1a. Objectives (from AD-416):
Objective 1: Enhance the overwintering health and survivorship of honey bees and alternative pollinators through the characterization and remediation of abiotic and biotic stressors, especially those in northern U.S. latitudes. Subobjective 1A: Characterize the physiological mechanisms of cold tolerance in stored Megachile rotundata and other important insects. Subobjective 1B: Characterize the sublethal effects of cold storage under field conditions and the effects of field conditions on progeny storability during diapause in Megachile rotundata and other important insects. Subobjective 1C: Characterize the sublethal effects of stress incurred during shipping and storage of honey bees and other important insects. Objective 2: Develop transferrable and quality proven germplasm cryopreservation technologies for honey bees, alternative pollinators and other insects of importance. Subobjective 2A: Improve cryopreservation protocols for male honey bee germplasm. Subobjective 2B: Development of a standardized embryo cryopreservation protocol for honey bees and other insects of economic importance . Subobjective 2C: Development of in vitro rearing technologies for honey bee.
1b. Approach (from AD-416):
In the United States the number of colonies has dropped by 61% since the 1940s. Managed bees are subjected to various stressors that, while not lethal in and of themselves, can induce developmental/behavioral abnormalities (sublethal effects) that decrease the availability and quality of the bees. What we currently don’t understand is which stressors are inducing these sublethal effects and which developmental stages are the most vulnerable. With the decline in the populations of the honey bee and non-Apis bees, there is the real risk of losing genetic diversity that is needed for conservation and breeding programs. Despite their agricultural importance, there is no germplasm repository for any bee species. The goals of this project are to deliver high quality pollinators to the end users, by reducing management-induced stressors and to establish user friendly cryopreservation techniques for honey bees and other non-Apis species. Specifically, we propose to address the following questions: 1) What are the molecular responses to management stress and do they change over the course of development? 2) What are the major stressors that are leading to sublethal effects in managed pollinators? 3) Can pollinator quality under field conditions be improved by ameliorating management stress? 4) Can the physiological effects of honey bee spermatozoa cryopreservation by ameliorated by technical improvements, and can said techniques be adapted to non-Apis bee species? 5) Can honey bee embryonic cryopreservation techniques, including recovery from cryopreservation and subsequent in vitro rearing be standardized into a user accessible protocol?
3. Progress Report:
Objective 1: Research continues on the effects of cold storage on pollinator health and survival. Pollinators including the alfalfa leafcutting bee often experience cold storage as a component of standard management practices. While it has been established that a fluctuating thermal regime (FTR), in which the bees are given a daily brief respite from low temperatures, improves survival when compared to storage at constant temperatures, the underlying mechanisms are not well defined. Using highly sensitive calorimetry, we are testing the hypothesis that the protective effects of FTR may be partially due to physiological repair during the warm periods, and that this can be observed as an increase in metabolic heat production. During the current reporting period, we have successfully performed calorimetry on bees undergoing both FTR and constant temperature storage, with unstored bees as controls, and analysis of this data is ongoing. Additionally, while the effects of FTR on survival has been well documented, how this procedure may affect pollinator quality is not well understood. This is especially important because sub-lethal effects, such as physical deformities and other impairments are a well-known consequence of cold exposure in insects. To test for these effects, we exposed bees to one week of either FTR or constant temperature treatments, a non-lethal treatment, and observed the bees upon emergence. We discovered that although alive, bees exposed to constant temperature storage were more likely to exhibit physical defects such as deformed wings. Furthermore, even bees without physical deformities were less likely to nest than their FTR counterparts, indicating additional damage as well. Surprisingly, the offspring of FTR treated bees were more likely to enter the overwintering state of diapause, demonstrating that the effects of management practices can remain well after the stress has been removed. Progress has been made on determining how nutritional resources in the field affect pollinator quality during overwintering. A pilot study involved alfalfa leafcutting bees foraging on alfalfa, canola, camelina, and a legume-dominated naturalized area. Offspring were successfully produced in all four field types, and successful overwintering was documented for all treatments as well. Deciphering improved overwintering success will require substantially higher numbers of offspring, which is planned for in subsequent experiments. Foundational work has also been conducted on the characterization of the effects of shipping and overwintering stress on managed pollinators. Honey bee hives were overwintered in cargo containers, mimicking a practice that is becoming increasingly