to Ag Data Commons and is provided at the following link: https://doi.org/10.15482/USDA.ADC/27898743.v1. A link is provided on the title page document to comply with the double-blind peer review process.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Our dataset has been submitted to Ag Data Commons and is provided at the following link: https://doi.org/10.15482/USDA.ADC/27898743.v1. A link is provided on the title page document to comply with the double-blind peer review process.