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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Insect Genetics and Biochemistry Research » Research » Research Project #428476

Research Project: Cryopreservation of Bee Germplasm Research

Location: Insect Genetics and Biochemistry Research

2019 Annual Report


Objectives
Objective 1. Develop means for long-term storage of bee germplasm, including cryopreservation of embryos. [NP 305, Component 2, Problem Statements 2A, 2B, 2C] Objective 2: Develop storage technologies for cryopreservation of insect germplasm (e.g., for screwworms, honey bees, moths, and tephritid fruit flies) and low-temperature storage of solitary pollinators. [NP304, Component 3, Problem Statements 3A2 and 3B2] Objective 3: Isolate biomarkers to monitor diapause development in managed solitary pollinators and to measure population diversity of economically important species (such as solitary bee pollinators and Lygus). [NP304, Components 1 and 3, Problem Statements 1A, 1C, and 3A2] Objective 4: For economically important species (such as tephritid fruit flies, screwworms, honey bees and solitary bee pollinators), develop quality control biomarkers through the characterization of sub-lethal effects (developmental and physiological) resulting from storage. [NP304, Component 3, Problem Statements 3A2 and 3B2]


Approach
Pollinating insects are important to the U.S. agricultural economy, contributing an estimated $16 billion to annual crop production. This critical component is currently at risk due to a dramatic decrease in managed and native pollinators, and will face additional challenges due to climate change in the future. Despite this importance, there is no organized germplasm biorepository (genebank) for bees. This project will develop protocols that support the establishment of a National Bee Germplasm Repository. Specifically, the project will focus on: 1) the development of an improved protocol for the cryopreservation of bee spermatozoa, 2) the development of a novel protocol for the cryopreservation of bee embryos, and 3) the development of a protocol for the in vitro rearing of embryos after storage into reproductively viable adults. This research will ultimately result in the preservation of elite and genetically diverse pollinator strains, the development of a cryogenically-based system for the safe importation of bee germplasm, and the systematic delivery of high-quality germplasm and insects to end users.


Progress Report
Objective 1: Substantial progress has been made towards the development of insect cryopreservation protocols. Previously, we had reported on the development of a slow freezing technique for the cryoprotection of honey bee sperm. Although this technique is highly effective, it requires the use of expensive, specialized equipment that is not readily available to bee keepers, and cannot be used under field conditions. To improve the accessibility of honey bee cryopreservation, we have developed a new technique that uses rapid freezing in liquid nitrogen. This new technique requires minimal equipment and can be conducted in the field, while still achieving 99% sperm viability. Hence, this technique shows promise in extending the accessibility of crucial honey bee lines, and preserving a wide range of honey bee genetic diversity. Progress was made on the cryopreservation of other insect species as well. In collaboration with the Centers for Disease Control Foundation, we have successfully screened and identified non-toxic cryoprotectants needed for mosquito larval cryopreservation. This is the first step in developing a germplasm repository for mosquito. When in place, this repository will benefit research of these important disease vectors by reducing the dependence on continuous culture of important mosquito strains. Objective 2: Research continues on the characterization of the consequences of insect cryobanking procedures. Specifically, while both slow and rapid freezing cryopreservation techniques for honey bee sperm have yielded high viability scores based on laboratory tests, whether the sperm can be used to produce viable larvae had not yet been determined. In the current reporting period, slow frozen spermatozoa were successfully used to artificially inseminate virgin queens that went on to produce brood and fully functional colonies. This clearly demonstrates that honey bee genetic diversity can be safe guarded by cryopreservation of honey bee sperm. How rapidly frozen semen performs during artificial insemination is currently being assessed. Objective 3: Progress has been made on improving storage techniques for the alfalfa leafcutting bee. During normal management protocols, developing bees of this species are often exposed to low temperatures to synchronize peak pollinator activity with crop bloom, a process that is expected to increase in frequency and duration as climates continue to change. Although it is well known that mortality occurs if the low temperature exposure is severe enough, less severe exposure can negatively impact bee morphology and physiology (collectively known as sublethal effects). We have previously demonstrated that survival rates can be improved by using a fluctuating thermal regime (FTR), although the sublethal effects were not investigated. Recent research indicates that although FTR significantly increase survival, sublethal effects were induced: after only 1 week in FTR, about 1 percent of the bees had abnormal mouth parts, and after 4 weeks of FTR, approximately 25% of the bees were not able to fly. Additionally, although the sublethal effects of temperature stress are normally thought of as negative impacts, we have discovered potentially positive effects as well. Bees exposed to 1 week of FTR during development are significantly more likely to produce offspring that will enter an overwintering state (diapause) than offspring produced by untreated bees. Because bees failing to enter diapause has been implicated in the spread of foul brood disease, controlling the diapause status of offspring by a simple thermal treatment of the parental generation could be a viable option towards controlling this important alfalfa leafcutting bee disease. The importance of temperature during pollinator management may extend into the field as well. Currently, there is a disparity whereby American alfalfa farmers have a rate of return of less than 1 (meaning that each female released into the field produces less than one viable offspring to be used the following year), while Canadian farmers regularly benefit from rates of return of 2-3 offspring per released female. This results in this species not being domestically sustainable, requiring the annual purchase of large quantities of alfalfa leafcutting bees from Canada. The mechanism for this disparity in yield between American and Canadian farmers is not known, but we have hypothesized that the temperature of nest material in the field may be a significant factor. To test this hypothesis, instead of following the common practice of facing our nesting material to the southeast to receive early morning sun so that bees will become active earlier in the day, we constructed nest sites with four sides. We demonstrated that alfalfa leafcutting bees prefer to nest on the cooler northeast and northwest sides of the structure, and that bees nesting in the northeast side produced more offspring than those on the southeast side. These results indicate that temperature manipulation of the bee nests in the field may help increase American yields. Objective 4: Research continues on characterizing the molecular mechanisms controlling insect dormancy. Managing dormancy (also known as diapause) is a key component in the management for the alfalfa leafcutting bee. These bees are active in the field for only three to four months of the year, while the rest of their time is spent as diapausing prepupae in overwintering storage. To develop a more thorough understanding of this important life stage, we had previously conducted RNA-seq experiments to identify genes that might regulate diapause. Based on the results of these experiments we have now conducted additional experiments to examine the potential diapause regulation role of genes in the insulin pathway. Initial experiments indicated that diapause regulation is plastic and varies depending on environmental conditions. Currently running follow-up experiments have been designed with more time points to give us a better view of what genes are being expressed when. The applied implication of these studies is that the overwintering storage conditions will affect the alfalfa leafcutting bee physiology and therefore will impact their ability to pollinate and reproduce. Objective 5: As previously mentioned for Objective 3, we are working on improving storage techniques for the alfalfa leafcutting bee. Although we have developed several techniques for improving pollinator quality and quantity, the underlying mechanisms of these improvements has not been well described. Using a RNA-seq approach, we have identified genes involved in the response to improved storage techniques, including those that respond in as little as one day of treatment. In the future, these genes can serve as molecular biomarkers accelerating the development of more optimal low-temperature storage conditions for spring developing alfalfa leafcutting bees.