popular in the northern regions of the United States. Hives were kept at three different temperatures, and two strains of bees were used: one that is considered an industry standard, and one that has been bred specifically for improved overwintering in northern climates. Measurements that were recorded throughout the overwintering period included hive weight, internal hive temperature, and whole-hive respiration rates (recorded as oxygen consumption) throughout the overwintering period. Additionally, individual worker bees were removed from the hives at monthly intervals for calorimetry recording in the lab. Taken together, this data will help to inform the decisions made by bee managers in the future. Preliminary work was also conducted on the construction of a "smart shroud" which will maintain user-defined carbon dioxide (CO2) concentrations for a specific hive. There is growing evidence that increased CO2 can be beneficial to overwintering hives, although much remains to be characterized. The construction of multiple smart shrouds will allow us to test several CO2 concentrations simultaneously in subsequent years. Objective 2: Efforts also continue towards the development of transferrable germplasm cryopreservation technologies for honey bees. Although honey bee sperm cryopreservation using a slow freezing technique has been well established, concern has been raised about the quality of the sperm after recovery from storage, and that certain cryoprotectants used during slow freezing could be detrimental to queen bees after artificial insemination. As a possible alternative, we have developed a protocol that implements vitrification, which rapidly freezes the spermatozoa into a glass-like state. While this protocol has resulted in live, seemingly healthy sperm after removal from storage, additional studies on both queen health after artificial insemination and the health of the resultant hive still need to be conducted. Work has continued on the cryopreservation of honey bee embryos as well. Although we previously reported on the cryopreservation of an embryo resulting in a viable larva, considerable work is still needed before this becomes a transferrable technology. Recent efforts have focused on selection of the optimal developmental stage for cryopreservation, which is widely regarded and the most critical parameter when developing an embryonic cryopreservation protocol. We have identified a narrow window of development that results in 20-25% viability after recovery from liquid nitrogen, thus providing a pivotal base for subsequent optimization experiments. Connected to these efforts is the development of a protocol for rearing reproductively viable queens from cryopreserved embryos. Preliminary studies that took larvae grown from cryopreserved embryos and grafting them into healthy hives proved problematic, leaving queen rearing in vitro as the only viable option. However, while worker bees in a hive feed individual larvae a custom diet that results in either worker or queen development, the exact mechanism remains unclear, which greatly complicates queen in vitro rearing. We have found that diet quantity, rather than diet components, is what is most important in determining "queenliness": larvae fed less food become worker bees, while larvae fed more food develop more queen characteristics. These findings will be crucial as we continue to develop a reliable in vitro queen rearing protocol.
1. Cold storage of alfalfa leafcutting bees can cause reduced pollinator quality under field conditions. Pollinators including the alfalfa leafcutting bee often experience cold storage as a component of standard management practices. While it has been established that a fluctuating thermal regime (FTR), in which the bees are given a daily brief respite from low temperatures, improves survival when compared to storage at constant temperatures; how this procedure may affect pollinator quality is not well understood. To test for these effects, scientists in Fargo, North Dakota, exposed developing bees to either FTR or constant temperature storage and observed the bees upon emergence. We discovered that although alive, bees exposed to constant temperature storage were more likely to exhibit defects such as deformed wings, and even those without deformities were less likely to nest than their FTR counterparts, indicating additional damage as well. Surprisingly, the offspring of FTR treated bees were more likely to enter the overwintering state of diapause than their untreated counterparts. This may be of critical importance to pollination services managers because the presence of nondiapausing bees are a key contributor to the transmission of diseases such as chalkbrood.
2. A simplified honey bee sperm cryopreservation technique. The current technique uses potentially harmful chemicals such as dimethyl sulfoxide (DMSO) and possible pathogen carriers such as egg yolk that could reduce the quality of the sperm as well as the inseminated queen. Additionally, the technique requires specialized equipment that limits the use of this technique by non-scientists. To solve these issues, researchers in Fargo, North Dakota, and Baton Rouge, Louisiana, have developed an improved technique that uses a sugar based anti-freeze and a manual freezing technique to preserve sperm in liquid nitrogen to create a glass-like vitrified state. After storage, the sperm exhibited viability and mobility similar to untreated controls. This is the first report of bee semen vitrification which will serve as the basis for a greatly simplified preservation protocol for the end user.
