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.
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?
Objective 1: Research continues on the effects of low-temperature storage on pollinator quality after storage. Previously, we demonstrated that giving the alfalfa leafcutting bee, Megachile rotundata, a daily one hour high-temperature pulse subjected during low-temperature storage (which is called a fluctuating thermal regime or FTR) significantly increases bee survival. More recently, we demonstrated that survival could be further improved by exposing the bees to more ecologically relevant thermoperiods. Our current working hypothesis for the underlying mechanism of this improved survival is that the time at the higher temperature enables the bees to repair any physiological damage that occurred during the low-temperature exposure, and that these repairs would be associated with an increase in the production of metabolic heat. To test this hypothesis, we are using high-sensitive calorimetry to measure metabolic heat before, during, and after FTR exposure. This data will help us identify when repair mechanisms have been activated and will inform us as to when to collect samples for RNA-seq experiments to characterize the underlying molecular mechanisms. During this reporting period we finished all calorimetry experiments and started data analysis. Although the FTR protocol increases survival, we have discovered some bees suffer from physiological defects (sublethal effects) that will decrease their ability to pollinate. We have hypothesized that the low-temperature period (cryophase) of the FTR causes the most damage, and that raising the temperature of the cryophase would decrease both lethal and sublethal effects. During this reporting period we finished analyses of experiments and demonstrated that pollinator quality can be improved over that seen in the FTR protocol with thermoperiods whose cryophase temperature was increased from 6°C to 12 or 15°C. Although we have extensive laboratory data on the effects of cold exposure on pollinator quality, observations may differ under field conditions. To investigate the possibility of cold exposure impacting pollination services, a series of field experiments were carried out in which bees were pretreated in the laboratory and then released into the field. Bees exposed to FTR were more likely to nest as compared to bees exposed to either control or constant 6°C bees. Interestedly, those bees from the constant 6°C treatment that did nest produce larger offspring than the control bees. During this reporting period we completed data analysis on this project. Objective 2: Progress has been made towards the development of transferrable and quality proven germplasm cryopreservation technologies, specifically for embryonic stage male germplasm of pollinating insects. For male germplasm storage, we deviated from our original plan to improve the quality of cryopreserved honey bee spermatozoa due to maximized telework constraints. Instead, our primary goal was to extend the procedure designed for the honey bees to other pollinators, especially the bumble bees and native bee pollinators. By making significant changes to our honey bee semen cryopreservation, including changing the semen extender medium as well as the seminal source(the seminal vesicle instead of collected ejaculate), we were able to cryopreserve Bombus impatiens spermatozoa, which represents the first germplasm cryopreservation for a bumble bee species. Embryonic studies continued in honey bees, with progress being made to improve the embryonic viability after permeabilization to allow uptake of cryoprotectants. This includes the development of a reliable in vitro rearing protocol, which is needed to attain the full commercial impact of embryonic cryopreservation. We improved our in vitro rearing protocol for honey bee larvae, and conducted comparative studies on the in vitro development of both the permeabilized and the untreated embryos and larvae.
1. Cryopreservation of bumble bee sperm. Bumble bees are not only critical pollinators of agriculturally important crops, but they also include approximately 54 species native to North America, many of which are in decline. Despite this importance, there has been no procedure to cryopreserve their germplasm. ARS researchers in Fargo, North Dakota, reported on the first successful cryopreservation and revival of bumble bee sperm cells, resulting in 55% survival, which is expected to be sufficient to artificially inseminate a queen bumble bee and obtain progeny. These results will serve as a foundation for the development of protocols that will be essential to safeguard important traits in domesticated pollinator species and to preserve the diversity of declining ones.
2. Ultra-low temperature storage of larval mosquitoes. Mosquitos are one of the world’s most dangerous disease vectors, causing significant negative impacts to society. A key limiting factor to the study of mosquito species is that rearing them is a costly and labor-intensive endeavor. Researchers at the ARS laboratories in Fargo, North Dakota, collaborating with scientists at the Centers for Disease Control in Atlanta, Georgia, have been working to develop low-temperature storage protocols to reduce the burden of mosquito rearing. With a goal of developing a cryopreservation protocol, they have determined the extent to which multiple cryoprotectants in over 20 different combinations could be used to successfully store mosquito larvae with minimal damage. The discovery of several combinations showing low toxicity will provide new opportunities for preserving and studying mosquitos.
3. Improved low-temperature storage of the alfalfa leafcutting bee. When managed for pollination services, the alfalfa leafcutting bee is commonly stored at constant low temperatures in the spring to slow their development so that peak nesting activity coincides with peak crop bloom. Interrupting the low temperature storage with a short daily high-temperature pulse (fluctuating thermal regime, FTR) significantly increases bees’ survival. However, commercial rearing chambers are not capable of rapidly changing the temperature as required for a FTR protocol, due to the large mass of bees commonly found in production agricultural settings. ARS researchers in Fargo, North Dakota, have developed new FTR protocols requiring significantly smaller temperature changes that don’t jeopardize the bees’ survival. These novel storge protocols will decrease energy use and stress on the chamber’s refrigeration system and ensure high quality bees.
Yocum, G.D., Rajamohan, A., Rinehart, J.P. 2021. Comparison of fluctuating thermal regimes and commercially achievable constant-temperature regimes for short-term storage of the alfalfa leafcutting bee (Hymenoptera: Megachilidae). Journal of Economic Entomology. https://doi.org/10.1093/jee/toab019.
Campbell, J.B., Dosch, A., Hunt, C.M., Dotson, E.M., Benedict, M.Q., Rajamohan, A., Rinehart, J.P. 2021. Physiological responses to cryoprotectant treatment in an early larval stage of the malaria mosquito, Anopheles gambiae. Journal of Cryobiology. https://doi.org/10.1016/j.cryobiol.2020.12.001.
Campion, C.C., Rajamohan, A., Rinehart, J.P. 2021. Comparative analysis of cryopreservation of seminal vesicle derived spermatozoa from Bombus impatiens and Apis mellifera - Implications for artificial insemination of bumble bees. Journal of Cryobiology. 102:136-139. https://doi.org/10.1016/j.cryobiol.2021.06.002.