Location: Insect Genetics and Biochemistry Research
2020 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. Most efforts focused on the honey bee, which at the beginning of this project plan was the only domesticated animal without a germplasm repository. Initial studies focused on improving upon existing honey bee spermatozoa slow freezing cryopreservation protocols. This led to the creation of Fargo Extender Medium, which was designed to accommodate honey bee specific sperm physiology, resulting in higher quality sperm after removal from storage in liquid nitrogen. However, concerns with the slow freezing technique include the fact that it uses potentially harmful chemicals that could reduce the fitness of artificially fertilized queens and requires specialized equipment that decreases the likelihood of widespread use. To improve the accessibility of honey bee cryopreservation, we developed a new technique in insects that uses rapid freezing in liquid nitrogen. This new technique requires minimal equipment, uses sugars as cryoprotectants, and can be conducted in the field, while still achieving 99% sperm viability. However, successful sperm cryopreservation is only part of a viable germplasm repository. To solve this problem, we also created the world's first cryopreserved honey bee embryo. The embryonic cryopreservation process was improved in subsequent years to improve embryonic survival rates after removal from storage. Taken together, these techniques show 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, which is the first step in developing a germplasm repository for mosquito. A mosquito spermatozoa cryopreservation protocol was developed as well, resulting in high levels of viable sperm after removal from storage. Once these protocols are refined, they will serve as the basis of a repository that will benefit research of these important disease vectors by reducing the dependence on continuous culture of important mosquito strains.
Objective 2: 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 transcriptomics approach, we have identified genes involved in the response to a variety of storage protocols, 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.
Objective 3: Substantial progress was made related to improving insect cold storage. First, we discovered that storing developing alfalfa leafcutting bees under a true thermoperiod (comprised of a thermophase/day temperature and a cryophase/night temperature) significantly improves survival over previously published pulse thermal regime (characterized by a daily brief exposure to high temperature). Although the original thermoperiod protocol resulted in improved survival, a substantial number of bees emerged during storage, which would be problematic for pollination service providers. In order to ameliorate this issue, we developed thermoperiods with lower cryophase temperatures and found that bees can survive exposure to thermoperiod with cryophase temperature as low as 23 degrees F, and premature emergence no longer occurred. Secondly, we investigated how both oxygen and carbon dioxide concentrations affect bees during storage. As cavity nesters, alfalfa leafcutting bees may experience atmospheric gas concentrations during development and overwintering significantly different from what is considered "normal." Previous data from our research group indicated that varying these gases during development can have profound implications on survival and pollinator quality. However, more recent work indicates that alfalfa leafcutting bee pupae exposed to wide range of oxygen and carbon dioxide ratios during low-temperature storage had no notable effect on survival. This would indicate that bee managers will not need to be concerned about the possible buildup of carbon dioxide or the depletion of oxygen during storage. Finally, we conducted experiments of the effects of transportation stress on honey bees. Modern apiculture creates a wide range of stressors that could negatively impact colony health. Our research team has taken particular interest in the stresses incurred during transportation. To characterize key stressors incurred during transportation, we developed "smart hive" sensors that monitor temperature, wind speed, barometric pressure, and vibrations during transportation. During road testing in North Dakota, preliminary data indicates that vibration is more problematic than temperature or barometric pressure during a short distance move. To identify underlying physiological processes activated during transportation, we worked with collaborators to collect RNA samples during industry-standard movement from North Dakota to California. Transcriptomic results indicate that transportation induced a significant differential gene expression, the physiological interpretation of which is currently underway.
Objective 4: Progress was made on the characterization of 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 first conducted RNA-seq experiments to identify genes that might regulate diapause. Based on the results of these experiments we then conducted additional experiments to examine the potential diapause regulation role of genes in the insulin pathway. Results from this experiment indicate that while the insulin pathway plays a role in diapause regulation in this species, other factors are also involved. Concurrent with these experiments was an investigation into how micro-environmental factors affect diapause progression. This was accomplished by following bees overwintering in a controlled environment with those overwintered under natural conditions, and conducting transcriptomic analysis of multiple timepoints for both treatments. The results from this experiment highlighted the fact that diapause regulation is plastic and varies substantially due to environmental conditions. Other studies involved the blue orchard bee, Osmia lignaria, a newly managed species that is native to North America and is a highly efficient pollinator of orchard crops. We sequenced the genome of this species, and a highly refined complete genome was released publicly during the final year of the project. This genome has been used to characterize a transcriptomics study to characterize diapause in this important alternative pollinator.
Objective 5: Progress has been made on improving storage techniques for the alfalfa leafcutting bee. 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% 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. Since 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.
This project expired in fiscal year 2020, and has been replaced by project #3060-21220-032-00D, in which many of the objectives are continuing.
Accomplishments
1. Complete genome of an orchard crops pollinator. The blue orchard bee, Osmia lignaria is a more efficient pollinator of orchard crops than the honey bee, Apis mellifera. But, unique regionally adapted biotypes of the blue orchard bee are facing possible extinction due to interbreeding with other biotypes brought in by commercial suppliers for pollination services. This loss of these regionally adapted biotypes will have a long-term negative impact on tree fruit production in regions including the Pacific Northwest. To address this problem, ARS researchers in Fargo, North Dakota, have developed a highly refined complete genome of the blue orchard bee. ARS researchers at Logan, Utah, are currently using this new genome to determine the geographic distribution of blue orchard bee biotypes. These effects will significantly contribute to the conservation of this commercially important pollinator.