3. Rearing queen bees in the laboratory. In a bee colony, worker bees can feed a larva a custom diet and cause the larva to develop either into a worker or a queen. If we are able to determine the differences in the worker or the queen diet, we could rear the queen bees in the lab and create a complete honey bee colony. ARS researchers at Fargo, North Dakota, collaborating with scientists at North Dakota State University, have demonstrated that it is the quantity, not the quality of the diet that is important: larvae fed less of the diet become worker bees, and those fed more of the same diet become queens. This new insight opens up new avenues of research into queen health and the development of new techniques of queen rearing.
5. Record of Any Impact of Maximized Teleworking Requirement:
The maximized telework established by the agency in response to the COVID-19 pandemic has impacted our research project in several ways. The most critically affected activities were those that involved collaborative activities with other locations, both within the Agricultural Research Service and with outside collaborators including those at universities and other government agencies. Two milestones were missed due to our collaborator's inability to conduct their own research activities and/or to provide material in support of our research. Travel restrictions have been less impactful for the majority of our field sites that are within close proximity to our center, but when more extensive travel was needed, we appreciated leadership's flexibility in allowing such activities and assistance in ensuring said travel was conducted in a safe manner. However, when safety could not be assured, we delayed travel, especially to collaborator locations, which will delay some research for at least a year. Additionally, some university partners have much more stringent travel restrictions in place, which included travel to field sites. This has resulted in projects associated with the project to be delayed for at least a year as well. Logistical considerations have affected our research. Conducting limited science in the lab while most computer work is conducted elsewhere severely affects efficiency. While we greatly appreciate the ability to conduct ongoing research as long as it is done is a safe manner, we have delayed the start of new research. We expect this to have long-term impacts on the output of our research group. Other logistical considerations have impacted us as well. For instance, one of our major aims of our project plan is to measure pollinator stress that is incurred during normal shipping. Shipping is far from normal during this pandemic, so that aspect of our research has been delayed for at least a year as well. Finally, there are the intangible impacts: reduced interactions, or interactions by Zoom or other means have been noticeably less productive, especially as the situation persists. This is not to say that we haven't experienced positive impacts as well. Training, notebooks, and data file annotation are up-to-date, and administrative tasks are being handled in a more attentive manner. Additionally, we have been revisiting older datasets, resulting in the development of new manuscripts, which will improve the group's long-term impact, and help minimize some of the negative impacts listed above.
Rajamohan, A., Danka, R.G., Hopkins, B.K., Rinehart, J.P. 2020. A non-activating diluent to prolong in vitro viability of Apis mellifera spermatozoa: Effects on cryopreservation and on egg fertilization. Journal of Cryobiology. 92:124-129. https://doi.org/10.1016/j.cryobiol.2019.11.045.
Torson, A.S., Yocum, G.D., Rinehart, J.P., Nash, S.A., Bowsher, J.H. 2019. Fluctuating thermal regimes prevent chill injury but do not change patterns of oxidative stress in the alfalfa leafcutting bee, Megachile rotundata. Journal of Insect Physiology. https://doi.org/10.1016/j.jinsphys.2019.103935.
Yocum, G.D., Rinehart, J.P., Rajamohan, A., Bowsher, J.H., Yeater, K.M., Greenlee, K.J. 2019. Thermoprofile parameters affect survival of Megachile rotundata during exposure to low-temperatures. Integrative & Comparative Biology. 59(4):1089-1102. https://doi.org/10.1093/icb/icz126.
Wilson, E.S., Murphy, C.E., Rinehart, J.P., Yocum, G.D., Bowsher, J.H. 2020. Microclimate temperatures impact nesting preference in Megachile rotundata. Environmental Entomology. https://doi.org/10.1093/ee/nvaa012.
Slater, G.P., Yocum, G.D., Bowsher, J.H. 2020. Diet quantity influences caste determination in honey bees (Apis mellifera). Proceedings of the Royal Society. 287:20200614. https://doi.org/10.1098/rspb.2020.0614.