Accomplishments
1. From frozen embryo to viable adult. As the honey bee faces ever increasing threats to its survival, the establishment of a National Honeybee Germplasm Repository is essential to the conservation of this critical insect species. ARS researchers in Fargo, North Dakota, and Baton Rouge, Louisiana, recently reported on the successful cryopreservation of honey bee embryos; however, recovering those embryos from storage and rearing them to the adult stage has remained elusive. By building upon successful techniques for embryonic cryopreservation, researchers have now developed a preliminary protocol to rear adult bees from cryopreserved embryos, thereby overcoming a key technical hurdle in establishing a vital repository for the bee industry.

2. Honey bee transportation stress. Although pollination services require honey bee colonies to be transported thousands of miles each year, little is known about the effect of transportation on bee health. To investigate the potential impact of transportation stress on honey bee health, ARS researchers in Fargo, North Dakota, monitored colony temperatures and individual bee gene expression during their fall transportation route between North Dakota and California. Colony temperatures varied significantly by position on the truck, and these temperature differences appeared to contribute to stress during transportation. Furthermore, molecular data demonstrated that genes associated with stress were upregulated by the cross-country move, and were still being differentially expressed 3 weeks later. These results indicate that transportation is a significant stressor affecting honey bee health, but also suggest that bee health issues may be mitigated by the pollinator services industry by improved truck design.


Review Publications
Bennett, M., Rinehart, J.P., Yocum, G.D., Yocum, I.S. 2019. A precise and autonomous system for the detection of insect emergence patterns. Journal of Visualized Experiments. 143:e58362. https://doi.org/10.3791/58362.
Melicher, D.M., Su, K., Meier, R., Bowsher, J.H. 2018. Comparative analysis reveals the complex role of histoblast nest size in the evolution of novel insect abdominal appendages in Sepsidae (Diptera). BMC Evolutionary Biology. 18:151. https://doi.org/10.1186/s12862-018-1265-3.
Melicher, D.M., Wilson, E.S., Bowsher, J.H., Peterson, S., Yocum, G.D., Rinehart, J.P. 2019. Long-distance transportation causes temperature stress in the honey bee, Apis mellifera (Hymenoptera: Apidae). Environmental Entomology. 48(3):691-701. https://doi.org/10.1093/ee/nvz027.
Melicher, D.M., Torson, A.S., Anderson, T.J., Yocum, G.D., Rinehart, J.P., Bowsher, J.H. 2019. Immediate transcriptional response to a temperature pulse under a fluctuating thermal regime. Integrative & Comparative Biology. 59(2):320-337. https://doi.org/10.1093/icb/icz096.